49 Annual Drosophila Research Conference • Program and Abstracts

49th Annual Drosophila Research Conference • Program and Abstracts Web site: www.genetics-gsa.org site: Web e-mail: [email protected] Conference site: www.drosophila-conf.org site: Conference The Genetics Society of America 9650 Rockville Pike, Bethesda, MD 20814-3998 Pike, 9650 Rockville Telephone: 301/634-7300 • Fax: 301/634-7079 Fax: 301/634-7300 • Telephone: 49TH ANNUAL DROSOPHILA RESEARCH CONFERENCE

April 2–6, 2008 Town and Country Hotel & Conference Center San Diego, California

Program and Abstracts Volume

• 2008 Meeting Organizers Nancy Bonini, University of Pennsylvania Susan Celniker, Lawrence Berkeley National Laboratory Brian Oliver, NIDDK, NIH, HHS John Tamkun, University of California, Santa Cruz

• 2007/2008 Drosophila Board of Directors Officers and Regional Representatives* President Utpal Banerjee University of California, Los Angeles President-Elect Carl Thummel University of Utah Past-President Trudy MacKay North Carolina State University Past-President & Elections Chair Mark Krasnow Stanford University Past-President Lynn Cooley Yale University Treasurer Michael Bender University of Georgia Canada Howard Lipshitz University of Toronto Great Lakes Amanda Simcox Ohio State University Northwest Jim Truman University of Washington Southeast Rebecca Kellum University of Kentucky California Graeme Davis University of California, San Francisco Heartland Susan Abmayr Stowers Institute for Medical Research New England Mitzi Kuroda Harvard University Medical School Mid-Atlantic Liz Gavis Princeton University Midwest Pam Geyer University of Iowa

• International Representatives Australia/Oceana Phil Batterham University of Melbourne Asia Vijay Raghavan The National Centre for Biological Sciences Europe Barry Dickson Research Institute of Molecular Pathology

*2008/2009 Board of Directors will be listed in the Program Addendum and take office following the 2008 Drosophila Research Conference.

• Sponsored by The Genetics Society of America 9650 Rockville Pike, Bethesda, MD 20814-3998 Telephone: 301/634-7300 Fax: 301/634-7079 e-mail: [email protected] Web site: www.genetics-gsa.org Conference site: www.drosophila-conf.org

TABLE OF CONTENTS 3

Schedule of Events ...... 4 Map: Town and Country Resort & Conference Center ...... 11 General Information ...... 12 Larry Sandler Memorial Lecture ...... 14 The DeLill Nasser Award for Professional Development in Genetics...... 14 Conversations in Genetics, Volume III...... 15 Guidelines for Making Platform Presentations ...... 16 Guidelines for Making Poster Presentations...... 17 Exhibitors...... 18 Floorplan: Posters and Exhibits, Grand Exhibit Hall ...... 21 Opening General Session and Plenary Sessions I and II ...... 22 Workshops ...... 23 Platform Sessions ...... 27 Poster Sessions ...... 39 Abstracts Introduction ...... 81 Platform Sessions...... 83 Poster Sessions ...... 142 Speaker and Author Index...... 394 Keyword Index ...... 409 FlyBase Genetic Index to Abstracts...... 416

FUTURE CONFERENCE DATES

2009 2012 March 4–8 March 7–11 Chicago, IL Chicago, IL

2010 2013 April 7–11 April 3–7 Washington, DC Washington, DC

2011 March 30–April 3 San Diego, CA 4 SCHEDULE OF EVENTS 49th Annual Drosophila Research Conference

WEDNESDAY, APRIL 2 12:00 noon - 6:00 pm Ecdysone Workshop Pacific Ballroom Organizers: Craig Woodard, Mt. Holyoke, South Salons 2-3 Hadley, Massachusetts; and Randall S. Hewes, University of Oklahoma, Norman 3:00 pm - 6:00 pm Drosophila Board of Directors Meeting Pacific Ballroom Salon 1 3:30 pm - 9:00 pm Registration and Book Pickup Open Atlas Foyer 7:00 pm - 9:00 pm Opening General Session Atlas Ballroom 7:00 pm Welcome and Opening Remarks Susan Celniker, Lawrence Berkeley National Laboratory, Berkeley, California 7:05 pm Introduction of Larry Sandler Memorial Lecture Helen Salz, Case Western Reserve University, Cleveland, Ohio 7:15 pm Larry Sandler Memorial Lecture 8:00 pm Memoriam to Seymour Benzer: the Father of Fly Neurogenetics Nancy Bonini, University of Pennsylvania, Philadelphia 8:05 pm Introduction of Historical Lecture William McGinnis, University of California, San Diego 8:15 pm Historical Lecture Antonio Garcia-Bellido, Facultad de Ciencias, Universidad Autonoma de Madrid, Spain 9:00 pm - 12:00 am Mixer/Reception Terrace In case of rain, reception will be held in the Pavilion/Poolside Golden Ballroom THURSDAY, APRIL 3 7:15 am - 8:30 am Continental Breakfast Atlas Foyer 8:00 am - 5:00 pm Registration Area and Book Pickup Atlas Foyer 8:30 am - 12:00 noon Plenary Session I Atlas Ballroom Moderators: John Tamkun, University of California, Santa Cruz; and Nancy Bonini, University of Pennsylvania, Philadelphia SCHEDULE OF EVENTS 5 49th Annual Drosophila Research Conference

8:30 am Image Award Presentation John Tamkun, University of California, Santa Cruz 8:35 am Trafficking and Polarity in the Control of Drosophila Growth David Bilder, University of California, Berkeley 9:00 am Ig-receptor Diversity in Insect Immunity and Neuronal Wiring Dietmar Schmucker, Harvard Medical School, Cambridge, Massachusetts 9:30 am Pathways of Anti-Viral Immunity Sara Cherry, University of Pennsylvania, Philadelphia 10:00 am Break Sponsored by Genetic Services, Inc. (booth 109) 10:30 am Cross-Regulation of HOX and Sex Determination Genes in Development and Evolution Artyom Kopp, University of California, Davis 11:00 am Inheritance of Polycomb Proteins through DNA Replication in Vitro Nicole Francis, Harvard University, Cambridge, Massachusetts 11:30 am MicroRNA Functions Stephen Cohen, Temasek Life Sciences Laboratory, Singapore 12:00 noon - 1:30 pm Networking Box Luncheon Golden Pacific Ballroom 1:00 pm - 6:00 pm FlyBase Demo Room Open for Tutorials and Royal Palm Discussion Ballroom 1-3 Presentations: 1:30 pm-2:00 pm: Bulk Data Tools for Biologists 3:00 pm-3:30 pm: Accessing Data from the 12 Drosophila Genomes 1:00 pm - 6:00 pm FlyMine Demonstrations Royal Palm Presentations at 2:30 pm and 4:00 pm Ballroom 4 2:00 pm - 4:00 pm Exhibits and Poster Viewing Grand Exhibit Hall 2:00 pm-3:00 pm: Even-Numbered Poster Authors 3:00 pm-4:00 pm: Odd-Numbered Poster Authors 3:30 pm - 4:30 pm "Meet the Board" Reception Atlas Foyer 6 SCHEDULE OF EVENTS 49th Annual Drosophila Research Conference

4:30 pm - 6:30 pm Concurrent Platform Sessions Immune System and Cell Death Golden West Neurophysiology and Behavior Town & Country Organogenesis California 8:00 pm - 11:00 pm Exhibits and Poster Viewing Grand Exhibit Hall 8:00 pm-9:00 pm: "A" Poster Authors 9:00 pm-10:00 pm: "B" Poster Authors 10:00 pm-11:00 pm: "C" Poster Authors FRIDAY, APRIL 4 8:30 am - 12:30 pm Concurrent Platform Sessions Regulation of Gene Expression Town & Country Evolution and Quantitative Genetics Golden West Cytoskeleton and Cell Biology California 8:30 am - 5:00 pm Registration Area and Book Pickup Atlas Foyer 9:00 am - 6:00 pm FlyBase Demo Room Open for Tutorials and Royal Palm Discussion Ballroom 1-3 Presentations: 1:30 pm-2:00 pm: Easy Tools for Power Searching 3:00 pm-3:30 pm: Bulk Data Tools for Biologists 9:00 am - 6:00 pm FlyMine Demonstrations Royal Palm Presentations at 2:30 pm and 4:00 pm Ballroom 4 10:15 am - 10:45 am Break Atlas Foyer 12:45 pm - 1:45 pm GSA Mentor Roundtable Luncheon Royal Palm Advanced Sign Up Required Ballroom 5-6 1:45 pm - 3:45 pm Concurrent Workshops Gases in Drosophila Physiology and Pacific Ballroom Development Salons 6-7 Organizers: Greg Beitel, Northwestern University, Evanston, Illinois; and Eric Johnson, University of Oregon, Eugene ModENCODE Pacific Ballroom Organizer: Kevin White, University of Chicago, Salon 1 Illinois Monoamines Pacific Ballroom Organizer: Kyung-An Han, Pennsylvania State Salon 2 University, University Park SCHEDULE OF EVENTS 7 49th Annual Drosophila Research Conference

Cell Cycle and Checkpoints Pacific Ballroom Organizers: Tin Tin Su, University of Colorado, Salon 3 Boulder; and Claudio Sunkel, Instituto de Biologia Molecular e Celular and Universidade do Porto, Portugal 2:00 pm - 4:00 pm Visit the Exhibits Grand Exhibit Hall 4:30 pm - 6:30 pm Concurrent Platform Sessions Gametogenesis Golden West Signal Transduction Town & Country Pattern Formation California 8:00 pm - 11:00 pm Exhibits and Poster Viewing Grand Exhibit Hall 8:00 pm-9:00 pm: "C" Poster Authors 9:00 pm-10:00 pm: "B" Poster Authors 10:00 pm-11:00 pm: "A" Poster Authors SATURDAY, APRIL 5 8:30 am - 10:15 am Concurrent Platform Sessions Drosophila Models of Human Diseases Town & Country RNA Biology Golden West Genome and Chromosome Structure California 8:30 am - 4:00 pm Registration Area and Book Pickup Atlas Foyer 9:00 am - 5:00 pm FlyBase Demo Room Open for Tutorials and Royal Palm Discussion Ballroom 1-3 Presentations: 1:30 pm-2:00 pm: Accessing Data from 12 Drosophila Genomes 3:00 pm-3:30 pm: Easy Tools for Power Searching 9:00 am - 6:00 pm FlyMine Demonstrations Royal Palm Presentations at 2:30 pm and 4:00 pm Ballroom 4 10:15 am - 10:45 am Break Atlas Foyer 10:45 am - 12:30 pm Concurrent Platform Sessions Drosophila Models of Human Diseases Town & Country

(continued) Techniques and Functional Genomics Golden West Chromatin and Gene Expression California 8 SCHEDULE OF EVENTS 49th Annual Drosophila Research Conference

12:45 pm - 1:45 pm Coalition for the Life Sciences Trellises Garden Limited attendance. Sign up by 5:00 pm on April Grill 4 at Drosophila registration desk. 1:30 pm - 3:30 pm Exhibits and Poster Viewing Grand Exhibit Hall 1:30 pm-2:30 pm: Odd-Numbered Poster Authors 2:30 pm-3:30 pm: Even-Numbered Poster Authors 4:00 pm - 6:00 pm Concurrent Platform Sessions Physiology and Aging Golden West Neurogenetics and Neural Development Town & Country Cell Division and Growth Control California 6:45 pm - 8:45 pm Concurrent Workshops The Maternal to Zygotic Transition: Pacific Ballroom Deciphering the Ultimate Genetic Switch Salons 6-7 Organizers: John Sisson, University of Texas, Austin; and Howard Lipshitz, University of Toronto, Canada Drosophila Research and Pedagogy at Pacific Ballroom Primarily Undergraduate Institutions (PUI) Salon 3 Organizers: Don Paetkau, Saint Mary's College, Notre Dame, Indiana; and Mark Hiller, Goucher College, Baltimore, Maryland Chromosome Pairing and Trans-sensing Pacific Ballroom Effects Salon 1

Organizer: Giovanni Bosco, University of Arizona, Tucson Extracellular Matrix Pacific Ballroom Organizer: Halyna Shcherbata, University of Salon 2 Washington, Seattle Drosophila Population Genomics Pacific Ballroom Organizers: Chuck Langley, University of Salons 4-5

California, Davis; Chip Aquadro, Cornell University; and Andy Clark, Cornell University 7:00 pm - 10:00 pm Open Poster Viewing Grand Exhibit Hall Last opportunity to view posters before program closes at 10:00 pm 9:30 pm - 11:30 pm Concurrent Workshops Longevity and Functional Senescence - Pacific Ballroom Catch the Rhythm Salons 6-7 Organizers: Larry Harshman, University of SCHEDULE OF EVENTS 9 49th Annual Drosophila Research Conference

Nebraska, Lincoln; John Tower, University of Southern California, Los Angeles; Mike Grotewiel, Virginia Commonwealth University, Richmond; and RJ Wessells, University of Michigan, Ann Arbor Cell Death Pacific Ballroom Organizers: Andreas Bergmann, M.D. Anderson Salon 1

Cancer Center, Houston, Texas; and Jamie Rusconi, University of Albany, New York RNA Control and Developmental Processes Pacific Ballroom

Organizer: Talila Volk, Weismann Institute, Israel Salon 2 Immunity and Pathogensis Pacific Ballroom Organizers: Brian Lazzaro, Cornell University, Salon 3

Ithaca, New York; and Louisa Wu, University of Maryland, College Park SUNDAY, APRIL 6 8:30 am - 12:00 noon Plenary Session II Atlas Ballroom Moderators: Brian Oliver, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland; and Susan Celniker, Lawrence Berkeley National Laboratory, Berkeley, California 8:30 am Poster Award Presentation Brian Oliver, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland 8:35 am Modeling Emotional Behavior in Drosophila David Anderson, California Institute of Technology, Pasadena, California 9:00 am Using Electrophysiology and Genetics to Dissect Neural Circuit Function Rachel Wilson, Harvard Medical School, Boston, Massachusetts 9:30 am Quantitative Analysis of the Transcription Network Controlling Early Embryo Patterning Mark Biggin, Lawrence Berkeley National Laboratory, Berkeley, California 10:00 am Break 10:30 am Regulatory Genomics in Drosophila Species Manolis Kellis, Massachusetts Institute of Technology, Cambridge, Massachusetts 10 SCHEDULE OF EVENTS 49th Annual Drosophila Research Conference

11:00 am Two or Four Bristles: Evolution of Regulation of the achaete-scute Genes Pat Simpson, University of Cambridge, United Kingdom 11:30 am Regulation Oogenesis: from Stem Cells to Steroids Allan Spradling, Carnegie Institute of Washington, Baltimore, Maryland 12:00 noon Meeting Close Susan Celniker, Lawrence Berkeley National Laboratory, Berkeley, California

Poster Award Competition for Students and Postdocs

Did you know that your poster could win an award? The Genetics Society of America is sponsoring a competition based on scientific merit and poster clarity. There will be a first ($500), second ($300) and third ($200) place award given in each category. Judging will take place throughout the Conference and winners will be announced at Sunday’s Plenary Session. Good Luck! TOWN AND COUNTRY RESORT & CONFERENCE CENTER 11 12 GENERAL INFORMATION

Badges Badges are required for admission to all sessions, the opening mixer, and the posters and exhibits in the Grand Exhibit Hall. Security officers will not allow individuals without badges to enter the Grand Exhibit Hall. If you lose your badge, a replacement may be requested at the Registration and Information Counters during posted registration hours. Badges may not be used by anyone other than the registered attendee. Each attendee must have his/her own badge.

Employment Opportunities/Seeking Employment Notices – Atlas Foyer Individuals and institutions offering or seeking employment may post notices and résumés on the “Employment Opportunities” bulletin boards set up near the Registration and Information Counters in the Atlas Foyer.

FlyBase Demonstrations – Royal Palm Ballroom 1-3 FlyBase invites all Conference registrants to come to the demo room to learn how to make the best use of the new FlyBase tools and features for your research and teaching. Throughout the day, other than the scheduled group presentations noted below, FlyBase personnel are available in the demo room for one-on-one tutorials, troubleshooting and discussions. Any thoughts on improvements we can make to FlyBase are gratefully appreciated.

Thursday, April 3 1:00 pm–6:00 pm Demo room open for tutorials and discussions Presentations 1:30 pm–2:00 pm: Bulk Data Tools for Biologists 3:00 pm–3:30 pm: Accessing Data from the 12 Drosophila Genomes

Friday, April 4 9:00 am–6:00 pm Demo room open for tutorials and discussions Presentations 1:30 pm–2:00 pm: Easy Tools for Power Searching 3:00 pm–3:30 pm: Bulk Data Tools for Biologists

Saturday, April 5 9:00 am–5:00 pm Demo room open for tutorials and discussions Presentations 1:30 pm–2:00 pm: Accessing Data from the 12 Drosophila Genomes 3:00 pm–3:30 pm: Easy Tools for Power Searching

FlyMine Demonstrations – Royal Palm Ballroom 4 FlyMine is an integrated database of genomics and proteomics data for Drosophila and other insects. Come and see us to find out how you can easily run a range of diverse queries across these data sources, construct your own data mining queries and find out how you can query FlyMine with your own experimental results.

FlyMine will be available throughout the meeting for questions, demonstrations, help with specific queries and user feedback. In addition we will give presentations at times listed below where we will provide an overview of all the main features of FlyMine and how you can query the datasets available.

Thursday, April 3 1:00 pm–6:00 pm Presentations: 2:30 pm–3:00 pm and 4:00 pm–4:30 pm

Friday, April 4 9:00 am–6:00 pm Presentations: 2:30 pm–3:00 pm and 4:00 pm–4:30 pm

Saturday, April 5 9:00 am–6:00 pm Presentations: 2:30 pm–3:00 pm and 4:00 pm–4:30 pm

GSA Mentor Roundtable Luncheon – Royal Palm Ballroom 5-6 This session will be held on Friday, April 4, from 12:45 pm–1:45 pm. Principal investigators will discuss their work and give valuable advice and input on career opportunities. For interested graduate and post docs. Fee. Limited attendance.

Coalition for the Life Sciences – Trellises Garden Grill On Saturday, April 5, from 12:45 pm–1:45 pm, Lynn Marquis of the Coalition for the Life Sciences will lead a discussion on why grants are in jeopardy and how scientists can tell their stories to congressional representatives. Sign up at the Drosophila registration desk by 5:00 pm on Friday, April 4. Limited attendance. GENERAL INFORMATION 13

Meals Please note that only three meals are included in the meeting registration: the Opening Mixer on Wednesday night (with heavy hors d’oeuvres and a cash bar), the Continental Breakfast on Thursday morning, and the networking luncheon on Thursday afternoon. All other meals are not part of your Conference registration. For all other meals you may choose from options within the hotel (including grab-and-go food kiosks located in the meeting area for breakfast and lunch) or at nearby locations. Fashion Valley Shopping Center is conveniently located via the foot bridge behind the Royal Palm Tower and features fast-food and a variety of restaurants.

Message Boards – Atlas Foyer Message boards will be located near the Registration and Information counters.

Parking Parking is available at the Town and Country Resort & Conference Center at a rate of $5 per day.

Poster Sessions and Exhibits – Grand Exhibit Hall All posters will be displayed in the Grand Exhibit Hall. The Hall will be open to Conference registrants on a 24-hour basis beginning at 5:00 pm, Wednesday, April 2, until 10:00 pm, Saturday, April 5. Security will be posted at the entrance to the Hall and only individuals with official Drosophila Conference registration badges will be admitted. All attendees are responsible for their own personal items and should not leave items unattended. Guest pass applications may be made at the registration counter during regular registration hours.

Exhibit representatives will be in their booths: Thursday, April 3: 2:00 pm–4:00 pm and 8:00 pm–11:00 pm Friday, April 4: 2:00 pm–4:00 pm and 8:00 pm–11:00 pm Saturday, April 5: 1:30 pm–3:30 pm and 7:00 pm–10:00 pm

Authors are expected to be present at their boards according to the following schedule: Thursday, April 3: 2:00 pm–3:00 pm Even-numbered posters 3:00 pm–4:00 pm Odd-numbered posters 8:00 pm–9:00 pm “A” posters 9:00 pm–10:00 pm “B” posters 10:00 pm–11:00 pm “C” posters Friday, April 4: 8:00 pm–9:00 pm “C” posters 9:00 pm–10:00 pm “B” posters 10:00 pm–11:00 pm “A” posters Saturday, April 5: 1:30 pm–2:30 pm Odd-numbered posters 2:30 pm–3:30 pm Even-numbered posters 7:00 pm–10:00 pm Open poster viewing (Authors not required to be present)

All posters must be removed from poster boards no later than 11:00 pm on Saturday, April 5. After that time, remaining posters will be removed by vendors and may be lost or thrown away. The GSA Administrative Office does not take responsibility for posters that are not removed by 11:00 pm on Saturday.

Registration and Book Pickup – Atlas Foyer Conference registration and book pickup will be open in the Atlas Foyer as follows: Wednesday, April 2 3:30 pm–9:00 pm Thursday, April 3 8:00 am–5:00 pm Friday, April 4 8:30 am–5:00 pm Saturday, April 5 8:30 am–4:00 pm Sunday, April 6 Closed Note that attendees must be registered before attending the Opening General Session on Wednesday, April 2, 7:00 pm in order to attend that session. Smoking Smoking is only allowed in designated outdoor areas. 14 SANDLER MEMORIAL LECTURE AND NASSER AWARD

Larry Sandler Memorial Lecture

The Larry Sandler Memorial Lecture was established in 1988 by the colleagues, friends and students of Dr. Larry Sandler after his untimely death in February 1987. The award serves to honor Dr. Sandler for his many contributions to Drosophila genetics and his exceptional dedication to the training of Drosophila biologists. Any student completing his Ph.D. in an area of Drosophila research in the calendar year preceding the annual Drosophila Research Conference is eligible and may be nominated by his/her dissertation advisor. The Selection Committee for 2007 includes chair Helen Salz, R. Scott Hawley, Mariana Federica Wolfner, and James W. Erickson. The Committee reviews nominations, reads dissertations of the finalists, and selects the awardee. Past recipients of this honor are:

Bruce Edgar, 1988 David Begun, 1995 James Wilhelm, 2001 Kate Harding, 1989 Chaoyong Ma, 1996 Matthew C. Gibson, 2002 Michael Dickinson, 1990 Abby Dernburg, 1997 Sinisa Urban, 2003 Maurice Kernan, 1991 Nir Hacohen, 1998 Sean McGuire, 2004 Doug Kellogg, 1992 Terence Murphy, 1999 Elissa Hallem, 2005 David Schneider, 1993 Bin Chen, 2000 Daniel Ortiz-Barrientos, 2006 Kendal Broadie, 1994 Yu-Chiun Wang, 2007

The DeLill Nasser Award for Professional Development in Genetics

The DeLill Nasser Award for Professional Development in Genetics was established by The Genetics Society of America in mid-2001 in honor of the late DeLill Nasser, who served for 22 years as the program director of the Eukaryotic Genetics Section at the National Science Foundation. The award fund, made possible by contributions from her family and friends, is growing steadily. It recognizes Dr. Nasser’s contributions to the field of genetics and her strong support of young scientists. Travel and tuition awards will be made annually to allow graduate students and postdoctoral trainees to attend meetings or enroll in laboratory courses. Since 2002, deserving young investigators have been awarded to attend genetics research conferences. 2002 Awardees 2006 Awardees Amy Rice, Indiana University Anjon Audhya, Ludwig Institute for Cancer Research Kristin L. Latham, Oregon State University Gil B. Carvalho, California Institute of Technology Joshua Chern, Baylor College of Medicine Atina G. Cote, Hospital for Sick Children, University of Tim Christensen, Cornell University Toronto Elissa P. Lei, The Johns Hopkins University 2003 Awardee Kirki Tsigari, Alexander Fleming Biomedical Research Sandra M. Leal, Saint Louis University Medical School Institute 2004 Awardee 2007 Awardees Sue L. Jaspersen, University of Colorado, Boulder William W. Ja, CalTech 2005 Awardees Ya-Chieh Hsu, Baylor Joshua C. Mell, University of California, Davis 2008 Awardees Honorable Mention Gilles R. Hickson, University of California, San Francisco Elena A. Repnikova, Texas A&M University Yen-Ping Hsueh, Duke University Roshan A. Jain, Princeton University Chanhee Kang, University of Texas Amanda M. Larracuente, Cornell University Kate M. O’Connor-Giles, University of Wisconsin Mara Schvarzstein, Stanford University Sarit Smolikov, Harvard University School of Medicine

The GSA strongly encourages all of its members and friends to donate. The current fund balance is over $70,000. Up to five percent of the fund amount will be awarded each year. Please make your check payable to The Genetics Society of America and send it to Elaine Strass, GSA, 9650 Rockville Pike, Bethesda, MD 20814-3998. Please write “Nasser Fund” on the bottom left of the check. 16 16 GUIDELINES FOR MAKING PLATFORM PRESENTATIONS

IMPORTANT: • Each platform presenter has a total of 15 minutes: 12 minutes to speak plus 3 minutes for questions/answers/discussion. • Each platform presenter MUST UPLOAD HIS/HER PRESENTATION TO THE MEETING DATABASE NO LATER THAN 24 HOURS BEFORE THE PRESENTATION. • Bring a copy of your presentation on a CD or drive.

Please follow the instructions below so that your talk will be presented accurately: 1. You must test/preview your computer-generated presentation at the Conference, in the meeting room, one hour prior to the beginning of the platform session (i.e., not the beginning time of your specific presentation but the beginning time of the platform session). 2. You must bring a back-up of the presentation on a portable CD or drive. 3. If you must cancel your presentation, or you wish to change presenters, please notify Suzy Brown at the GSA Administrative Office ([email protected]) no later than February 15. Updated information received by that date will be included in the program addendum. 4. Please pay close attention to your presentation time. There are other presenters during the same time slot and we want all presenters to have their full 12 minutes of presenting time. If your Q&A time runs over, please meet later with participants in the lobby outside the meeting room to continue your conversation so that the program can stay on schedule.

If you are unable to upload your presentation in advance, please contact Suzy Brown at [email protected] no later than March 7 and note the following: You must supply your own laptop computer. A data projector (with necessary cables) will be supplied. IMPORTANT: Those using a Macintosh computer must bring their own MAC to VGA adaptor (usually supplied with the computer). If a presenter’s laptop fails, the presenter is responsible to find a replacement, perhaps by borrowing a friend’s laptop. Please disable any password protection or automatic timeout on your laptop.

HELPFUL TIPS FOR EFFECTIVE PRESENTATIONS Your presentation should help clarify ideas, emphasize key points, show relationships, and provide the visual information your audience needs to understand your message. Please consider the following suggestions as you plan your presentation: A. Keep visuals clear and easy to read. Abbreviate your message. Simple graphs, charts and diagrams are much more meaningful to an audience than complex, cluttered ones. When preparing your presentation, limit the information on each screen to a single point or idea, and ideally, not more than 5 lines of text per screen. Keep each screen simple with plenty of open space. B. Avoid using too many patterns and graphics in one frame. C. Use a minimum of words for text and title frames. Five to eight lines per frame and five to seven words per line are the maximum–fewer is better. D. Choose upper and lower case lettering, which is more legible than all capital letters. E. Vary the size of lettering to emphasize headings and subheadings, but avoid using more than three sizes per frame. F. Select sans serif type (example: Arial), which projects better and is easier to read than serif type. G. Maintain the same or similar type sizes from frame to frame, even if some frames have less copy than others. H. Keep all type horizontal, even in charts. I. Consider color with care. A dark background with highly contrasting text and graphics is most readable. Cool colors (example: deep blue, turquoise, purple) appear to recede and make white or light colored text more readable. In one study, blue was found to be the most effective background color for projection. Do not use red for text; it is extremely difficult to read. J. Highlight your main point or heading with a dominant color (example: yellow for the heading, white for body text). Avoid the use of intensely bright or saturated colors that compete with the text. K. Maintain a consistent color scheme. Use no more than six colors throughout your presentation. L. Select backgrounds to enhance your text or graphics. A background that transitions smoothly from lighter to darker shades of the same hue can be effective. Some software packages permit the gradation from one color to another. A textured background can be effective, but it should not detract from or compete with text or images. M. Consider photographs for added interest. Combined with simple, straightforward graphics, illustrations, cartoons and artwork, or photos can bring another dimension to your presentation. N. Remember the basics of good design: Plan a template. Use colors consistently with light fonts on a dark background. Keep text clear and easy to read. GUIDELINES FOR MAKING POSTER PRESENTATIONS 17

All posters will be located in the Grand Exhibit Hall of the Town and Country Resort & Conference Center. Authors may mount their posters on Wednesday, April 2, from 5:00 pm until 11:00 pm. You must be wearing your official meeting badge to gain entry to the Exhibition Hall.

IMPORTANT: POSTER BOARD SIZE AND FORMAT

Each author is allotted one-half of a 4’h x 6’w board, or net space of 3’8" (111.8 cm) HIGH by 2’10" (86.36 cm) WIDE. Posters should be formatted in “PORTRAIT” format. Posters using more space than allotted will be removed. Posters will be displayed throughout the duration of the meeting and may be viewed 24 hours a day beginning Wednesday, April 2, at 5:00 pm until Saturday evening. All posters must be removed from the boards no later than 11:00 pm, Saturday, April 5. The GSA Administrative Office staff, the hotel staff, and the personnel breaking down the boards will not be responsible for posters left on boards by their authors. Work crews will remove posters that remain on the poster boards after 11:00 pm on Saturday.

Poster sessions are scheduled as follows: Thursday, April 3 Poster Session 2:00 pm–3:00 pm Authors of even-numbered posters must be at boards 3:00 pm–4:00 pm Authors of odd-numbered posters must be at boards Poster Session 8:00 pm–9:00 pm “A” poster authors must be at boards 9:00 pm–10:00 pm “B” poster authors must be at boards 10:00 pm–11:00 pm “C” poster authors must be at boards

Friday, April 4 Poster Session 8:00 pm–9:00 pm “C” poster authors must be at boards 9:00 pm–10:00 pm “B” poster authors must be at boards 10:00 pm–11:00 pm “A” poster authors must be at boards

Saturday, April 5 Poster Session 1:30 pm–2:30 pm Authors of odd-numbered posters must be at boards 2:30 pm–3:30 pm Authors of even-numbered posters must be at boards Open Viewing 7:00 pm–10:00 pm

As shown in the list above, for each of the poster sessions, authors have been assigned a one-hour time slot during which they are expected to be at their posters for discussion. Additional poster time also has been scheduled on Saturday evening, during which authors may choose to be present at their posters.

Poster Design and Preparation The poster should be designed to summarize current research in graphic forms, i.e., charts, tables, graphs, and pictures. Simple use of color can add emphasis. Remember that the poster must be readable from a distance of at least 3 feet. Presentations should be self-explanatory so that the author is free to supplement and discuss particular points. For easy identification, provide a poster heading, listing its title and author(s). Lettering for the title should be at least 1" in height.

Poster materials may be mounted on thin poster paper or cardboard and attached to the poster board with push pins. GSA will provide approximately 25 pins per board, and they will be available near the entrance to the Grand Exhibit Hall. Do not mount your poster on heavy or thick backing, as it may be difficult to fasten to the board. Do not write or paint on the board. If you require assistance with mounting or removing your poster, please notify the GSA staff at the registration desk. 18 EXHIBITORS

As exhibitors at the 49th Annual Drosophila Research Conference, the following companies have contributed to the support of this Conference. Registrants are encouraged to visit the exhibits in the Grand Exhibit Hall and to take advantage of this opportunity to see the new products, publications and services available from these companies. Booth numbers follow company names.

■ Applied Precision, LLC 107 ■ Cold Spring Harbor Laboratory Press 208 1040 12th Ave., NW 500 Sunnyside Blvd. Issaquah, WA 98027 Woodbury New York, NY 11797 Tel: 425/657-1425 Tel: 516/422-4100 Fax: 425/657-1425 Fax: 516/422-4097 E-mail: [email protected] E-mail: [email protected] URL: www.appliedprecision.com URL: www.cshlpress.com

Applied Precision, LLC, is a leading manufacturer in high- Featured books include: Invertebrate Neurobiology precision, high-resolution imaging products including the (Greenspan); Genetic Variation: A Laboratory Manual DeltaVision® Core and personal DV High-Resolution (Weiner, Stephens, Gabriel); Single-Molecule Techniques: Imaging Systems designed for live-cell microscopy A Laboratory Manual (Selvin and Ha); The TGF- Family imaging. (Derynck and Miyazono); Molecular Biology of Aging (Guarente, Partridge, Wallace); Career Opportunities in ■ Biologix Research Company 308 Biotechnology and Drug Development (Freedman); 9876 Pflumm Rd. Evolution (Barton et al.). Lenexa, KS 66215 Tel: 913/648-8578 ■ Genesee Scientific 213/215/312/314 Fax: 913/648-6973 8430 Juniper Creek Lane E-mail: [email protected] San Diego, CA 92126 URL: www.BiologixResearch.com Tel: 858/536-8044 ex 101 Fax: 858/536-8087 Biologix Research Company is committed to the E-mail: [email protected] manufacture and supply of high quality plastic laboratory URL: www.flystuff.com supplies. We offer numerous general use products, as well as specialty products, such as Drosophila bottles Don’t miss the new additions to Genesee’s and Drosophila vials. Our manufacturing facilities are ISO comprehensive “Flystuff™” catalog (available worldwide): 9000 certified and comply with GMP regulations. Genesee-exclusive Droso-Plugger (by LMI/FlyTabs/ Genesee) which plugs 100 vials with proprietary mite- ■ Clever Sys., Inc. 306 proof foam in just seconds! Superfly low temperature 11425 Isaac Newton Square, Suite 202 incubator (Shel Lab). And the Ultimate Flypad. Come by Reston, VA 20190 our multi-booth “island” for interactive demonstrations and Tel: 703/787-6946 expert consultation. We look forward to meeting with you Fax: 703/757-7467 in Genesee’s home town of San Diego. E-mail: [email protected] URL: www.cleversysinc.com ■ Genetic Services, Inc. 109 One Kendall Square, Bldg. 300 Headquartered in the metropolitan DC area, Clever Sys., Cambridge, MA 02139 Inc., (CSI) develops and sells products and services for Tel: 617/872-3135 lab animal behavior analysis including rodents, E-mail: [email protected] Drosophila, zebra fish, primates, etc. CSI’s products are URL: www.geneticservices.com built with technologies of next generation, utilizing information of animal full body and body parts, finding Genetic Services, Inc., offers a variety of services for the what complex behaviors the animal is doing, (not just Drosophila community including injections, P-element tracking them), thus providing measurements of novel and site-directed transgenic production, genetic behavioral paradigms and new parameters that have screening, stock maintenance and more. Let us help never been available before. speed up your research and make your lab more competitive! To learn more, contact Genetic Services, Inc. at 617/872-3135, [email protected] or www.geneticservices.com EXHIBITORS 19

■ Intavis, Inc. 216 ■ Percival Scientific, Inc. 207 3105 N Ashland Ave., #249 505 Research Drive Chicago, IL 60657 Perry, IA 50220 Tel: 773/271-7185 Tel: 515/465-9363 Fax: 773/305-1845 Fax: 515/465-9464 E-mail: [email protected] E-mail: [email protected] URL: www.intavis.com URL: www.percival-scientific.com

Intavis automates complex procedures in peptide Percival Scientific, Inc., continues to set the standard of synthesis, proteomics and functional genomics. Our excellence for the environmental control industry, InsituPro Vsi fully automates in situ detection on whole producing over 70 different models of growth chambers, mounts and sections. The InsituStain automates most special application chambers, low temperature chambers, steps of in situ detection. Our AutoSpot and MultiPep RS environmental rooms and biological incubators. perform solid phase peptide synthesis or peptide synthesis on membranes. ■ Powers Scientific, Inc. 214 P O Box 268 ■ Leica Microsystems 115/117 Pipersville, PA 18947 2345 Waukegan Tel: 215/230-7100 Bannockburn, IL 60015 Fax: 973/663-0028 Tel: 800/248-0123 and 847/405-0123 E-mail: [email protected] Fax: 847/405-0164 URL: www.powersscientific.com E-mail: [email protected] URL: www.leica-microsystems.com Powers Scientific, Inc., offering Drosophila growth chambers with coated coils in 6 sizes (from 6-72 c.f. Leica Microsystems will display imaging solutions capacity) with four levels of temperature, humidity and designed to improve research. The world’s most powerful lighting control to fit the application and price range. stereofluorescence microscope will be on display, the Mosquito and C. elegans growth chambers also available. Leica MZ16 FA, which conducts time lapse, image overlay, and movie making. Also on display: Leica’s new ■ Prater Medical Products 316 MZ20.5 stereomicroscope, uiniquely designed with 17015 Kingsview Ave. Fusion Optics, which allows high resolution imaging over Carson, CA 90746 the entire zoom range. Tel: 805/627-1759 Fax: 805/627-1759 ■ Noldus Information Technology 209 E-mail: [email protected] 1503 Edwards Ferry Rd., Suite 201 URL: www.partermedical.com Leesburg, VA 20176 Tel: 800/355-9541 Parter Medical is the leading manufacturer of high quality Fax: 703/771-0441 placticware for Drosophila research. Our disposable/ E-mail: [email protected] reusable vials and bottles are made in a wide choice of URL: www.noldus.com sizes, materials, and packaging. All are manufactured for optimum durability and clairty of contents. Our Noldus Information Technology (www.noldus.com) products are available through national and regional offers systems for the recording and analysis of fly distributors. behavior. Several of our new tools for Insect Behavior Research include EthoVision XT, The Observer XT, and Systems for tracking insect flight in 3D. Please visit Noldus at booth 209 for more detailed information. 20 EXHIBITORS

■ Roboz Surgical Instrument Company 212 ■ Union Biometrica 113 P O Box 10710 84 October Hill Rd. Gaithersburg, MD 20898 Hilliston, MA 01746 Tel: 800/424-2984 Tel: 617/799-1928 Fax: 888/424-3121 Fax: 508/893-8044 E-mail: [email protected] E-mail: [email protected] URL: www.roboz.com URL: www.unionbio.com

Roboz Surgical Instrument Company is the leading name Union Biometrica designs, manufactures and distriburtes for high quality microdissecting instruments, general large-bore flow cytometers optimized for automated surgical instruments and other surgical devices for analysis, sorting, and dispensing of small model biomedical research. Our catalog includes microorganisms such as Drosophila embryos and larvae, dissecting scissors and forceps, microvascular clips, bone based on size and florescent properties. Our proprietary cutting instruments, cannulas, suture material, wound COPAS technology greatly increases throughput, closing clip systems, instrument care and handling sensitivity and reproducibility in such research products, and much more. applications as genetic screening and sample preparation. ■ Sable Systems International 206 6340 S Sandhill Rd., Suite 4 Las Vegas, NV 89120 Tel: 702/269-4445 Fax: 702/269-4446 E-mail: [email protected] URL: www.sablesys.com

Sable Systems International is the widely cited, international standard in turn-key, research grade respirometry systems capable of measuring Drosophila gas exchange in real time. Technical support is by published experts in insect respirometry. We invite you to drop by our booth and discuss what we can do for your research.

The 49th Annual Drosophila Research Conference gratefully acknowledges Genetic Services, Inc., sponsor of Thursday’s refreshment break. FLOORPLAN: POSTERS AND EXHIBITS 21

Atlas Ballroom →

GRAND EXHIBIT HALL ENTRANCE

Town and Country Resort & Convention Center Grand Exhibit Hall San Diego, CA 22 OPENING GENERAL SESSION AND PLENARY SESSIONS I AND II

WEDNESDAY, APRIL 2 7:00 pm–9:00 pm 11:00 am Inheritance of Polycomb Proteins through DNA Replication in Vitro. Nicole Francis. OPENING GENERAL SESSION Harvard University, Cambridge, Room: Atlas Ballroom Massachusetts. Presentations: 11:30 am MicroRNA Functions. Stephen Cohen. 7:00 pm Welcome and Opening Remarks. Susan Temasek Life Sciences Laboratory, Celniker. Lawrence Berkeley National Singapore. Laboratory, Berkeley, California. 7:05 pm Introduction of Larry Sandler Memorial Lecture. Helen Salz. Case Western Reserve SUNDAY, APRIL 6 8:30 am–12:00 noon University, Cleveland, Ohio. PLENARY SESSION II 7:15 pm Larry Sandler Memorial Lecture. Room: Atlas Ballroom Moderators: Brian Oliver, National Institute of 8:00 pm Memoriam to Seymour Benzer: the Father Diabetes, Digestive and Kidney of Fly Neurogenetics. Nancy Bonini. Diseases, Bethesda, Maryland; and University of Pennsylvania, Philadelphia. Susan Celniker, Lawrence Berkeley National Laboratory, Berkeley, 8:05 pm Introduction of Historical Lecture. William California McGinnis. University of California, San Diego. Presentations: 8:15 pm Historical Lecture. Antonio Garcia-Bellido. Facultad de Ciencias, Universidad Autonoma 8:30 am Poster Award Presentation. Brian Oliver. de Madrid, Spain. National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland. 8:35 am Modeling Emotional Behavior in THURSDAY, APRIL 3 8:30 am–12:00 noon Drosophila. David Anderson. California PLENARY SESSION I Institute of Technology, Pasadena, California. Room: Atlas Ballroom 9:00 am Using Electrophysiology and Genetics to Moderators: John Tamkun, University of Dissect Neural Circuit Function. Rachel California, Santa Cruz; and Nancy Wilson. Harvard Medical School, Boston, Bonini, University of Pennsylvania, Massachusetts. Philadelphia 9:30 am Quantitative Analysis of the Transcription Presentations: Network Controlling Early Embryo 8:30 am Image Award Presentation. John Tamkun. Patterning. Mark Biggin. Lawrence Berkeley University of California, Santa Cruz. National Laboratory, Berkeley, California. 8:35 am Trafficking and Polarity in the Control of 10:00 am Break Drosophila Growth. David Bilder. University 10:30 am Regulatory Genomics in Drosophila of California, Berkeley. Species. Manolis Kellis. Massachusetts 9:00 am Ig-receptor Diversity in Insect Immunity Institute of Technology, Cambridge, and Neuronal Wiring. Dietmar Schmucker. Massachusetts. Harvard Medical School, Cambridge, 11:00 am Two or Four Bristles: Evolution of Massachusetts. Regulation of the achaete-scute Genes. 9:30 am Pathways of Anti-Viral Immunity. Sara Pat Simpson. University of Cambridge, Cherry. University of Pennsylvania, United Kingdom. Philadelphia. 11:30 am Regulation Oogenesis: from Stem Cells 10:00 am Break to Steroids. Allan Spradling. Carnegie Sponsored by Genetic Services, Inc., booth Institute of Washington, Baltimore, Maryland. 109 12:00 noon Meeting Close. Susan Celniker. Lawrence 10:30 am Cross-Regulation of HOX and Sex Berkeley National Laboratory, Berkeley, Determination Genes in Development and California. Evolution. Artyom Kopp. University of California, Davis. WORKSHOPS 23

WEDNESDAY, APRIL 2 12:00 noon–6:00 pm FRIDAY, APRIL 4 1:45 pm–3:45 pm ECDYSONE WORKSHOP ModENCODE Room: Pacific Ballroom Salons 2-3 Room: Pacific Ballroom Salon 1 Organizers: Craig Woodard, Mt. Holyoke, Organizer: Kevin White, University of Chicago, South Hadley, Massachusetts; and Illinois Randall S. Hewes, University of Summary: The modENCODE workshop will introduce the Oklahoma, Norman model organism ENCODE project that endeavors to Summary: The Ecdysone Workshop welcomes all those comprehensively identify the sequence-based functional interested in the biochemistry, molecular biology, and elements in the D. melanogaster genome. The project is physiology of insect hormones. As such, we will discuss organized and conducted by a research network called mechanisms of 20-hydroxyecdysone, juvenile hormone, the modENCODE Consortium. and peptide hormone signaling in Drosophila, Manduca, Aedes, and other insect species. Topics Include: Transcriptome Topics Include (but are not limited to): Regulatory elements Receptor complex/target gene interactions Chromosomal proteins Downstream tissue-specific responses Small and microRNAs Global effects of hormone exposure. Origins of replication and data coordination Programs will be supplied upon request by email ([email protected] or [email protected]) after Preliminary Program: March 1. Comprehensive characterization of the Drosophila transcriptome. FRIDAY, APRIL 4 1:45 pm–3:45 pm Identification of transcription factor binding sites by high- throughput ChIP-chip experiments in Drosophila. GASES IN DROSOPHILA PHYSIOLOGY AND Genome-wide mapping of chromosomal proteins in DEVELOPMENT Drosophila. Room: Pacific Ballroom Salons 6-7 Organizers: Greg Beitel, Northwestern Genome-wide profiling of histone variants in Drosophila University, Evanston, Illinois; and and Caenorhabditis Eric Johnson, University of Oregon, Annotation of the small RNA/microRNA component of the Eugene Drosophila genome. Summary: Gases including O2, NO and CO2 have critical The systematic identification and analysis of replication roles in physiology and development. Presenters in this origins in Drosophila workshop will detail investigations of these roles as well A data coordinating center for modENCODE as the molecular mechanisms by which the concentrations of specific gases are sensed and transduced to cellular processes. FRIDAY, APRIL 4 1:45 pm–3:45 pm MONOAMINES Topics Include: Room: Pacific Ballroom Salon 2 Nitric oxide and wiring of the eye to the brain Organizer: Kyung-An Han, Pennsylvania State A fly model of hypercapnia: The effects of elevated CO2 University, University Park on the Drosophila immune system Summary: The monoamines dopamine, serotonin, and Tracheal cells as oxygen sensors octopamine regulate a wide variety of brain functions. Recent advances in transgenic manipulation and genetic Metabolomics and metabolic modeling of hypoxia in mutations in monoamine biosynthetic enzymes, Drosophila transporters, and receptors have greatly enhanced our Oxygen-sensitive guanylyl cyclases and taste perception understanding of diverse, yet selective, roles of Hypoxia and heart function monoamine systems in Drosophila behaviors. The goal Globins in Drosophila: respiratory proteins, oxygen of this workshop is to bring together those who work on scavengers or else? various aspects of monoamine functions for sharing ideas, Regulation of the response to hypoxia resources and information on recent progress in their studies. This would help clarify how different monoamine The cellular basis of respiration in adult Drosophila systems act on particular brain structures or neural circuits 24 WORKSHOPS to mediate relevant behavioral processes and would be SATURDAY, APRIL 5 6:45 pm–8:45 pm of great interests to many neurobiology researchers. THE MATERNAL TO ZYGOTIC TRANSITION: Topics Include: DECIPHERING THE ULTIMATE GENETIC SWITCH Room: Pacific Ballroom Salons 6-7 Octopamine and serotonin in aggressive behavior Organizers: John Sisson, University of Texas, Serotonin and dopamine in aggression and awakefullness Austin; and Howard Lipshitz, Dopamine in sleep University of Toronto, Canada Dopamine in rhythmicity Summary: This workshop is intended to review and synthesize some recent advances in our understanding Dopamine and octopamine in learning and memory of the molecular events governing the maternal to zygotic Vesicular monoamine transporter in locomotor, courtship, transition (MZT) in Drosophila embryos. The presentations and drug-induced behavior and discussion will focus largely on the molecular mechanisms controlling four aspects of the MZT: maternal FRIDAY, APRIL 4 1:45 pm–3:45 pm mRNA stability, translational regulation, nuclear division cycle control, and transcriptional activation of the zygotic CELL CYCLE AND CHECKPOINTS genome. The goal of the workshop is to foster productive Room: Pacific Ballroom Salon 3 exchange of ideas and promote potential collaborations Organizers: Tin Tin Su, University of Colorado, between participants. Boulder; and Claudio Sunkel, Instituto de Biologia Molecular e Topics Include: Celular and Universidade do Porto, Regulation of the Drosophila MZT by SMAUG Portugal Regulation of the MZT by a maternal clock and the DNA Summary: This workshop will focus on the mechanisms replication checkpoint that drive the cell division cycle normally and in response to checkpoint activation. The mechanism of dFMRP activity required for cellular morphogenesis during the MZT Topics Include: Timing zygotic transcription and maternal RNA turnover Kinetics of mitotic progression: the role of the kinetochore in the syncytial and cellular blastoderm and SAC proteins Primary sex determination: life within the MZT The role of Dpp as a growth factor in the wing imaginal disc SATURDAY, APRIL 5 6:45 pm–8:45 pm The anaphase promoting complex/cyclosome (APC/C) DROSOPHILA RESEARCH AND PEDAGOGY AT is required for re-replication control in endoreplication PRIMARILY UNDERGRADUATE INSTITUTIONS (PUI) cycles Room: Pacific Ballroom Salon 3 De novo CoA biosynthesis is required to maintain DNA Organizers: Don Paetkau, Saint Mary’s College, integrity and to prevent neurodegeneration in Drosophila Notre Dame, Indiana; and Mark Adversarial Epigenetic Regulation of Polo Kinase by Myb, Hiller, Goucher College, Baltimore, Lin-9/Mip130, and E2F2/RBF Maryland It is not what it looks like: TTF2 is not encoded by lodestar Summary: This workshop focuses on increasing visibility but by another gene of research performed at primarily undergraduate institutions and to facilitate faculty and students in their Cortical microtubules and the regulation of cytokinesis endeavors. The goals include: MicroRNA and inr-pathway regulate GSC cell cycle 1) encouraging undergraduate research by providing a through dacapo 3’UTR forum in which undergraduate students make oral Functional analysis of a novel family of ERM protein presentations; partners during cell division 2) connecting people interested in this career path with Cell cycle control in the developing eye people already in primarily undergraduate institution (PUI) faculty positions; 3) connecting PUI faculty for discussions and support on professional issues that differ from those at large institutions; WORKSHOPS 25

4) sharing ideas for using Drosophila as teaching tools in SATURDAY, APRIL 5 6:45 pm–8:45 pm the classroom and laboratory; EXTRACELLULAR MATRIX and 5) providing resources for PUI investigators. Room: Pacific Ballroom Salon 2 The workshop is divided into two components: Research Organizer: Halyna Shcherbata, University of presentations made by undergraduate students followed Washington, Seattle by breakout discussion groups. Further details may be Summary: Recent findings, discussed in this workshop, found at the dPUI Web page and on the dPUI information will show that the extracellular matrix proteins do not act sheet available at the conference registration desk. simply as structural proteins or as migratory substrates, but also as sources of signaling information that regulate Topics Include: cell morphology, cytoskeletal rearrangements, polarity, Undergraduate student research presentations morphogenesis, and signal transduction. The PUI faculty-led breakout discussion groups presentations in this workshop will focus on extracellular matrix molecules and their cell-surface receptors that play Several discussions sessions for graduate students and important roles in a broad array of developmental postdocs interested in a career at a PUI processes. Genomics education partnership discussion group Topics Include (but are not limited to): SATURDAY, APRIL 5 6:45 pm–8:45 pm Mechanisms of heart assembly CHROMOSOME PAIRING AND TRANS-SENSING Components of the apical extracellular matrix in cell shape EFFECTS remodeling Room: Pacific Ballroom Salon 1 The role of Dystroglycan-Dystrophin complex in different Organizer: Giovanni Bosco, University of tissues Arizona, Tucson Egfr/Ras signaling regulates DE-cadherin/Shotgun Pairing is important for meiotic chromosome segregation localization to control vein morphogenesis as well as somatic cell gene expression and gene silencing, and Drosophila has been a excellent model SATURDAY, APRIL 5 6:45 pm–8:45 pm system for studying the mechanisms that govern chromosome pairing and pairing sensitive phenomenon. DROSOPHILA POPULATION GENOMICS This workshop explores a broad range of topics relevant Room: Pacific Ballroom Salons 4-5 to chromosome pairing. It will feature nine presentations Organizers: Chuck Langley, University of that describe the molecular players regulating pairing and California, Davis; Chip Aquadro, pairing sensitive phenomenon such as meiotic and Cornell University; and Andy Clark, somatic pairing, recombination, polytene chromosomes, Cornell University heterochromatin, Polycomb-group effects, transvection, Summary: Acquisition of full genome sequence data for small RNAs and cosuppression. multiple individuals has been a dream of population and quantitative geneticists for many years. Modern theoretical Topics Include: population genetics makes a rich array of predictions Condensin II subunits antagonize somatic and germline about patterns of nucleotide variation and their chromosome pairing and alter meiotic recombination dependence on mutation, demography, migration, and Understanding the relationship between meiotic and selection. Small gene-specific resequencing data sets, somatic pairing and recombination and ascertainment-plagued SNP genotype data will soon give way to full genome sequence, thanks to the Heterochromatic threads that connect segregating plummeting costs brought about by short-read homologs at meiosis technologies. The challenge of this workshop is to discuss Cohesins, recombination and synaptonemal complex designs for acquisition of multiple genome sequences to Homolog pairing and cohesion in male meiosis meet the needs of the communities working on the genetics of complex traits and on population genetics. Interrelationships of transgene cosuppression, pairing dependent silencing and small RNA pathways Questions to be addressed include: What are the Chromatin structure of genes silenced by heterochromatin tradeoffs between sequence quality and sequence depth in trans of the different platforms? What are the best ways to treat Chromosome kissing phenomena dependent on short-read data to obtain accurate sequence variants? Polycomb-group proteins What is the level of interest in obtaining finished-quality Using compound chromosomes to study the initiation of data for targeted regions in multiple lines? How do false somatic pairing in embryos positive and false negative errors in SNP calling trade 26 WORKSHOPS off? What samples should be the first to be done? How between individuals working in the area. Topics may should data be made available and distributed? Is there include all aspects of apoptosis regulation and the need for a genome-wide SNP platform? How should importance of apoptosis for Drosophila biology. SNPs be selected for it? What lines of flies should be made available to leverage complex trait analysis? How SATURDAY, APRIL 5 9:30 pm–11:30 pm do we assure that young investigators feel that these research areas are wide-open to them? RNA CONTROL AND DEVELOPMENTAL PROCESSES Room: Pacific Ballroom Salon 2 SATURDAY, APRIL 5 9:30 pm–11:30 pm Organizer: Talila Volk, Weismann Institute, Israel LONGEVITY AND FUNCTIONAL SENESCENCE - Summary: The workshop on RNA and developmental CATCH THE RHYTHM control will focus on different modes of regulation of RNA Room: Pacific Ballroom Salons 6-7 metabolism that are essential for embryonic development. Organizers: Larry Harshman, University of These include mechanisms involved in RNA localization, Nebraska, Lincoln; John Tower, translation, stabilization, and splicing. University of Southern California, Los Angeles; Mike Grotewiel, Virginia Topics Include: Commonwealth University, Richmond; and RJ Wessells, Global analysis of mRNA localization pathways University of Michigan, Ann Arbor Drosophila PTB/hnRNI promotes formation of high-order Summary: The aging phenotype is expressed at the level oskar RNP complexes and represses oskar translation of specific organs and tissues as functional senescence, Bicaudal-C regulates nos expression during oogenesis as well as at the level the whole animal as altered behavior Combinatorial assembly of Bruno binding sites and mortality. Our ever-improving ability to assay fly cells Regulation of CyclinB mRMNA by Pumiio and behaviors as a function of time has revealed how a deterioration of the normal rhythm of cellular events Tissue development and RNA control – the role of Held characterizes aging phenotypes, including altered Out Wing transcription, metabolism, activity, sleep and heartbeat. The talks in this workshop illustrate how analysis of SATURDAY, APRIL 5 9:30 pm–11:30 pm Drosophila at every level of biology from molecular to behavioral is informing on basic mechanisms of aging. IMMUNITY AND PATHOGENSIS Room: Pacific Ballroom Salon 3 Topics Include: Organizers: Brian Lazzaro, Cornell University, Lamin processing and Progeria: studies in Drosophila Ithaca, New York; and Louisa Wu, University of Maryland, College Park Nuclear-mitochondrial signaling and life span Summary: This workshop will address a diversity of topics The role of reduced glutathione and the transsulfuration related to pathogenesis and immune function in pathway in extension of life span by dietary restriction Drosophila. Topics covered will include heritability of Effects of aging on sleep:wake cycles immune performance, how environmental factors and host Nutrient signaling and aging in Drosophila condition may affect resistance, regulation and modulation of signaling pathways in the Drosophila immune system, Modeling age-related locomotor impairment and the genetics of Drosophila-virus interactions. Cardiac functional decline: what can it tell us about mechanisms of aging Topics Include: Sensing of Gram-positive bacteria in Drosophila immunity SATURDAY, APRIL 5 9:30 pm–11:30 pm ATP-sensitive potassium channels mediate survival during CELL DEATH viral infection in Drosophila Room: Pacific Ballroom Salon 1 Intralocus sexual conflict, sex-biased gene expression and Organizers: Andreas Bergmann, MD Anderson the cost of immunity Cancer Center, Houston, Texas; and Trancriptional architecture of age-specific variation in Jamie Rusconi, University of Albany, immune function New York Feedback regulation in the IMD pathway Summary: The Cell Death Workshop is a forum for the discussion of Drosophila apoptosis. The workshop is The infection-induced proteolysis of the receptor PGRP- intended to highlight recent advances in apoptosis LC activates the IMD pathway and melanization cascades research and to foster communication and collaboration in Drosophila PLATFORM SESSIONS 27 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

THURSDAY, APRIL 3 4:30 pm–6:30 pm 8 - 6:15 Golden West Different caspases control distinct mechanisms of apoptosis- induced compensatory proliferation. Yun Fan, Andreas Immune System and Cell Death Bergmann. Department of Biochemistry & Molecular Biology, UT MD Anderson Cancer Center, Houston, TX. Moderator: Kristin White, Massachusetts General Hospital, Boston

1 - 4:30 THURSDAY, APRIL 3 4:30 pm–6:30 pm Immune response to tumors in Drosophila. José C. Pastor- Town & Country Pareja, Ming Wu, Tian Xu. Howard Hughes Medical Institute, Dept. of Genetics, Yale University School of Medicine, New Neurophysiology and Behavior Haven, CT. Moderator: Tom Clandinin, Stanford University, Palo Alto, 2 - 4:45 California The bacterial symbiont Wolbachia confers resistance to viruses in D. melanogaster. Luís Teixeira, Álvaro Ferreira, Michael 9 - 4:30 Ashburner. Department of Genetics, University of Cambridge, Molecular mechanisms of odor receptor expression and Cambridge, United Kingdom. function. Anandasankar Ray1, Wynand van der Goes van Naters2, John R. Carlson2. 1) Department of Entomology, 3 - 5:00 University of California, Riverside, CA; 2) MCDB, Yale University, Sensing of gram positive bacteria in Drosophila immunity. Lihui New Haven, CT. Wang, Petros Ligoxygakis. Dept Biochemistry, Univ Oxford, Oxford, United Kingdom. 10 - 4:45 The Drosophila sex peptide receptor mediates the post-mating 4 - 5:15 switch in female reproductive behavior. Nilay Yapici, Young- The Infection-Induced Proteolysis of The Receptor PGRP-LC Joon Kim, Carlos Ribeiro, Barry J. Dickson. Molecular Activates the IMD Pathway and Melanization Cascades in Biology and Genetics, Institute of Molecular Pathology, Vienna, Drosophila. Amy Tang1,2, Rebecca Schmidt2, Francesca Austria. Rinaldo1, Shayla Hesse1, Theodore Trejo1, Zachary Ortiz1, Masakazu Hamada2, Timothy Plummer1, Andrea Page- 11 - 5:00 McCaw3, Jeffery Platt1, Amy Tang1,2. 1) Department of Surgery, A novel tetraspanin required for synaptic endocytosis. Chi- Mayo Clinic College of Medicine, Rochester, MN 55902; 2) Kuang Yao1,3, Yong Qi Lin1,3, Tomoko Ohyama1, Cindy Ly2, Department of Biochemistry and Molecular Biology, Mayo Clinic Patrik Verstreken1, Karen L. Schulze1,3, Hugo J. Bellen1,2,3. Cancer Center, Mayo Clinic College of Medicine, Rochester, 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, MN 55902; 3) Department of Biology and Center for TX; 2) Department of Neuroscience, Baylor Col Medicine, Biotechnology and Interdisciplinary Studies, Rensselaer Houston, TX; 3) Howard Hughes Medical Institute, Baylor Col Polytechnic Institute, Troy, NY 12180. Medicine, Houston, TX.

5 - 5:30 12 - 5:15 Developmentally-regulated cell death of Drosophila salivary Genetic Analysis of AMP-Activated Protein Kinase. Jay glands utilizes ER stress-linked apoptosis. Robert Farkas1, Brenman1, Nevzat Kazgan1, Paul Medina1, Vincent Mirouse2, Lucia Mentelová1,2, Peter Low3, Gabor Juhasz3, Miklos Sass3. Daniel St. Johnston2, Linsay Williamson3, Erik Johnson3. 1) Institute of Experimental Endocrinology, Slovak Academy of 1) UNC Chapel Hill School of Medicine, Chapel Hill, NC USA; Sciences, 83306 Bratislava, Slovakia; 2) Department of 2) University of Cambridge, UK; 3) Wake Forest University, NC Genetics, Comenius University, Bratislava, Slovakia; 3) USA. Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary. 13 - 5:30 Functional and Genomic Analyses of Genes Regulated by 6 - 5:45 Fruitless in Adult Head and Central Nervous System Tissues. Wts function in autophagic cell death of salivary glands. Thomas Goldman, Michelle Arbeitman. Molecular and Sudeshna Dutta1,2, Eric Baehrecke1. 1) Cancer Biology, Compuational Biology, USC, Los Angeles, CA. University of Massachusetts Medical School, Worcester, MA; 2) Molecular & Cell Biology Program, University of Maryland, 14 - 5:45 College Park, MD 20742. Dynamic range compression by feedback inhibition in the olfactory system. Cory M. Root1, Kaoru Masuyama1, Lina 7 - 6:00 Enell2, Dick R. Nässel2, Jing W. Wang1. 1) Neurobiology Physiological apoptosis in the Drosophila ovarian polar cell Section, Div. of Biological Sciences, University of California, lineage involves Hid-mediated activation of a Diap1/Dronc/Drice San Diego, La Jolla, CA; 2) Department of Zoology, Stockholm cascade. Anne-Marie Pret, Asma Khammari, François University, Svante Arrhenius vag 14S-106 91 STOCKHOLM, Agnès, Pierre Gandille, Elisabeth Boissonneau. Centre de Sweden. Genetique Moleculaire UPR2167, CNRS/Pierre and Marie Curie University Paris VI, Gif-sur-Yvette, France. 28 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

15 - 6:00 22 - 5:45 A novel vesicular neurotransmitter transporter expressed in the Dynamic Regulation of the Eye Specification Network. Claire mushroom bodies and central complex is required for learning Salzer, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, and female receptivity. Elizabeth S. Brooks1, Bac T. Nguyen1, IN. Christopher J. Tabone2, J. Steven de Belle2, David E. Krantz1. 1) Semel Institute for Neuroscience, UCLA, Los Angeles, CA; 23 - 6:00 2) Department of Biological Sciences, UNLV, Las Vegas, NV. Regulation of sexually dimorphic gonad development in D. melanogaster. Nicole Camara, Mark Van Doren. Department 16 - 6:15 of Biology, Johns Hopkins University, Baltimore, MD. Regulation of Drosophila male courtship by complex integration of sensory information. Werner Boll1, Dimitrije Krstic1,2, 24 - 6:15 Markus Noll1. 1) Institute of Molecular Biology, University of The Regulation and Function of Cad74A in Drosophila Zürich, Zürich, Switzerland; 2) Ph.D. Program in Molecular Life Oogenesis. Jeremiah J. Zartman, Nir Yakoby, Chris A. Sciences, Zürich, Switzerland. Bristow, Stanislav Y. Shvartsman. Lewis Sigler Institute and Department of Chemical Engineering Princeton University, Princeton, NJ.

THURSDAY, APRIL 3 4:30 pm–6:30 pm California

Organogenesis Moderator: Justin Kumar, University of Indiana, Bloomington

17 - 4:30 Control of stem cell self-renewal and differentiation in the adult Drosphila intestine. Allison Bardin, Carolina Perdigoto, François Schweisguth. Dept Biol, Ecole Normale Superieure, Paris, France.

18 - 4:45 FoxK mediates TGF-beta signaling during midgut differentiation. Sergio Casas-Tinto1,2, Melisa Gomez-Velazquez1, Begoña Granadino-Goenechea2, Pedro Fernandez-Funez1. 1) Dept Neurology, UTMB, Galveston, TX; 2) Centro de Investigaciones Biologicas, CSIC, 28040 Madrid (Spain).

19 - 5:00 The combinatorial control of muscle identity. Jonathan Enriquez, Laurence Dubois, Virginie Daburon, Michèle Crozatier, Alain Vincent. centre de biologie du developpement, Toulouse, France.

20 - 5:15 A key role of Pox meso in somatic myogenesis of Drosophila. Cheng Zhang1,5, Hong Duan1,3,5, Jianming Chen1,4, Helen Sink2, Erich Frei1, Markus Noll1. 1) University of Zurich, Institute of Molecular Biology, Zurich, Zurich, Switzerland; 2) Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016; 3) Present address: Sloan-Kettering Institute, Department of Developmental Biology, 1275 York Avenue, New York, NY 10021; 4) Present address: Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037; 5) These authors contributed equally to this work.

21 - 5:30 Two distinct progenitor populations remodel the Drosophila tracheal system during metamorphosis. Molly Weaver1,2, Mark Krasnow1,2. 1) Department of Biochemistry, Stanford University School of Medicine, Stanford, CA; 2) Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA. PLATFORM SESSIONS 29 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

FRIDAY, APRIL 4 8:30 am–12:30 pm 31 - 10:00 Town & Country Regulation of muscle identity by homeodomain transcription factors. Brian Busser1, Aditi Singhania1, Savina Jaeger2, Regulation of Gene Expression Anton Aboukhalil2, Michael Berger2, Caitlin Gamble1, Stephen Gisselbrecht2, Martha Bulyk2, Alan Michelson1. 1) Moderator: David Arnosti, Michigan State University, LDSB, NHLBI/NIH, Bethesda, MD; 2) Division of Genetics, East Lansing Brigham & Women’s Hospital, Boston, MA.

10:15 am - Break 25 - 8:30 The zinc finger protein Zelda is a key activator of the zygotic 32 - 10:45 genome in Drosophila. Hsiao-Lan Liang1, Chung-Yi Nien1, Sequential developmentally programmed steps at target Hsiao-Yun Liu1, Mark Metzstein2, Nikolai Kirov1, Christine promoters reverse repression by Polycomb for terminal Rushlow1. 1) Biology, New York Univ, New York, NY; 2) Human differentiation in a stem cell lineage. Xin Chen, Jose Morillo, Genetics, Univ. of Utah, Salt Lake City, UT. Chenggang Lu, Margaret Fuller. Dev Biol, Stanford Univ, Stanford, CA. 26 - 8:45 Application of a cis-regulatory grammar for functional 33 - 11:00 characterization of transcriptional regulatory elements in the dHCF is required for Myc-dependent transcription regulation Drosophila embryo. David N. Arnosti1, Ahmet Ay2, Chichia in Drosophila. Michael Furrer, Mirjam Balbi, Peter Gallant. Chiu2, Evan Dayringer2, Rupinder Sayal1, Walid Fakhouri1. Zoological Institute, University of Zürich, Zürich, Switzerland. 1) Dept Biochemistry & Molec Biol, Michigan State Univ, East Lansing, MI; 2) Dept of Mathematics, Michigan State Univ, East 34 - 11:15 Lansing, MI. Integration of inputs from multiple modular elements generates a gradient of transcriptional repression in response to the Dpp 27 - 9:00 morphogen. Rahul Warrior1, Yao Li-Chin1, Phin Sopheap1, Enhancer identification by comparative genomics relies on Rushlow Christine2, Arora Kavita1. 1) Dept Developmental & abundant non-conserved DNA. Brant Peterson1, Emily Hare1, Cell Biol, Univ California, Irvine, Irvine, CA; 2) Department of Venky Iyer1, Rick Kurashima3, Eric Jang3, Michael Eisen1,2. Biology, New York University, New York, NY 10003. 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA; 2) Lawrence Berkeley National Lab, Berkeley, 35 - 11:30 CA; 3) USDA Agricultural Research Service - PBARC, Hilo, HI. The Mechanism of Dscam Mutually Exclusive Splicing. Brenton Graveley1, Sara Olson1, Marco Blanchette2,5, Jung Park1, 28 - 9:15 Yiannis Savva1, Gene Yeo3, Joanne Yeakley4, Donald Rio2. Sepsid even-skipped enhancers are functionally conserved in 1) Dept Genetics & Dev Biol, Univ Connecticut Health Ctr, Drosophila despite lack of sequence conservation. Emily Hare1, Farmington, CT; 2) Department of Molecular and Cell Biology, Brant Peterson1, Venky Iyer1, Rudolf Meier2, Michael Eisen1,3. Center for Integrative Genomics, University of California, 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA 94720-3204; 3) 4Crick-Jacobs Center for Berkeley, CA; 2) Department of Biological Sciences, National Theoretical and Computational Biology, Salk Institute, LaJolla, University of Singapore, Singapore; 3) Genomics Division, CA 92037; 4) Illumina Inc., 9885 Towne Centre Drive, San Ernest Orlando Lawrence Berkeley National Laboratory, Diego, CA 92121-1975; 5) Stowers Institute for Medical Berkeley, CA. Research, Kansas City, MO 64110. 29 - 9:30 36 - 11:45 Cell fate specification in the blastoderm embryo involves The role of site accessibility in microRNA target recognition. developmental regulation of transcription elongation. Peter Nicola Iovino1,3, Michael Kertesz2,3, Ulrich Unnerstall1, Eran Gergen1, Lisa Prazak1, Xiaoling Wang1, Kevin Celestrin1, Segal2,3, Ulrike Gaul1,3. 1) Laboratory of Developmental Hyowon Choi2, Giorgio Medranda1. 1) Department of Neurogenetics, Rockefeller University, New York, NY; 2) Biochemistry & Cell Biology, Stony Brook University, Stony Weizmann Institute, Rehovot, Israel; 3) equal contributors. Brook, NY; 2) Mount Holyoke University, South Hadley, MA. 37 - 12:00 30 - 9:45 Mextli, a novel eIF4E-binding protein from Drosophila. Greco A comprehensive catalog of homeodomain DNA-binding Hernandez1,2, Gritta Tetweiler1,2, Mathiu Miron1,2, Paul Lasko1, specificities from D. melanogaster. Michael Brodsky1, Marcus Nahum Sonenberg2. 1) Dept. of Biology, McGill University; 2) Noyes1, Ryan Christensen2, Atsuya Wakabayashi1, Gary Dept. of Biochemistry. McGill University. Stormo2, Scot Wolfe1. 1) Program in Gene Function & Expression, Univ Massachusetts Medical School, Worcester, 38 - 12:15 MA; 2) Department of Genetics, Washington University, School Dorsal interacting protein 3 potentiates activation by Drosophila of Medicine, St. Louis, MO. Rel-homology domain proteins. Girish Ratnaparkhi, Albert Courey. Chemistry & Biochemistry, UCLA, Los Angeles, CA. 30 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

FRIDAY, APRIL 4 8:30 am–12:30 pm 47 - 11:00 Golden West Genetic and genomic analyses of the wing polyphenism and polymorphism in the pea aphid (Acyrthosiphon pisum). Jennifer Evolution and Quantitative Genetics Brisson1, Greg Davis2, David Stern2, Sergey Nuzhdin3. 1) Section of Ecology & Evolution, University of California, Davis, Moderator: Trisha Wittkopp, University of Michigan, Davis, CA; 2) Princeton University; 3) University of Southern Ann Arbor California.

48 - 11:15 39 - 8:30 Do insecticides alter natural host-parasitoid interactions in Concerted cis-regulatory evolution at multiple neuroectodermal Drosophila? Neil Milan, Todd Schlenke. Dept. of Biology, loci. Justin Crocker, Albert Erives. Biology, Dartmouth Emory University, Atlanta, GA. College, Lebanon, NH. 49 - 11:30 40 - 8:45 Multiple infections of Spiroplasma bacteria in Drosophila. Estimating the fraction of sites under positive and negative Tamara S. Haselkorn, Therese A. Markow, Nancy A. Moran. selection via explicit population genetic hidden Markov models. Dept Ecology & Evolutionary Biol, University of Arizona, Tucson, Andrew Kern, David Haussler. Center For Biomolecular AZ. Science and Engineering, UC Santa Cruz, Santa Cruz, CA. 50 - 11:45 41 - 9:00 The phenotypic effects of interspecific cytonuclear interactions. cis-regulatory variation is typically poly-allelic in Drosophila. Colin Meiklejohn, Kristi Montooth, Dawn Abt, David Rand. Jonathan Gruber, Anthony Long. Dept Ecology & Evol Biol, Dept EEB, Brown Univ, Providence, RI. Univ California, Irvine, Irvine, CA. 51 - 12:00 42 - 9:15 Satellite sequence de-condensation as a cause of hybrid Evolutionary constraint and adaptation in the metabolic network lethality. Patrick M. Ferree, Daniel A. Barbash. Department of 12 Drosophila species. Anthony Greenberg, Sarah of Molecular Biology and Genetics, Cornell University, Ithaca, Stockwell, Andrew Clark. Dept Molec Biol & Genetics, Cornell NY. Univ, Ithaca, NY. 52 - 12:15 43 - 9:30 Adaptive evolution of the aging gene Insulin-like Receptor. Population transcriptomics of host shifts in the cactophilic D. Annalise Paaby1, Mark Blacket2, Ary Hoffmann2, Paul mojavensis. Luciano Matzkin, Therese Markow. Dept Ecology Schmidt1. 1) Department of Biology, University of Pennsylvania, & Evolutionary Biol, Univ Arizona, Tucson, AZ. Philadelphia, PA; 2) Centre for Environmental Stress and Adaptation Research, University of Melbourne, Melbourne, 44 - 9:45 Australia. The evolution of non-coding RNAs from insect Hox complexes. Matthew Ronshaugen. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

45 - 10:00 FRIDAY, APRIL 4 8:30 am–12:30 pm Structure and evolution of gene network involved in specification California and function of extraembryonic membranes in insects. Yury Goltsev1, Gustavo Rezende1,2, Denise Valle2, Gregory Cytoskeleton and Cell Biology Lanzaro3, Mike Levine1. 1) UC Berkeley; 2) Instituto Oswaldo Cruz, Brazil; 3) UC Davis. Moderator: Brooke McCartney, Carnegie Mellon University, Pittsburgh, Pennsylvania 10:15 am - Break 53 - 8:30 46 - 10:45 The role of cell surface internalization in epithelial polarity and Engineering the genomes of wild insect populations. Bruce proliferation control. Sarah L. Windler, David Bilder. Molecular Hay1, Chun-Hong Chen1, Haixia Huang1, Catherine Ward1, and Cell Biology, University of California, Berkeley, Berkeley, Jessica Su1, Ming Guo2. 1) Deptment of Biology, MC156-29, CA. California Institute of Technology, Pasadena, CA; 2) Departments of Neuology and Pharmacology, The David Geffen 54 - 8:45 School of Medicine at UCLA, Los Angeles, CA 90095. Centrosomin is regulated by multiple kinases at mitotic centrosomes during development in D. melanogaster. Robert Eisman, Lei Gong, Melissa Phelps, Thomas Kaufman. Dept Biol, Jordan Hall A505, Indiana Univ, Bloomington, IN. PLATFORM SESSIONS 31 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

55 - 9:00 Biochemistry and Molecular Biology, The University of Texas In vivo quantitative imaging of coordinate cell movements within MD Anderson Cancer Center, Houston, TX. developing Drosophila embryos. Amy McMahon1, Willy Supatto2, Scott Fraser2, Angela Stathopoulos1. 1) Biology, 65 - 12:00 California Institute of Technology, Pasadena, CA; 2) Beckman The Frizzled and Fat/Dachsous pathways control wing Imaging Center, California Institute of Technology, Pasadena, topography. Simon Collier, Kristy Doyle, Justin Hogan, Eric CA. Aten. Dept Biological Sci, Marshall Univ, Huntington, WV.

56 - 9:15 66 - 12:15 Drosophila APC2 APC1 null epithelial clones exhibit wingless Stripe non-autonomously controls the orientation of actin-based pathway dependent cell shape changes and epithelial protrusions. Stacie Dilks, Stephen DiNardo. University of misfolding. Sandra G. Zimmerman, Lauren M. Thorpe, Pennsylvania, Philadelphia, PA. Carolyn A. Mallozzi, Vilma R. Medrano, Brooke M. McCartney. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA. FRIDAY, APRIL 4 4:30 pm–6:30 pm 57 - 9:30 Golden West Hook-like is a negative regulator of endocytic trafficking. Adam Haberman, Sanchali Ray, Helmut Kramer. Cntr Basic Gametogenesis Neurosci, UT Southwestern Med Cntr, Dallas, TX. Moderator: Xin Chen, Johns Hopkins University, Baltimore, 58 - 9:45 Maryland Diaphanous, a regulator of cell contractility and motility. Catarina Homem, Mark Peifer. Dept Biol, Univ North Carolina, 67 - 4:30 Chapel Hill, NC. Transition of male primordial germ cells to functional germline stem cells. Matthew Wawersik1, Rebecca Sheng2, Trevor 59 - 10:00 Posenau1, Juliann Gumulak-Smith1, Erika Matunis2, Mark DRhoGEF2 regulates contractile force during segmental groove Van Doren3. 1) Biology Dept., College of William & Mary, morphogenesis. Shai Mulinari, Mojgan Padash Barmchi, Udo Williamsburg, VA; 2) Dept. of Cell Biology, Johns Hopkins School Häcker. Experimental Medical Science, Lund University, Lund, of Medicine, Baltimore, MD; 3) Dept. of Biology, Johns Hopkins Sweden. University, Baltimore, MD. 10:15 am - Break 68 - 4:45 Live imaging of asymmetric centrosome migration and spindle 60 - 10:45 orientation in Drosophila male germline stem cells. Jun Cheng1, The golgi SNARE, Gos28, is essential for rhodopsin transport Nahid Hemati2, Yukiko M. Yamashita2,3, Alan J. Hunt1. 1) and photoreceptor survival. Erica E. Rosenbaum, Natalia S. Department of Biomedical Engineering; 2) Center for Stem Cell Rozas, Nansi Jo Colley. Dept. of Ophth. & Vis. Sci., Dept. of Biology, Life Sciences Institute; 3) Department of Cell and Genetics Univ. Wisconsin, Madison, WI. Developmental Biology, University of Michigan, Ann Arbor, MI. 61 - 11:00 69 - 5:00 Eating yourcellf into shape: Atg1 and autophagy in cell shape Live Imaging of Drosophila Spermatogonia Dedifferentiating changes. Pavan Kadandale, Amy Kiger. Dept. of Cell & into Germline Stem Cells. Xuting Rebecca Sheng, Crista Developmental Biology, UCSD, La Jolla, CA. Brawley, Erika Matunis. Dept Cell Biol, Johns Hopkins Univ, Baltimore, MD. 62 - 11:15 Dynamics of sarcomere assembly in the flight muscles. John 70 - 5:15 Sparrow, Zacharias Orfanos. Dept Biol, Univ York, York, United Asymmetric activation of Rac in germ line stem cells of the Kingdom. ovary controls both the plane of division and the response to BMP signals. Wen Lu1, M. Olivia Casanueva2, Chip 63 - 11:30 Ferguson1,2,3. 1) Committee on Genetics; 2) Committee on Genetic control of cell morphogenesis during formation of the Developmental Biology; 3) Dept. of Molecular Genetics and Cell Drosophila cardiac tube. Caroline Medioni1, Martine Astier1, Biology, University of Chicago, Chicago, IL. Monika Zmojdzian2, Krzysztof Jagla2, Michel Semeriva1. 1) CNRS-UMR6216, IBDML, MARSEILLE, France; 2) INSERM 71 - 5:30 U384, Faculté de Médecine, Clermont-Ferrand, France. Insulin signals regulate GSC maintenance via the control of niche size. Hwei-Jan Hsu, Daniela Drummond-Barbosa. 64 - 11:45 Department of Cell and Developmental Biology, Vanderibilt steamer duck inhibits epidermal cell-cell fusion in Drosophila Univerisity Medical Center, Nashville, TN. larvae. Yan Wang, Michael Galko. The Department of 32 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

72 - 5:45 80 - 5:45 Stem Cell Maintenance Through Competition in the Follicle A cell autonomous requirement for the Dally-like core protein Stem Cell Niche. Todd Nystul, Allan C. Spradling. Embryology in Hedgehog signaling. Elizabeth H. Williams1, William N. Dept, Carnegie Inst, Baltimore, MD. Pappano2, Philip A. Beachy1. 1) HHMI, Dept of Developmental Biology, Stanford Univ Sch Med, Stanford, CA; 2) HHMI, Dept 73 - 6:00 of Molecular Biology & Genetics, Johns Hopkins Univ Sch Med, A Bam complex mediates a cap-dependent translational switch Baltimore, MD. to promote stem cell differentiation. Jean Maines, Yun Li, Dennis McKearin. Department of Molecular Biology, UT 81 - 6:00 Southwestern Medical Center, Dallas, TX. Sequential actions of feedforward and feedback loops pattern Drosophila egg: genetic experiments and computational 74 - 6:15 modeling. Nir Yakoby1,2, Jessica Lembong1,2, Trudi Role of Drosophila Ime4 and Ime2 in the Initiation of Meiosis. Schüpbach3, Stanislav Y. Shvartsman1,2. 1) Lewis-Sigler Cintia Hongay, Gerald Fink, Terry Orr-Weaver. Fink and Orr- Institute for Integrative Genomics, Princeton University, Weaver Labs, Whitehead Inst, Cambridge, MA. Princeton, NJ; 2) Dept. of Chemical Engineering, Princeton University, Princeton, NJ; 3) Howard Hughes Medical Institute and Dept. of Molecular Biology, Princeton University, NJ.

FRIDAY, APRIL 4 4:30 pm–6:30 pm 82 - 6:15 Town & Country Analysis of synthetic interactions in Drosophila by RNAi. Thomas Horn1, Elin Axelsson2, Wolfgang Huber2, Michael Signal Transduction Boutros1. 1) Signaling & Functional Gen, German Cancer Research Ctr, Heidelberg, Baden-Wuerttemberg, Germany; 2) Moderator: Scott Barolo, University of Michigan Medical EMBL/EBI - European Bioinformatics Institute, Wellcome Trust School, Ann Arbor Genome Campus, Cambridge CB10 1SD, UK.

75 - 4:30 Specificity of Signaling by Drosophila Fibroblast Growth Factors. Angelike Stathopoulos, Phoebe Tzou, Snehalata Kadam. FRIDAY, APRIL 4 4:30 pm–6:30 pm Div Biol, MC 114-96, Caltech, Pasadena, CA. California

76 - 4:45 Pattern Formation Endosomal Entry Regulates Notch Receptor Activation in Drosophila. Thomas Vaccari1, Han Lu1, Ritu Kanwar2, Mark Moderator: Richard Mann, Columbia University, Fortini2, David Bilder1. 1) Molecular & Cell Biology, University New York of California, Berkeley, Berkeley, CA; 2) Center for Cancer Research, National Cancer Institute, Frederick, MD. 83 - 4:30 From parts to pattern: deciphering pair rule seven stripe 77 - 5:00 formation with a completed cis-regulatory blueprint. Mark Pentagone, a novel BMP/Dpp target gene, involved in patterning Schroeder, Ulrike Gaul. Rockefeller University, New York, NY. and growth in Drosophila. Robin Vuilleumier1, Markus Affolter2, George Pyrowolakis1. 1) Developmental Biology 84 - 4:45 Unit, Biology I, University of Freiburg, Freiburg, Germany; 2) Reading between the lines: pair-rule regulation of cell adhesion Dept. of Cell Biology, Biozentrum, University of Basel, Basel, molecules. W. Ray Anderson1,2, Leslie Pick2. 1) Dept. of Cell Switzerland. Biology and Molecular Genetics and; 2) Dept. of Entomology, University of Maryland, College Park, MD 20742. 78 - 5:15 A translational block to HSPG synthesis permits BMP signalling 85 - 5:00 in the early Drosophila embryo. Douglas Bornemann1, Regulation of Ft-Ds signaling by the DHHC transmembrane Sangbin Park2, Sopheap Phin1, Rahul Warrior1. 1) Dept protein Approximated. Hitoshi Matakatsu, Seth Blair. Zoology, Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; University of Wisconsin-Madison, Madison, WI. 2) Department of Developmental Biology, Stanford University School of Medicine, Stanford, California. 86 - 5:15 Drosophila glypican Dally-like acts in FGF-receiving cells to 79 - 5:30 modulate FGF signaling during tracheal morphogenesis. Dong Interactions between the retinal determination protein Eyes Yan 1,2, Xinhua Lin1,2. 1) Div Developmental Biol, Cincinnati absent and the Abelson tyrosine kinase suggest a novel function Children’s Hosp, Cincinnati, OH; 2) The Graduate Program in for Eya in cytoskeletal regulation during axonogenesis. Wenjun Molecular and Developmental Biology, University of Cincinnati Xiong, Noura Dabbouseh, Ilaria Rebay. Ben May Dept. for College of Medicine, Cincinnati, OH. Cancer Research, The Univ. of Chicago, Chicago, IL. PLATFORM SESSIONS 33 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

87 - 5:30 The homeotic genes labial and Deformed regulate decapentaplegic expression restricted to the peripodial epithelium and required for the formation of the adult head. Brian Stultz1, Mark Mortin2, Deborah Hursh1. 1) DCGT, FDA/ CBER, Bethesda, MD; 2) LMG, NICHD/NIH, Bethesda, MD.

88 - 5:45 The anterior-posterior gradient of microtubule organization in the oocyte depends on Par-1-induced Tau phosphorylation. Ai- Guo Tian, Wu-Min Deng. Dept Biological Sci, Florida State Univ, Tallahassee, FL.

89 - 6:00 Drosophila EGFR signaling is modulated by differential compartmentalization of Rhomboid intra-membrane proteases. Shaul Yogev, Eyal Schejter, Benny Shilo. Molecular Genetics, Weizmann Institut, Rehovot, Israel.

90 - 6:15 Regeneration genes affect the position, time and amount of blastema formation. Anne Sustar1, Kim McClure1,2, Gerold Schubiger1. 1) Dept Biology, Univ Washington, Seattle; 2) Dept Anatomy, UC San Francisco. 34 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

SATURDAY, APRIL 5 8:30 am–10:15 am SATURDAY, APRIL 5 8:30 am–10:15 am Town & Country Golden West

Drosophila Models of Human Diseases RNA Biology Moderator: Ethan Bier, University of California- Moderator: A. Javier Lopez, Carnegie Mellon University, San Diego, La Jolla Pittsburgh, Pennsylvania

91 - 8:30 105 - 8:30 A prion-like domain in Drosophila fragile X protein is essential Global analysis of mRNA localization reveals a prominent role for regulating synaptic plasticity. Paromita Banerjee1, Brian P. in the organization of cellular architecture and function. Eric Schoenfeld2, Sean M. J. McBride2, Thomas C. Dockendorff1. Lecuyer1, Hideki Yoshida1, Christina Alm1, Neela 1) Dept Zoology, Miami Univ, Oxford, OH; 2) Molecular Parthasarathy1, Tomas Babak1, Pavel Tomancak2, Henry Cardiology, Albert Einstein College of Medicine, Bronx, NY. Krause1. 1) Donnelly CCBR, University of Toronto, Toronto, Canada; 2) Max Planck Institute of Molecular Cell Biology and 92 - 8:45 Genetics, Dresden, Germany. De novo CoA biosynthesis is required to maintain DNA integrity in a Drosophila model of Pantothenate Kinase-Associated 106 - 8:45 Neurodegeneration. Ody Sibon, Floris Bosveld, Anil Rana, Fragile X Protein controls the efficacy of mRNA transport in Harm Kampinga. Dept Cell Biol, Univ Groningen, Groningen, neurons. Daniela C. Zarnescu, Patty Estes, Michelle O’Shea. Netherlands. Dept Molecular & Cell Biol, Univ Arizona, Tucson, AZ.

93 - 9:00 107 - 9:00 Using Drosophila to probe the activities of anthrax toxins. A genetic screen for asymmetrically localized RNAs in Annabel Guichard1, Shauna Mc Gillivray2, Beatriz Cruz- Drosophila tracheal cells. Jayan N. Nair1, Maria Leptin1, Paolo Moreno1, Victor Nizet2, Ehan Bier1. 1) Division of Biological Filardo2, Veit Riechmann2, Elizabeth R. Gavis3. 1) Institute Sciences, UCSD, La Jolla, CA; 2) Division of Pediatric for Genetics, University Of Cologne, Cologne, NRW, Germany; Pharmacology & Drug Discovery UCSD School of Medicine 2) Institute for Developmental Biology,University of La Jolla, CA. Cologne,NRW, Germany; 3) Dept. of Molecular Biology, Princeton University, Princeton 08544. 94 - 9:15 Drosophila easily-shocked: phosphatidyl-ethanolamine 108 - 9:15 metabolism and cardiac disorders. Hui-Ying Lim1, Robert J. RNA silencing influences gypsy chromatin insulator function Wessells2, Rolf Bodmer1. 1) Burnham Inst. for Medical and nuclear organization. Elissa P. Lei, Nellie Moshkovich, Research, La Jolla, CA; 2) U.Mich, Ann Arbor, MI. Patrick J. Boyle. Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD. 95 - 9:30 Regulation of ER stress induced apoptosis by the Unfolded 109 - 9:30 Protein Response. Min-Ji Kang, Hyung Don Ryoo. Analysis of the role of splicing and cis-acting elements in oskar Department of Cell Biology, NYU, School of Medicine, New mRNA localization in Drosophila oocyte. Sanjay Ghosh, York, NY. Virginie Marchand, Anne Ephrussi. Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, 96 - 9:45 Germany. Genetic Interaction between Survival of Motor Neuron Gene (SMN) and BMP Signaling Pathway. Howard Chang1, Yokokura 110 - 9:45 Takakazu1, Dimlich Douglas1, Kankel Mark1, Mukherjee Control of alternative splicing by regulatory networks in Ashim2, Walker Amy3, Harris Jevede3, Duckworth April1, Hart Drosophila. Britta Hartmann1, R. Castelo1, S. Boue1, M. Anne3, Van Vactor David1, Artavanis-Tsakonas Spyros1. 1) Blanchette2, E. Peden3, R. RioSingh3, D. Rio2, J. Valcarcel1. Dept Cell Biology, HMS, Harvard Medical School, Boston, MA; 1) Centre de Regulació Genòmica, Barcelona, Spain; 2) 2) Department of Molecular and Human Genetics Banaras University of California, Berkeley, USA; 3) University of Hindu University Varanasi-221005 India; 3) MGH Cancer Colorado, Boulder. Center, Building 149,Charlestown, MA. 111 - 10:00 97 - 10:00 Mir-3 and mir-318 regulate Drosophila nautilus gene expression. Regulation of phosphoinositide phosphates in Drosophila Anandarao Ravulapalli, Wei Qin, Bruce Paterson. Laboratory morphogenesis. Inês Ribeiro, Jared Dennis, Amy Kiger. Div of Biochemistry and Molecular Biology, National Cancer Biological Sciences, Univ California, San Diego, La Jolla, CA. Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Session continued on page 35. PLATFORM SESSIONS 35 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

SATURDAY, APRIL 5 8:30 am–10:15 am SATURDAY, APRIL 5 10:45 am–12:30 pm California Town & Country

Genome and Chromosome Structure Drosophila Models of Human Diseases (continued from page 34) Moderator: Roger Hoskins, Lawrence Berkeley National Laboratory, Berkeley, California Moderator: Ethan Bier, University of California-San Diego, La Jolla 112 - 8:30 Efficient identification of Drosophila Y-chromosome sequences 98 - 10:45 by short-read sequencing. Bernardo Carvalho1, Andrew Modeling human brain cancer in Drosophila. Renee D. Read, Clark2. 1) Dept de Genetica, Univ Fed Rio de Janeiro, Rio de John B. Thomas. Molecular Neurobiology Laboratory. Salk Janeiro, Brazil; 2) Molecular Biology and Genetics, Cornell Institute for Biological Studies, San Diego, CA 92037. University, USA. 99 - 11:00 113 - 8:45 Mutations in the gene clueless cause mitochondrial Evolution of nested genes in Drosophila and vertebrates. mislocalization and Parkinson-like phenotypes in the Drosophila Fyodor A. Kondrashov1, Raquel Rassis2, Alexey ovary and muscle. Rachel Cox1,2, Megan Kutzer1, Shelley Kondrashov2, Eugene Koonin3. 1) Section of Ecology, Paterno1,2, Allan C. Spradling1,2. 1) Dept Embryology, Carnegie Behavior & Evolution, UCSD, La Jolla, CA; 2) Center for Inst of Washington, Baltimore, MD; 2) HHMI. Computational Medicine and Biology and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109; 100 - 11:15 3) National Center for Biotechnology Information, National ubiquilin antagonizes presenilin, stabilizes APP and promotes Library of Medicine, National Institutes of Health, Bethesda, neurodegeneration. Ming Guo, Atish Ganguly, Renny MD. Feldman. Dept Neurology & Pharmacology, Univ California, Los Angeles, Los Angeles, CA. 114 - 9:00 Repetitive Elements and the Rise of Chimeric Genes in the D. 101 - 11:30 melanogaster Subgroup. J. Roman Arguello1, Shuang Yang2,3, Genomic regulation of lipid storage. Mathias Beller1,2, Carole Xin Li2,3, Yun Ding2,3, Qi Zhou2,3, Ying Chen1, Yue Zhang2, Sztalryd1,3, Herbert Jaeckle2, Brian Oliver1. 1) Laboratory of Ruoping Zhao2, Frédéric Brunet4, Lixin Peng2, Manyuan Cellular and Developmental Biology, National Institute of Long1, Wen Wang2. 1) Dept Ecology & Evolution, Univ Chicago, Diabetes and Digestive and Kidney Diseases, National Chicago, IL; 2) Kunming Institute of Zoology, Kunminng, China; Institutes of Health, Bethesda MD 20892; 2) Max-Planck-Institut 3) Graduate School of Chinese Academy of Science, Beijing, fuer biophysikalische Chemie, Abt. fuer Molekulare China; 4) Ingénieur de Recherche en Bioinformatique Equipe Entwicklungsbiologie, Am Fassberg 11, 37077 Goettingen, Génomique Evolutive des Vertébrés IGFL, France. Germany; 3) GRECC/Geriatrics , Veterans Affairs Medical Center 10, North Greene Street, Baltimore MD 21201. 115 - 9:15 Highly asymmetrical rates of evolution between paralogs 102 - 11:45 following the duplication of testes expressed genes from neo- Genetic modifiers of MeCP2 function in Drosophila. David X-chromosomes to autosomes. Richard Meisel1, Mira Han2, Mittelman1, Holly Cukier1, Ann Collins1, Alma Perez1, Matthew Hahn2. 1) Penn State University; 2) Indiana University. Zhaolan Zhou1, Huda Zoghbi1,2, Juan Botas1. 1) Departemnt of Molecular and Human Genetics, Baylor College of Medicine, 116 - 9:30 Houston, TX; 2) Howard Hughes Medical Institute, Baylor Age-related changes in double-strand break repair. William College of Medicine, Houston TX. Engels, Christine Preston, Dena Johnson-Schlitz, Carlos Flores. Dept Genetics, Univ Wisconsin, Madison, WI. 103 - 12:00 Generation of Neurotoxic Prion Protein Isoforms and the Role 117 - 9:45 for Hsp70 in Prion Protein Conversion. Sergio Casas-Tinto, Evolutionary genetics of hybrid sterility in Drosophila. Nitin Melisa Gomez-Velazquez, Claudio Soto, Pedro Fernandez- Phadnis, Allen Orr. Dept Biol, Univ Rochester, Rochester, NY. Funez, Diego E. Rincon-Limas. Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555. 118 - 10:00 Drosophila histone variant H2Av localizes to centromeres and 104 - 12:15 regulates normal localization of centromeric histone H3 variant Rhomboid-7 and Omi are components of the Pink1/Parkin CENP-A/CID. Weiguo Zhang1,2, Gary H. Karpen1,2. 1) the Life pathway which affects mitochondrial membrane dynamics. Sciences Division, the Lawrence Berkeley National Laboratory, Alexander J. Whitworth1, Jeffrey Lee2, Angela Poole3, Ruth Berkeley, CA; 2) Department of Molecular and Cellular Biology, Thomas3, Venus Ho1, Leo Pallanck3, Angus McQuibban2. 1) University of California-Berkeley, Berkeley, CA. Dept of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; 2) Dept of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 3) Department of Genome Sciences, University of Washington, Seattle. 36 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

SATURDAY, APRIL 5 10:45 am–12:30 pm 125 - 12:15 Golden West A Morphology and Gene Expression Atlas of Drosophila Embryogenesis. Cris L. Luengo Hendriks1, Soile V. E. Techniques and Functional Genomics Keränen1, Pablo Arbelaez2, Gunther H. Weber1, Charless C. Fowlkes3, Clara N. Henriquez1, David W. Kaszuba1, Bernd Moderator: Bernard Mathey-Prevot, Duke University Hamann4, Jitendra Malik2, Mark Biggin1, David W. Knowles1. Medical Center, Durham, NC 1) Lawrence Berkeley National Laboratory, Berkeley, CA; 2) University of California, Berkeley, CA; 3) University of California, Irvine, CA; 4) University of California, Davis, CA. 119 - 10:45 A toolkit for high-throughput gene engineering in flies. Radoslaw K. Ejsmont1, Mihail Sarov2, Pavel Tomancak1. 1) Tomancak Lab, MPI-CBG, Dresden, Germany; 2) BAC Facility, MPI-CBG, Dresden, Germany. SATURDAY, APRIL 5 10:45 am–12:30 pm California 120 - 11:00 PhiC31-mediated cassette exchange in Drosophila. Jack R. Chromatin and Gene Expression Bateman, Anne M. Lee, Lillian Merriam, Laura Stadelmann, C.-ting Wu. Department of Genetics, Harvard Medical School, Moderator: Elissa Lei, National Institutes of Diabetes, Boston, MA. Digestive and Kidney Diseases; Bethesda, Maryland

121 - 11:15 126 - 10:45 The Drosophila ORF Collection: a high quality resource for Understanding the function of insulators in the Drosophila proteomic and functional genomic studies. Mark Stapleton, genome. David J. Marion1, Alexey A. Soshnev2, Kate Charles Yu, Ken Wan, Soo Park, Bhaveen Kapadia, Bayan Appleton3, Xingguo Li3, Misty D. Wehling3, Ryan M. Baxley2, Parsa, Joseph W. Carlson, Susan E. Celniker. Genome and Pamela K. Geyer1,2,3. 1) Genetics Program; 2) Molecular and Computational Biology, Lawrence Berkeley National Lab, Cellular Biology Program; 3) Department of Biochemistry, Berkeley, CA. University of Iowa, Iowa City, IA 52242.

122 - 11:30 127 - 11:00 Genome-wide mapping and annotation of protein expression Bhringi, a highly conserved regulator of Twist transcription factor and interaction in D. melanogaster, using a hybrid PiggyBac/P- activity. Scott J. Nowak1, Katie Gonzalez2, Mary Baylies1. 1) element YFP gene trap system with tandem affinity tags. Ed Dept. of Developmental Biology, Sloan-Kettering Institute, New Ryder1, Helen Spriggs1, Emma Drummond1, Laura Harris1, York, NY; 2) Scripps Research Institute, La Jolla, CA. Jane Webster1, Glynnis Johnson1, John Roote1, Nick Lowe3, Kathryn Lilley2, Svenja Hester2, Julie Howard2, Johanna 128 - 11:15 Rees2, Steve Russell1,2, Daniel St. Johnston3. 1) Dept Genetic analyses of cofactors that cooperate with the Brahma Genetics, Cambridge Univ, UK; 2) Cambridge Systems Biology (SWI/SNF) chromatin remodeling complex in the regulation of Centre, Cambridge Univ, Cambridge, UK; 3) Gurdon Institute, target genes. Chhavi Chauhan1, Claudia Zraly1,2, Manuel Dept Genetics, Cambridge Univ, Cambridge, UK. Diaz1,2, Andrew Dingwall1,2,3. 1) Molecular Biology Program, Loyola University Chicago, Stritch School of Medicine, 123 - 11:45 Maywood, IL; 2) Oncology Institute; 3) Department of Pathology. Identification of compounds that modulate lipid droplet storage in S3 cells using qHTS. Douglas Auld1, Ya-Qin Zhang1, Noel 129 - 11:30 Southall1, Mathias Beller3, James Inglese1, Christopher Paused Polymerase in the Drosophila Embryo. Michael S. Austin1, Brian Oliver2. 1) NIH Chemical Genomics Center, NIH, Levine1, Julia Zeitlinger2, Joung-Woo Hong1, Jess Piel1, Rockville, MD; 2) National Insitutes of Diabetes and Digestive Dave Hendrix1, Richard A. Young3. 1) MCB, UC Berkeley, and Kidney Diseases, NIH, Bethesda MD, 20892; 3) Max- Berkeley, CA; 2) Stowers Institute for Medical Research, Kansas Planck-Institut for Biophysical Chemistry, Göttingen, Germany. City, MO; 3) Whithead Institue, MIT, Cambridge, MA.

124 - 12:00 130 - 11:45 Systems model of ATP-generating metabolic network in Chromatin-remodeling by Kismet in Transcription and Drosophila flight muscle. Jacob D. Feala1, Laurence Coquin2, Development. Kristel M. Dorighi1, Shrividhya Srinivasan1,2, Andrew D. McCulloch1, Giovanni Paternostro1,2. 1) Dept. of John W. Tamkun1. 1) MCD Biology, UC Santa Cruz, Santa Cruz, Bioengineering, University of California - San Diego, La Jolla, CA; 2) Developmental Biology, Stanford University, Stanford, CA; 2) Burnham Institute for Medical Research. CA.

131 - 12:00 Regulation of Myc-induced cell growth by the histone H3K4 demethylase Lid. Julie Secombe, Ling Li, Robert Eisenman. Div Basic Sci, Fred Hutchinson Cancer Res Ctr, Seattle, WA. PLATFORM SESSIONS 37 Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

132 - 12:15 139 - 5:30 P element repression by an epigenetic telomeric trans-silencing Regulation of metabolism, organ senescence, and lifespan by involving RNA silencing and heterochromatin formation. the nutrient sensing TOR pathway. Sean Oldham, Claire Stéphane Ronsseray, Thibaut Josse, Laure Teysset, Anne- Davies, Nancy Luong, Ryan Birse, R. J. Wessells, Suzanne Laure Todeschini, Augustin de Vanssay, Clara Sidor, Valérie Graham, Rolf Bodmer. Burnham Institute for Medical Delmarre, Dominique Anxolabéhère. Dynamique du Génome Research, La Jolla, CA, 92037. et Evolution, Inst Jacques Monod, Paris, France. 140 - 5:45 A genetic screen implicates the ER translocon and TRiC/CCT cytoplasmic chaperone complex as novel regulators of SATURDAY, APRIL 5 4:00 pm–6:00 pm autophagy. Andrew M. Arsham, Thomas P. Neufeld. Dept of Golden West Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN. Physiology and Aging Moderator: Daniela Drummond-Barbosa, Vanderbilt University, Nashville, Tennesee SATURDAY, APRIL 5 4:00 pm–6:00 pm Town & Country 133 - 4:00 The TOR pathway couples nutrition and developmental timing Neurogenetics and Neural Development in Drosophila. Pierre Leopold, Sophie Layalle. CNRS/ University of Nice, Nice, France. Moderator: Marc Freeman, University of Massachusetts Medical School, Worchester 134 - 4:15 Stem cell aging is controlled both intrinsically and extrinsically 141 - 4:00 in the Drosophila ovary. Lei Pan1,2, Ting Xie1. 1) Stowers Inst, dfezl encodes a novel regulator of neural stem cell self-renewal Kansas City, MO; 2) Inst of Biophysics,Chinese Academy of in Drosophila. Mo Weng1,2, Shufen Situ2, Caitlin Gamble2, Sciences,Beijing,China. Cheng-Yu Lee1,2. 1) Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, 135 - 4:30 MI 48109; 2) Center for Stem Cell Biology, Life Sciences Crosstalk between the Insulin and Toll pathways shifts nutrient Institute, University of Michigan, Ann Arbor, MI 48109. metabolism in response to infection. Justin DiAngelo1, Sara Cherry2, Morris Birnbaum1. 1) Institute for Diabetes, Obesity 142 - 4:15 and Metabolism, Univ Pennsylvania/HHMI, Philadelphia, PA; Dephosphorylation of Bazooka by PP2A is required for proper 2) Department of Microbiology and The Penn Genomics apical-basal polarity in embryonic neuroblasts. Michael P. Institute, Univ Pennsylvania, Philadelphia, PA. Krahn, Andreas Wodarz. Department of Stem Cell Biology, University of Goettingen, Germany. 136 - 4:45 Rescue of the flightless phenotype of a Glutathione S- 143 - 4:30 transferase S1 (GstS1) null mutant. Oksana Litvinova1, Sarah From stem cell to unique neuron: subdivision of the Castor Dauback2, Ashis Mondal3, Piotr Zimniak2, Helen Benes1. 1) temporal window by a feedforward loop involving Squeeze, Nab Neurobiol & Develop Sci; 2) Pharmacol & Toxicol; 3) Internal and Collier/Knot. Magnus Baumgardt1, Daniel Karlsson1, Medicine, Univ of Arkansas for Med Sci, Little Rock, AR. Javier Terriente2, Fernando J. Díaz-Benjumea2, Stefan Thor1. 1) Dept. of Clinical and Experimental Medicine, Linköping 137 - 5:00 University Medical School, Linköping, Sweden; 2) Centro de The regulation of Drosophila lifespan by falafel. Brian Sage1, Biología Molecular-Severo Ochoa/C.S.I.C., Universidad Xi Lou2, Li Qian3, Rolf Bodmer3, Heinrich Jasper2, Marc Autónoma-Cantoblanco, Madrid, Spain. Tatar1. 1) Dept Ecol & Evol Biol, Brown Univ, Providence, RI; 2) Dept of Biology, Univ of Rochester, Rochester, NY; 3) Center 144 - 4:45 for Neurosciences and Aging, The Burnham Institute, La Jolla, Temporal transcription factors schedule the end of neurogenesis CA. via cell cycle exit or apoptosis. Louise Cheng, Cedric Maurange, Alex Gould. National Institute for Medical 138 - 5:15 Research, London, United Kingdom. Genetic and environmental regulation of metabolism, behavior and lifespan by specific nutrient components. Danielle A. 145 - 5:00 Skorupa1,3, Azra Dervisefendic1, Scott D. Pletcher1,2,3. 1) Structural Basis for Robo Receptor Control of Lateral Huffington Center on Aging, Baylor College of Medicine, Positioning: An Unexpected Role for Robo Extracellular Houston, TX; 2) Department of Molecular & Human Genetics, Domains. Timothy A. Evans1, Barry J. Dickson2, Greg J. Baylor College of Medicine, Houston, TX; 3) Interdepartmental Bashaw1. 1) Dept of Neuroscience, University of Pennsylvania Program in Cell & Molecular Biology, Baylor College of School of Medicine, Philadelphia, PA; 2) Research Institute of Medicine, Houston, TX. Molecular Pathology, Vienna, Austria. 38 PLATFORM SESSIONS Program number is in bold above the title. The first author is the presenter. Abstracts begin on page 81.

146 - 5:15 153 - 5:00 Down Syndrome Cell Adhesion Molecules as multifunctional Drosophila short neuropeptide F signaling regulates growth by Netrin receptors required for midline crossing. Gracie L. ERK mediated insulin signaling. Kweon Yu1, Kyu-Sun Lee1, Andrews, Thomas Kidd. Biology Department/ms 314, Kyung-Jin Min2, Marc Tatar2. 1) Centre for Regenerative University of Nevada, Reno, NV 89557, USA. Medicine, Korea Res Inst of Bioscience & Biotechnology, Daejeon, Korea; 2) Dept of Ecology & Evolutionary Biology, 147 - 5:30 Brown University, Providence, RI. DOUBLESEX establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by 154 - 5:15 directing cell number. Laura Sanders, Michelle Arbeitman. GTRs: small GTPases implicated as novel regulaTORs of Molecular and Computational Biology, University of Southern growth. Pankuri Goraksha, Thomas Neufeld. Molecular, California, Los Angeles, CA. Cellular, Developmental Biology & Genetics, University of Minnesota, Minneapolis, MN. 148 - 5:45 Expression of vestigial in the Drosophila embryonic central 155 - 5:30 nervous system. Kirsten Guss1, Hemlata Mistry1,2, James alpha-Endosulfine is a conserved protein required for meiotic Skeath2. 1) Dept Biol, Dickinson College, Carlisle, PA; 2) Dept maturation that interacts with an E3 ubiquitin ligase and Genetics, Washington University School of Medicine, St. Louis, regulates Twine/CDC25 levels. Jessica Von Stetina, Susanne MO. Tranguch, Sudhansu Dey, Ethan Lee, Laura Lee, Daniela Drummond-Barbosa. Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN.

SATURDAY, APRIL 5 4:00 pm–6:00 pm 156 - 5:45 California Mutation in the Ubiquitin Activating Enzyme, E1, can cause tissue overgrowth by upregulating Ras pathway activity by both Cell Division and Growth Control cell-autonomous and non-autonomous means. Hua Yan1, Mei- Ling Chin2, Cathie Pfleger1. 1) Department of Oncological Moderator: Amy Kiger, University of California-San Diego, Sciences, Mount Sinai School of Medicine, New York, NY; 2) La Jolla Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, NY. 149 - 4:00 Insulin Receptor pathway regulates cell division through miRNAs and p21/dacapo in the Drosophila germ line stem cells. Jenn-Yah Yu, Steve Hatfield, Halyna Shcherbata, Karin Fischer, Hannele Ruohola-Baker. Department of Biochemistry, University of Washington, Seattle, WA.

150 - 4:15 The anaphase promoting complex/cyclosome (APC/C) is required for re-replication control in endoreplication cycles. Norman Zielke1,2, Silvia Querings2, Frank Sprenger2,3. 1) Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA; 2) University of Cologne, Institute for Genetics, Zuelpicherstrasse 47, 50674 Koeln, Germany; 3) University of Zuerich, Institute of Zoology, Winterthurerstrasse 190, CH-8057 Zuerich, Switzerland.

151 - 4:30 The role of Misato in Drosophila spindle assembly. Violaine Mottier, Giovanni Cenci, Fiammetta Vernì, Maurizio Gatti, Silvia Bonaccorsi. Genetica Biol Molecolare, Univ di Roma La Sapienza, Rome, Italy.

152 - 4:45 Functional analysis of a novel family of ERM protein partners during cell division. Sébastien Carreno1,2, Hélène Foussard2, Jérome Miailhe2, Cédric Polesello2, Philippe Valenti2, Pierre Ferrer2, Francois Payre2. 1) Université de Montréal, IRIC, Montreal, QC, Canada; 2) Centre de Biologie du Développement, CNRS University ToulouseIII, Toulouse France. POSTER SESSIONS 39 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

Cell Division and Growth Control 167B Roles for the Drosophila E3 Ligase, Neuralized in Hematopoiesis. 157A Chiyedza Small1, Jemila Caplan1, Vassilis Baoussis2, Cell competition and growth control during Drosophila Christos Delidakis2, Shubha Govind1. 1) Department of development. Francisco A. Martin, Salvador C. Herrera, Gines Biology, The City College of CUNY, New York, NY, 10031; 2) Morata. CBMSO (CSIC-UAM), UAM (Cantoblanco), Madrid, Institute of Molecular Biology and Biotechnology, FoRTH, P.O. Spain. Box 1527, GR 711 10, Heraklion, Crete, Greece.

158B 168C Requirements for cell competition during Drosophila Functional studies of the Mitf gene in Drosophila. Tianyi Zhang, development. Ricardo M. Neto da Silva1, Laura A. Johnston2. Francesca Pignoni. Dept Ophthalmology, Harvard Medical Sch/ 1) Gulbenkian PhD Program in Biomedicine, Instituto Gulbenkian MEEI, Boston, MA. de Ciencia, Oeiras, Portugal; 2) Department of Genetics & Development, Columbia University, 701 West 168th Street, 169A HHSC704, New York, NY 10032. DmSGT1 is required to maintain spindle bipolarity through a mechanism that involves Polo stabilization. Torcato J. Martins1, 159C Andre F. Maia1, Sören Steffensen1, Claudio Sunkel1,2. 1) Identification of factors that mediate Myc-induced competition Molecular Genetics, Institute for molecular and cell biology, between cells. Nanami Senoo-Matsuda, Laura A. Johnston. Porto,Portugal; 2) ICBAS, Instituto de Ciências Biomédicas Abel Deptartment of Genetics & Development, College of P&S, Salazar, Universidade do Porto, Porto, Portugal. Columbia University, New York, NY 10032. 170B 160A Determinants of CNN targeting and MTOC regulation at The regulation and function of Drosophila Atg1 in autophagy. centrosomes. Timothy L. Megraw, Jiuli Zhang, Ling-Rong Kao. Yu-Yun Chang, Thomas Neufeld. Genetics and Cell Green Ctr/Pharmacology, Univ Texas Southwestern Med Ctr, Development, University of Minnesota, Minneapolis, MN. Dallas, TX.

161B 171C Drosophila rictor and raptor in cell growth control. Sekyu Choi, Developmental Regulation of S-Phase Coupled Destruction of Gina Lee, Jongkyeong Chung. Department of Biological E2F1. Jean M. Davidson1, Shu Shibutani1, Robert J. Duronio1,2. Sciences, Korea Advanced Institute of Science and Technology, 1) Department of Biology, University of North Carolina - Chapel Yusong-Gu, Taejon, Korea. Hill, Chapel Hill, NC; 2) Curriculm of Genetics and Molecular Biology, University of North Carolina - Chapel Hill, Chapel Hill, 162C NC. ATG1, an autophagy regulator, negatively regulates S6 kinase. Wonho Kim, Sung Bae Lee, Sunhong Kim, Jongkyeong 172A Chung. Department of Biological Sciences, Korea Advanced Two microRNAs, bantam and miR-7, play roles in the regulation Institute of Science and Technology, Yusong-Gu ,Taejon, Korea. of Drosophila Germline Stem Cell cell cycle regulation. Steven H. Reynolds, Ellen J. Ward, Jenn-Yah Yu. Biochemistry, 163A University of Washington, Seattle, WA. An Essential Role for Gp93, the Endoplasmic Reticulum Hsp90 Chaperone, in Growth Control. Jason C. Maynard1, Eric Spana2, 173B Christopher V. Nicchitta1. 1) Department of Cell Biology,Duke Investigating the role of Nuf, a Rab11 effector, in cytokinetic UniversityMedical Center; 2) Department of Biology, Model furrow formation. Justin Crest, William Sullivan. Dept MCD Systems Genomics, DukeUniversity, Durham, NC. Biol, Univ California,Santa Cruz, CA.

164B 174C Vesicle Recognition and Trafficking in Autophagy Regulation. Cell division requires a direct interaction between microtubule- Keith Naps, Thomas Neufeld. Genetics, Cell Biology & associated RacGAP and the contractile ring componenent, Development, University of Minnesota, Minneapolis, MN. Anillin. Stephen Gregory1, Saman Ebrahimi1, Joanne Milverton1, Whitney Jones2, Amy Bejsovec2, Robert Saint3. 165C 1) Centre for the Molecular Genetics of Development, University JAK/STAT signaling regulates cellular growth in Drosophila. of Adelaide, Adelaide, SA, Australia; 2) Department of Biology, Aloma Rodrigues, Erika Bach. Pharmacology, NYU School of Duke University, Durham, NC; 3) Centre for the Molecular Medicine, New York, NY. Genetics of Development, Research School for Biological Sciences, Australian National University, Canberra, ACT, 166A Australia. Structure/Function analysis of the dMyc protein in vivo. Daniela Schwinkendorf, Peter Gallant. Zoological Institute, University of Zürich, Zürich, Switzerland. 40 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

175A 185B Proteomic analysis of Drosophila Fragile X mutant cleavage Regulation of MEI-S332 Centromere Localization. Cristina stage embryos. Kate Monzo1, Susan R. Dowd2, Jonathan S. Nogueira, Hannah Cohen Koyfman, Lisa Deng, Lynn Young, Minden2, John C. Sisson1. 1) The Section of MCD Biology and Andrea Page-McCaw, Terry Orr-Weaver. Whitehead Institute The Institute for Cellular and Molecular Biology, The University for Biomedical Research, Cambridge, MA. of Texas at Austin, Austin, TX; 2) The Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA. 186C Loss of chiasma maintenance with age. Sharon E. Bickel, 176B Vijayalakshmi Subramanian. Dept. of Biological Sciences, Role of DMYPT in Incomplete Cytokinesis During Drosophila Dartmouth College, Hanover, NH. Oogenesis. SengKai Ong, Change Tan. Biological Science, University of Missouri-Columbia, Columbia, MO. 187A Complex genetic interactions control male meiosis in Drosophila. 177C Silvia Bongiorni1, Silvia Volpi1, Giorgio Prantera1, Barbara Enabled and its kinase Abelson provide a secondary input into Wakimoto2. 1) Dept. Agrobiology and Agrochemistry University the Pebble-Rho1-Diaphanous pathway initiating cytokinesis. of Tuscia, Viterbo,Italy; 2) Dept. of Biology University of Sergei Prokopenko, William Chia. Drosophila Development Washington,Seattle. Group, Temasek Lifesciences Laboratory, Singapore. 188B 178A Wispy, the Drosophila homolog of GLD-2, is required during Specialized lipids act to couple actomyosin ring to the plasma oogenesis and egg activation. Jun Cui, Katharine Sackton, membrane during meiotic cytokinesis in Drosophila males. Edith Vanessa Horner, Kritika Kumar, Mariana Wolfner. Department Szafer-Glusman, Margaret Fuller. Developmental Biology, of Molecular Biology and Genetics, Cornell University,Ithaca, NY. Stanford School of Medicine, Stanford, CA. 189C 179B A conserved pachytene checkpoint is linked to meiotic crossover PLC and MLCK regulate actin dynamics during cytokinesis. formation in Drosophila. Eric F. Joyce, Kim S. McKim. Waksman Raymond Wong1, Lacramioara Fabian2, Julie Brill1,2. 1) Inst, Rutgers Univ, Piscataway, NJ. Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 2) Developmental Biology, Hospital for Sick 190A Children, Toronto, Ontario, Canada. Z2-4034 Identifies a Gene Required for Chromosome Segregation. Apple G. Long, Sarah J. Radford, Kim S. McKim. 180C Dept. of Genetics, Waksman Institute, Rutgers University, A distinctive basal promoter architecture defines the entire Piscataway, NJ. Drosophila Ribosome Biogenesis regulon. Albert Erives1, Seth Brown2, Michael Cole2. 1) Dept. of Biological Sciences, 191B Dartmouth College, Hanover, NH; 2) Dartmouth Medical School, MCM related genes in the precondition class promote crossover Lebanon, NH. repair early in meiotic prophase. Shree N. Tanneti, Eric F. Joyce, Kim S. McKim. Waksman Inst, Rutgers Univ, Piscataway, NJ. 181A Genetic and biochemical studies of the Mre11/Rad50/Nbs 192C complex in Drosophila. Guanjun Gao, Germana Colazzo, Analysis of dRING function during Drosophila oogenesis. Pei Xiaolin Bi, Cassie Rauser, Yikang Rong. LBMB, National Zhou, Sharon E. Bickel. Department of Biological Sciences, Cancer Institute, NIH, Bethesda, MD. Dartmouth College, Hanover, NH.

182B 193A The role of Drosophila myb in cell proliferation and differentiation. Analysis of the dynamics of the condensin subunit Cap-G. Margritte K. Rovani, Carrie A. Fitzpatrick, Gary Ramsay, Alisa Sabine Herzog, Sonal Nagarkar, Stefan Heidmann. Genetics, L. Katzen. Department of Biochemistry & Molecular Genetics, University of Bayreuth,Bayreuth, Germany. University of Illinois at Chicago, Chicago, IL. 194B 183C Differential requirements for mitotic sister-chromatid cohesion Coordination of larval and imaginal growth in Minutes. Meng- in the soma and in the germ-line. Ana Marques1, Rui Tostões1, Ping Tu, Laura A. Johnston. Dept Genetics & Development, Thomas Marty2, 2R Screen Team2, Rui Martinho1. 1) Instituto ColumbiaUniv, New York, NY. Gulbenkian da Ciência, Oeiras, Portugal; 2) Skirball Institute, NYU-Medical Center. 184A Characterization of a novel conserved cyclin in Drosophila. 195C Dongmei Liu. Dept CMMG, Wayne State Univ, Detroit, MI. Defining the cell cycle roles of fly Sgt1. Lucia Mentelová1, Gonçalo Costa1, Fátima Pereira1, Claudia Florindo1, Álvaro Tavares1,2. 1) Cell Division Group , Inst. Gulbenkian de Ciência, Oeiras,Portugal; 2) Chemical Eng. Inst. Superior Tecnico, Lisboa, Portugal. POSTER SESSIONS 41 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

196A 206B Drosophilano poles encodes an E3 ubiquitin ligase required for Insulin-signaling and the developmental regulation of allometry genomic stability in the early embryo. Julie Merkle, Laura Lee. in Drosophila. Alexander Shingleton. Department of Zoology, Department of Cell & Developmental Biology, Vanderbilt MichiganState University,East Lansing, MI. University MedicalCenter, Nashville, TN. 207C 197B Imaginal disc growth regulates critical size for metamorphosis Cyclin J is required for normal oocyte development. Govindaraja in Drosophila. Alexander Shingleton, Bradley Stieper, Michael Atikukke1, Russell L. Finley, Jr.1,2. 1) Dept Biochem & Molec Driscoll. Department of Zoology, Michigan StateUniversity, East Biol, Wayne State Univ, Detroit, MI; 2) Center for Molecular Lansing, MI. Medicine and Genetics, Wayne State Univ, Detroit, MI. 208A 198C Regulation of regenerative growth in imaginal discs. Rachel K. A genetic system to study compensatory growth in the imaginal Smith-Bolton, Melanie Worley, Hiroshi Kanda, Iswar discs of D. melanogaster. Abigail Gerhold, Adrian Halme, Iswar Hariharan. Molecular and Cell Biology, University of Hariharan. Molecular and Cell Biology, University of California, California,Berkeley, CA. Berkeley,Berkeley, CA. 209B 199A The role of dMyc in pattern-directed growth during development Functional and Regulatory Analysis of SUBITO in D. of the Drosophila wing. Christine Wu1, Laura A. Johnston2. 1) melanogaster. Jeffry Cesario, Kim McKim. Waksman Inst, Biological Sciences, Columbia University, New York, NY; 2) Rutgers Univ, Piscataway, NJ. Genetics & Development, Columbia University, New York, NY.

200B 210C Understanding the mechanisms involved in the activation of the p53-independent apoptosis limits aneuploidy following irradiation. Hippo pathway by the tumor suppressor fat. Caroline Badouel1, Laura M. McNamee, Michael Brodsky. Program in Gene Laura Gardano2, Helen McNeill1. 1) Samuel Lunenfeld Function and Expression, University of Massachusetts Research Institute, Toronto, Ontario, Canada; 2) Wellcome Trust MedicalSchool, Worcester, MA. Centre for Cell Biology, Edinburgh, Scotland. 211A 201C Identification of novel tumor suppressor genes through DCP-1 Local and systemic responses to ionizing irradiation in Drosophila with GMR-GAL4 mediated modifier screening. JuHyun Shin, larvae. Adrian Halme, Abigail Gerhold, Iswar Hariharan. OokJoon Yoo. Dept Life Sci, KAIST, DaeJeon, Korea. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA. 212B The Rab11 GTPase controls somatic cell fate decisions in the 202A Drosophila ovary and behaves as a neoplastic tumor suppressor The E1-ubiquitin-activating enzyme uba1 in Drosophila controls in follicle epithelial cells. Jiang Xu, Lan Lan, Nicholas Bogard, apoptosis autonomously and tissue growth non-autonomously. Robert S. Cohen. Department of Molecular Biosciences, Tom Lee, Tian Ding, Zhihong Chen, Vani Rajendran, Heather University of Kansas, Lawrence, KS. Scherr, Melinda Lackey, Clare Buldoc, Andreas Bergmann. Biochemisty & Molecular Biology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. Cytoskeleton and Cellular Biology 213C 203B The cadherin Fat2 is required for planar cell polarity and organ To Specify or to Proliferate: That is the Question in the Early Fly shape in Drosophila. Christian Dahmann, Tina König, Ivana Retina. Shera Lesly, Justin Kumar. Dept Biol, Indiana University, Viktorinova. Max Planck Institute of Molecular Cell Biology and Bloomington, IN. Genetics, Dresden, Germany. 204C 214A Multitasking: How Dpp manages to regulate patterning and Characterization of a-catenin using a structure-function growth. Gerald Schwank, Simon Restrepo, Konrad Basler. approach. Ridhdhi Desai, Milena Pellikka, Ritu Sarpal, Ulrich Institute of Molecular Biology, University of Zuerich, Zuerich, Tepass. Cell and Systems Biology, University of Toronto,Toronto, Zuerich, Switzerland. Ontario, Canada. 205A 215B The influence of nutrition on growth and insulin signalling in D. Cdc42 promotes adherens junction integrity by negatively melanogaster.Gerhard Seisenbacher, Hugo Stocker, Ernst regulating apical endocytosis. Kathryn Harris, Ulrich Tepass. Hafen. Institute of Molecular Systems Biology, ETH Zurich, Cell & Systems Biology, University of Toronto,Toronto, ON, Switzerland. Canada. 42 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

216C 225C Characterization of lumen morphogenesis in the Drosophila Rap1's role in Drosophila morphogenesis. Nathan Harris, retina. Nicole Husain1, Milena Pellikka1, Kwang-Min Choe2, Jessica Sawyer, Mark Peifer. Biology, UNC - Chapel Hill, Chapel Thomas R. Clandinin2, Ulrich Tepass1. 1) Cell & Systems Hill, NC. Biology, University of Toronto, Toronto, ON, Canada; 2) Department of Neurobiology, Stanford University, Stanford, CA. 226A Roles of Spectrins in Drosophila photoreceptor morphogenesis. 217A Sang-Chul Nam1, Tony Chen1, Kwang-Wook Choi2. 1) Interactions between the FERM protein Yurt and septate junction Department of Biology, Baylor University, Waco, TX; 2) proteins define a novel basolateral epithelial polarity pathway. Department of Molecular and Cellular Biology, Baylor College Patrick Laprise1, Sarah Paul3, Kathryn Harris2, Greg Beitel3, of Medicine, Houston, TX. Ulrich Tepass2. 1) CRC-HDQ, Laval University, Quebec, QC, Canada; 2) Department of Cell and Systems Biology, University 227B of Toronto, Ontario, ON, Canada; 3) Department of Biochemistry, Enabled and Capping Protein Regulation of the Actin Molecular Biology and Cell Biology, Northwestern University, Cytoskeleton in Drosophila Development. Stephanie Evaneston, IL. Nowotarski1, Julie Gates2, Mark Peifer1. 1) Biology, University of North Carolina, Chapel Hill, NC; 2) Biology, Bucknell University, 218B 701 Moore Avenue, Lewisburg, PA 17837. Dynein-mediated apical localization of crumbs transcripts is required for effective Crb activity in epithelial polarity. Zhouhua 228C Li1, Liwei Wang1, Thomas S. Hays2, William Chia1,3, Yu Cai1,3. The function of the PDZ-GEF Dizzy in ventral furrow formation. 1) Germ Cell Development Group, Temasek Life Sciences Alice Ott1, Martina Rembold2, Sam J. Mathews2, Maria Leptin2, Laboratory,National University of Singapore, Singapore; 2) Rolf Reuter1. 1) Division of Animal Genetics, University of Department of Genetics, Cell Biology, and Development, Tübingen, Tübingen, B-Württemberg, Germany; 2) Inst. for University of Minnesota, Minneapolis, MN; 3) Department of Genetics, Developmental Genetics Unit, University of Cologne, Biological Science, National University of Singapore, Singapore. Cologne, Germany.

219C 229A muscleblind gene regulates microtubule organization and Second-site noncomplementation screen to identify genes that epithelial integrity in follicle cells. Gouthami Nallamothu, Tien interact with Rho1. Kistie Patch, Shannon Stewart, Aaron Hsu. Dept Pathology & Lab Medicine, Medical Univ South Welch, Robert Ward. Dept Molecular Biosciences, University Carolina, Charleston, SC. of Kansas, Lawrence, KS.

220A 230B The role of the oocyte nucleus in grk mRNA localization during Microtubule-driven transformations in ER morphology during oogenesis. Amanda Norvell, Carolyn Gray, Joshua oogenesis. Nancy Pokrywka1, Anna Payne-Tobin1, Tulle Schoenfeld, Jing-jing Feng. Dept Biol, Col of New Jersey, Hazelrigg2. 1) Dept Biology, Vassar College, Poughkeepsie, NY; Ewing, NJ. 2) Dept Biol Sci, Columbia Univ, New York, NY.

221B 231C Function of Drosophila a-catenin in adherens junction formation. The roles of Abelson Kinase in cytoskeletal regulation during Ritu Sarpal, Milena Pellikka, Ulrich Tepass. Cell and Systems embryonic morphogenesis in Drosophila. Edward M. Rogers1, Biology, University of Toronto,Toronto, ON, Canada. Donald T. Fox2, Mark Peifer1. 1) Biology Department, UNC- Chapel Hill, Chapel Hill, NC; 2) Carnegie Institute, Baltimore, 222C MD. Awd, the Homologue of the Human Metastasis Suppressor Gene, Regulates Epithelial Integrity of Follicle Cells. Julie 232A Woolworth1, Tien Hsu2. 1) Dept Molecular & Cellular Biol, WASP and SCAR/WAVE play distinct roles in activating the Arp2/ Medical Unif South Carolina, Charleston, SC; 2) Dept Pathology 3 complex during Drosophila myoblast fusion. Gritt Schäfer1, & laboratory Med, Medical Unif South Carolina, Charleston, SC. Susanne Berger1, Anne Holz2, Lothar Beck1, Renate Renkawitz-Pohl1, Susanne-Filiz Önel1. 1) Dept. for Biology, 223A Philipps-University Marburg, Germany; 2) Institute for Allgemeine Local regulation of F-actin during the first step of Drosophila und Spezielle Zoologie, Justus-Liebig-University Giessen, myoblast fusion. Christine Dottermusch, Gritt Schaefer, Germany. Renate Renkawitz-Pohl, Susanne-Filiz Oenel. Biology, Philipps-University, Marburg, Germany. 233B Scar Coordinates with Solitary to Regulate Actin Cytoskeletal 224B Dynamics During Myoblast Fusion. Kristin Sens, Elizabeth Control of nuclei positioning in contractile muscle cells. Hadas Chen. MBG, Johns Hopkins Sch Medicine, Baltimore, MD. Elhanany, Talila Volk. Molecular Genetics, Weizmann, Rehovot, Israel. POSTER SESSIONS 43 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

234C 242B Rho-Kinase is required for epithelial morphogenesis in the A unified biophysical model for the dynamics of antero-posterior Drosophila embryo. Robert Simone, Stephen DiNardo. Dept and terminal systems: diffusion and nucleocytoplasmic shuttling Cell & Developmental Biol, Univ Pennsylvania, Philadelphia, PA. in the syncytium. Matthieu Coppey1,2, Alistair N. Boettiger3, Yoosik Kim1,2, Alexander M. Berezhkovskii4, Stanislav Y. 235A Shvartsman1,2. 1) Carl Ichan Lab, Princeton Univ, Princeton, Characterization of phosphatidylinositol 4-kinase IIIa during NJ; 2) Department of Chemical Engineering,Princeton Univ, Drosophila development. Julie Tan1,2, David Hipfner3, Julie Princeton, NJ; 3) Department of Molecular and Cell Biology, Brill1,2. 1) Program in Developmental and Stem Cell Biology, University of California, Berkeley, USA; 4) Mathematical and Hospital for Sick Children, Toronto, ON, Canada; 2) Dept of Statistical Computing Laboratory, Division of Computational Molecular Genetics, University of Toronto, Toronto, ON, Canada; Bioscience, Center for Information Technology, National Institutes 3) Dept of Epithelial Cell Biology, Institut de Recherches Cliniques of Health. de Montreal, Montreal, QC, Canada. 243C 236B In vivo measurement of kinesin-1 and dynein stalling forces: Cloning and characterization of uninflatable, a gene required how does motor number affect transport? Steven P. Gross1, for tracheal inflation. Liang Zhang, Aaron Olson, Robert Ward. George Shubeita1, Susan Tran2, Jing Xu1, Michael Welte2. 1) Dept. Molecular Biosciences, Univ. Kansas, Lawrence, KS. Dev. and Cell Biology, UC Irvine, Irvine, CA; 2) Department of Biology, University of Rochester, Rochester, NY 14627. 237C Investigating the mechanisms of the cortical localization of APC2. 244A Meng-Ning Zhou, Andrea Blitzer, Brooke McCartney. Dept Vegetable, a GPI mannosyltransferase II, is required by Biological Sci, Carnegie Mellon Univ, Pittsburgh, PA. rhodopsin and for photoreceptor viability. Suraiya Haroon, Erica E. Rosenbaum, Katie L. Caillouette, Nansi Jo Colley. Dept. 238A of Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin, Madison, The endosomal SM protein dVps45 is necessary for cell signaling WI. and proliferation. Mohammed Akbar, Sanchali Ray, Helmut Kramer. Dept of Neuroscience , Univ Texas SW Medical Ctr, 245B Dallas, TX. Identification and characterization of Drosophila UNC-76 binding proteins. Rebecca Josowitz, Jordan Cox, Monica Zapata, 239B Joseph Gindhart. Department of Biology, University of Trafficking of the Drosophila vesicular monoamine transporter Richmond, Richmond, VA. is required for a subset of amine-dependent behaviors. Anna Grygoruk, David E. Krantz. Psychiatry & Biobehavioral 246C Sciences, School of Medicine, Brain Research Institute, UCLA, The dynamics of nanos mRNA incorporation into nascent pole Los Angeles, CA. cells. Dorothy A. Lerit, Timothy T. Weil, Elizabeth R. Gavis. Department of Molecular Biology, Princeton University, 240C Princeton, NJ. Rabenosyn and Vps45 regulate cell polarity and early endosomal entry. Holly A. Morrison1, Heather Dionne1, Tor Erik Rusten2, 247A Andreas Brech2, Bill Fisher3, Barret Pfeiffer3, Susan Celniker3, A role for the RNA-binding protein Lark in oskar RNA localization Harald Stenmark2, David Bilder1. 1) Dept of Molecular & Cell to the posterior pole during oogenesis. Gerard McNeil, Sheryl Biology, University of California, Berkeley, CA; 2) Centre for Purrier, Manpreet Kaur, Ruth Kang. Department of Biology, Cancer Biomedicine, University of Oslo, and Department of York College/CUNY, Jamaica, NY. Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway; 3) Genome 248B Sciences Department, Life Sciences Division, Lawrence Sec23-IP is required for rhodopsin transport, lipid metabolism Berkeley National Lab, Berkeley, CA. and photoreceptor viability. Anne C. Muller, Erica E. Rosenbaum, Katie L. Caillouette, Nansi Jo Colley. Dept. of 241A Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin,Madison, Phosphatidylinositol 4-kinase and Sac1 regulate trafficking to WI. lysosome-related organelles in Drosophila. Jason Burgess1,2, Ho-Chun Wei1, Helmut Kramer3, Julie Brill1,2. 1) Program in 249C Developmental and Stem Cell Biology, The Hospital for Sick A cellular basis for Wolbachia transmission in the maternal Children, Rm 13-401L, 101 College St, TMDT East Tower, germline. Laura Serbus, William Sullivan. MCD Biology, Toronto, ON, Canada; 2) Department of Molecular Genetics, University of California, Santa Cruz, CA. University of Toronto; 3) Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, 250A TX 75390-9111. Protein Phosphatase 2A negatively regulates Smoothened protein trafficking to modulate Hedgehog signaling outcomes. Ying Su, Jason Ospina, Andrew Michelson, Alan Zhu. Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH. 44 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

251B 261C Myotubularin Phosphoinositide Phosphatase Regulation of Atlastin promotes homotypic fusion of ER membranes. Genny Lysosomal Fusion and Cellular Morphogenesis. Michaella Orso1, Diana Pendin2,3, Jessica Tosetto2,3, Andrea Daga2,4. 1) Velichkova, Inês Ribeiro, Amy Kiger. Div Biological Sci, Univ Scientific Institute E. Medea, Conegliano, Italy; 2) Dulbecco California, San Diego, La Jolla, CA. Telethon Institute at the E. Medea Scientific Institute, Conegliano, 31015 Italy; 3) Department of Pharmacology, University of 252C Padova, Padova, 35131 Italy; 4) Department of Neurology, The GSK3/Shaggy is required for axonal transport. Carole Weaver, David Geffen School of Medicine, University of California, Los Lawrence S. B. Goldstein. Department of Cellular & Molecular Angeles, California 90095. Medicine, University of California at San Diego, La Jolla, CA. 262A 253A Developmentally controlled sequestration of Importin a2 to lipid Rab-regulated vesicle trafficking in Development. Jun Zhang, droplets. Naina Phadnis, Michael Welte. Department of Biology, Matthew Scott. Dept. of Developmental Biology, HHMI, Stanford Univeristy of Rochester,Rochester, NY 14627. University, Stanford, CA. 263B 254B Mononuclear muscle cells of the ovarian epithelial sheath. Akemi Bcr-Abl interacts with Rho1 to alter cell migration during J. Tanaka, Andrew M. Hudson, Lisa N. Petrella, Lynn Cooley. Drosophila development. Nicholas Artabazon, Sara Tittermary, Department of Genetics, Yale UniversitySchool of Medicine, New Traci Stevens. Biol Dept, Randolph-MaconCollege, Ashland, Haven, CT. VA. 264C 255C Water channels, oviduct osmolarity and egg activation in Two Studies of Transcriptional Control of Development. Adam Drosophila. Ido Apel1, Menachem Moshelion2, Yael Heifetz1. Bousum, Jesse Hogan, Hilary Price, Thomas Kidd. University 1) Dept. of Entomology; 2) Institute of Plant Sciences and of Nevada, Reno,Reno, NV. Genetics, The Hebrew University,Rehovot, Israel.

256A 265A Domain Analysis to Dissect the Role of Drosophila PINCH in The transcriptional control of secretion in the Drosophila Epithelial Adhesion and Migration. Maria C. Elias1,3, Julie L. embryonic salivary gland. Rebecca M. Fox, Monique Marshall, Kadrmas1,3, Mary C. Beckerle1,2,3. 1) Department of Oncological Deborah J. Andrew. Department of Cell Biology, Johns Hopkins Sciences, University of Utah, Salt Lake City, UT; 2) Department University,School of Medicine,Baltimore, MD. of Biology, University of Utah, Salt Lake City, UT; 3) Huntsman Cancer Institute, University of Utah, Salt Lake City, UT. 266B Characterization of sec61a during embryogenesis. Xiaochen 257B Wang, Elspeth Pearce, Robert Ward. Dept Molecular Function of singed during Drosophila development: a new role Biosciences, Univ Kansas,Lawrence, KS. in blood cell migration. Jennifer Zanet1, Brian Stramer2, Tom Millard2, Paul Martin2, Francois Payre1, Serge Plaza1. 1) Centre de Biologie du Dévelopement, Toulouse, France; 2) Department Genome and Chromosome Structure of Physiology, University of Bristol,Bristol, UK. 267C Chromosome segregation and meiosis I progression in 258C Drosophila oocyte. Ounissa Aït-Ahmed, Régis Meyer, Michèle Requirements for epidermal wound repair: Lessons from Delaage, Roland Rosset, Michèle Capri. Institut de Génétique Drosophila genetics. Michelle Juarez, Efren Sandoval, William Humaine UPR 1142, CNRS, Montpellier, France. McGinnis. Dept Cell & Developmental Biol, Univ California, San Diego, La Jolla, CA. 268A Evidence for Common Fragile Sites in Drosophila. Matthew C. 259A LaFave1, Lewis J. Overton2, Jeff Sekelsky1,2. 1) Curriculum in Analysis of mutants in Strn-mlck and CG1776, two Drosophila Genetics and Molecular Biology, University of North Carolina, myosin light chain kinase (MLCK) genes. Andrew Kreuz1, Ivan Chapel Hill, NC; 2) Department of Biology, University of North Tesic2, Deyra Rodriguez2, Rafael Acosta1, Amanda Simcox2. Carolina, Chapel Hill, NC. 1) Dept Biol, Villa Julie Col, Baltimore, MD; 2) Dept. Molecular Genetics, The Ohio State University, Columbus, OH. 269B Nucleoskeletal proteins EAST and CP60 modulate activity of 260B the gypsy insulator in D. melanogaster. Pavel Georgiev, Anton Drosophilamessy mitochondria, and fragmented mitochondria Golovnin, Larisa Melnikova, Ivan Krivega, Margarita are required for mitochondrial morphology and imaginal disc Kostuchenko, Ilya Volkov. Inst Gene Biology RAS, Moscow, development. Dennis LaJeunesse. Dept Biol, Univ North Russia. Carolina, Greensboro, NC. POSTER SESSIONS 45 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

270C 279C The Genomics Education Partnership: the dot chromosome, a Effect of 2L TAS deficiencies on telomere-telomere associations. comparative genomics investigation by undergraduates. Sarah Radmila Capkova Frydrychova1, Trevor Archer2, James Elgin1, Anya Goodman2, Christopher Jones3, Gary Kuleck4, Mason1. 1) LMG, NIEHS, RTP, NC; 2) LMC, NIEHS, RTP, NC. Gerald McNeil5, Ken Saville6, Joyce Stamm7, David Lopatto8. 1) Washington U, St Louis MO; 2) Cal Poly St U, San Luis Obispo 280A CA; 3) Moravian C, Bethleham PA; 4) Loyola Marymount U, Los Unprotected Drosophila telomeres activate the spindle assembly Angeles CA; 5) York C/CUNY, New York NY; 6) Albion C, Albion checkpoint. Giovanni Cenci1, Mariarosaria Musaró1,2, Laura MI; 7) U Evansville, Evansville IN; 8) Grinnell C, Grinnell IA. Ciapponi2, Barbara Fasulo3, Maurizio Gatti2. 1) DISTeBA, Univ Lecce-Ecotekne, Leece, Italy; 2) Dept. of Genetics and Molecular 271A Biology, Univ. "La Sapienza," Rome; 3) Molecular Cell and A search for locus producing RNAi through a high-throughput Developmental Biology Dept., Univ. of California, Santa Cruz. analysis of heterochromatin transcription. Danielle Nouaud1, Clémentine Vitte1, Dominique Anxolabéhère1, Hadi 281B Quesneville2. 1) Dynamique du Génome et Evolution, Institut Mapping of the Telomere elongation gene. James Mason, Jacques Monod - CNRS -Universités Paris6 et Paris7, Paris, Radmila Capkova Frydrychova. Lab Molec Genetics, D3-01, France; 2) URGI, INRA unit 1164, Evry, France. NIH/NIEHS, Res Triangle Pk, NC.

272B Heterochromatin variations in evolution of malaria mosquitoes. Regulation of Gene Expression Igor Sharakhov1, Maria Sharakhova1, Irina Brusentsova2. 1) 282C Department of Entomology, Virginia Tech, Blacksburg, VA; 2) MEF-2 and CF-2 collaborate in activation of the myogenic Institute of Cytology and Genetics, Novosibirsk, Russia. program in Drosophila. Anton Bryantsev, Kathleen Kelly- Tanaka, Thai Lee, Richard Cripps. Department of Biology, 273C University of New Mexico, Albuquerque, NM. Arginine kinase isoforms from D. melanogaster. Sohini Ghosh, Glen Collier. Dept Biological Sci, Univ Tulsa, Tulsa, OK. 283A Weckle is required for the transcriptional activities of Dorsal in 274A Drosophila. Dechen Fu, Mike Levine. Molecular and Cell Analysis of chromatid segregation in Drosophila. Amber Hohl1, Biology, U. C. Berkeley, Berkeley, CA. 94720. Ruth Griffin1,2, Jack R. Bateman1, C.-ting Wu1. 1) Genetics, Harvard MedicalSchool, Boston,MA; 2) Centre National de la 284B Researche Scientifique, UMR 5092, Biochimie et Biophysique Atonal and Senseless regulate rhomboid expression during des Systemes Integres, Grenoble. embryonic chordotonal organ development. David Li-Kroeger, Lorraine Witt, Brian Gebelein. Division of Developmental 275B Biology, Cincinnati Childrens' Hospital, Cincinnati, OH. Condensin II complex inhibits pairing in polyploid nurse cells and female meiotic chromosomes. Helen F. Smith1, Justin 285C Blumenstiel2, Tom Hartl1, Scott Hawley2, Giovanni Bosco1. Analysis of the DNA binding activity of the Drosophila Zic family 1) MCB, University of Arizona, Tucson, AZ; 2) Stowers Institute, member, odd-paired (opa). Aditya Sen, Heuijung Lee, Brian Kansas City, MO. Stultz, Deborah Hursh. Division of Cell and Gene Therapies, CBER/FDA, Bethesda, MD. 276C Single-gene duplications and transpositions in the Drosophila 286A genome. Edwin C. Stephenson, Ajinkya C. Inamdar. Identification of Nuclear Localization Signals in the Drosophila Department of Biological Sciences, University of OVO Protein. Akram Abou-Zied1, Mark Garfinkel2, Anthony Alabama,Tuscaloosa, AL. Mahowald3. 1) Suez Canal University, Department of Zoology, Faculty of Science, Ismailia, Egypt; 2) University of Alabama at 277A Birmingham, Department of Environmental Health Sciences, A new example of trans-inactivation phenomenon in D. Birmingham, AL; 3) Stanford University School of Medicine, melanogaster. Mikhail V. Kibanov, Sergey A. Lavrov, Yuri A. Department of Developmental Biology, Stanford, CA. Abramov, Vladimir A. Gvozdev. Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian 287B Academy of Sciences, Moscow, Kurchatov Sq. 2, 123182, Transcription elongation controls expression of Hox genes during Russia. Drosophila development. Vivek S. Chopra, Michael Levine. Center for Integrative Genomics, University of California, 278B Berkeley, Berkeley, CA. Not Too Much of a Good Thing: Under-replicated Regions in Polytene Salivary Gland Cells. Noa Sher1, Helena Kashevsky1, 288C Thomas Eng1, David MacAlpine2, Terry Orr-Weaver1. 1) A screen for transcription factors that coordinate cell fate Whitehead Institute, Cambridge,MA 02142; 2) Duke determination and patterning of photoreceptors in the Drosophila UniversityMedical Center,Durham, NC 27710. eye. Hui-Yi Hsiao, Robert Johnston, David Jukam, Claud Desplan. Biology, New York University, NY. 46 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

289A 297C Regulation of toy, a Pax6 gene in Drosophila. Linn Jacobsson1, Hox and Senseless competition forms a molecular switch to Jesper Kronhamn2, Åsa Rasmuson-Lestander1. 1) Molecular regulate gene expression in the PNS. Brian Gebelein, David Biology, Umea,Sweden; 2) UCMP, Umea University,Umea, Li-Kroeger, Lorraine Witt. Division of Developmental Biology, Sweden. Cincinnati Children's Hospital MedicalCenter, Cincinnati, OH.

290B 298A Twoleg-arista-wing complex mutations disrupt different coding Molecular dissection of the IAB5 cis-regulatory module in regions of TBP related factor 2 in D. melanogaster. Simonova Drosophila. Sara E. Goetz, John M. Allen, Robert A. Drewell. B. Olga1,2, Burdina Natalia.1, Vorontsova Yulia2, Cherezov Biology Department, Harvey Mudd College, Claremont, CA. Roman1, Modestova Elena1, Kulikova Dina1,2, Mertsalov Ilya1,2. 1) Koltzov Institute of Developmental Biology, Russian Academy 299B of Sciences, Moscow, Russian Federation; 2) Institute of Gene Characterization of Ultrabithorax-responsive regulatory Biology, Russian Academy of Sciences, Moscow, Russian sequences. Bradley Hersh, Jamie Wood. Dept Biological Federation. Sciences, Clemson University, Clemson, SC.

291C 300C The Role of Paused Polymerase in the Regulation of Drosophila Functional activity of rapidly evolving cis regulatory modules in Developmental Genes. Jessica Piel, Joung-Woo Hong, the Drosophila bithorax complex.Margaret C. W. Ho1, Holly Michael Levine. Dept Molecular & Cell Biol, Univ California, Johnsen1, Esther Bae1, Ben Schiller1, Jason W. H. Chan1, Berkeley, Berkeley, CA. William Fisher2, Susan E. Celniker2, Robert A. Drewell1. 1) Biology, Harvey Mudd College, Claremont, CA; 2) Berkeley 292A Drosophila Genome Project, Lawrence Berkeley National Characterization of tfiia-s-2, a germline-specific homolog of the Laboratory, Berkeley, CA. small subunit of the General Transcription Factor, TFIIA. Margaret Wood, Cynthia Maddox, Haley Adams, Leah Cohen, Rebekah 301A Turk, Mark Hiller. Biology, Goucher College,Baltimore, MD. The vnd/nk-2 homeobox gene is activated in different neuroblasts by different combinatorial sets of enhancers in the 5’-upstream 293B enhancer region. Andrey Ivanov, Siuk Yoo, Marshall Nirenberg. Characterizing the transcriptional regulation of the Drosophila Lab Biochemical Genetics, NIH/NHLBI, Bethesda, MD. E75 gene through analysis of upstream non-coding DNA. Travis Bernardo1, Habiba Jannat1, Bill Maughan1, Veronica 302B Dubrovskaya1, Edward Berger2, Edward Dubrovsky1. 1) Analysis of cis-regulatory elements controlling repo transcription Biology, Fordham University,Bronx, NY; 2) Biology, Dartmouth in Drosophila.Robert W. Johnson, Jamie L. Wood, William C. College, Hanover, NH. Colley, Bradley W. Jones. Biology, University of Mississippi, Oxford, MS. 294C The TCF Helper site, a new cis-regulatory element in the 303C Wingless signaling pathway. Mikyung Chang, Jinhee Chang, Systems biology analysis of the cis-regulatory control of the Anu Gangopadhyay, Andrew Shearer, Kenneth Cadigan. expression of Drosophila even-skipped stripes 2 and 3. Ah-Ram MCDB, University of Michigan, Ann Arbor, MI. Kim1, Shuling Hou2, David Sharp3, John Reinitz1. 1) Department of Applied Mathematics and Statistics and Center 295A for Developmental Genetics, Stony Brook University, Stony Characterization of Enhancer of split regulatory region Brook, NY 11794-3600; 2) Theoretical Division, Los Alamos sequences conserved in multiple Drosophila species. Deborah National Laboratory, Los Alamos, NM 87545; 3) Chief Science Eastman, Morgan Maeder. Biology, Connecticut Col, New Office, Los Alamos National Laboratory, Los Alamos, NM 87545. London, CT. 304A 296B Functional Analysis of the nerfin-1 Neuroblast Promoter. Mukta A cellular resolution atlas of gene expression in D. Kundu, Alexander Kuzin, Thomas Brody, Ward Odenwald. pseudoobscura reveals interspecies variation in embryonic Neural Cell Fate Determinants, NINDS, Bethesda, MD. patterning. Michael Eisen2, Cris Luengo2, Charless Fowlkes4, Soile Keränen2, Clara Henriquez2, Lisa Simirenko2, Gunther 305B Weber2, Oliver Rübel5, Min-Yu Huang5, Jitendra Malik3, David Transition from Notch-dependent to Notch-independent Knowles2, Mark Biggin2, Angela DePace1. 1) Department of regulation of high-level Suppressor of Hairless expression in Systems Biology, Harvard Medical School, Boston, MA; 2) the socket cell. Feng Liu, James W. Posakony. Division of Genome Sciences Department, Genomics Division, Lawrence Biological Sciences/CDB, University of California San Diego, Berkeley National Laboratory, Berkeley, CA; 3) Department of La Jolla, CA. Computer Science, UC Berkeley, CA; 4) Department of Computer Science, UC Irvine, CA; 5) Computer and Data Visualization Department, UC Davis, CA. POSTER SESSIONS 47 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

306C 315C Transcription factors bind thousands of active and inactive Combinatorial regulation of Drosophila muscle enhancers - a elements in the Drosophila blastoderm. Stewart MacArthur1, systems-level approach. Robert P. Zinzen, Julien Gagneur, Xiaoyong Li1, Michael Eisen1,2, Mark Biggin1. 1) Genomics Charles Girardot, Eileen E. Furlong. Dev Biology Unit, EMBL, Division, Lawrence Berkeley National Laboratory, Berkeley, Heidelberg, Germany. California; 2) Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, 316A California. Oligomeric state of Drosophila CtBP plays a role in transcriptional regulation of Wingless signaling targets. Chandan Bhambhani, 307A Jinhee Chang, Mikyung Chang, Kenneth Cadigan. Molecular, A Cell Non-Autonomous Feedback Loop Regulates neuralized Cellular & Developmental Biology, Univeristy of Michigan, Ann Expression and Function Downstream of the Proneural Proteins Arbor, MI. During Sensory Organ Precursor Selection. Steven W. Miller, James W. Posakony. Division of Biological Sciences/CDB, UC 317B San Diego, La Jolla, CA 92093-0349. Dicing Sex. Jamila Horabin, Ursula Olcese. Dept Biomedical Sciences, Florida State Univ, Tallahassee, FL. 308B The Homeotic selector genes and the NK homeodomain 318C transcription factor Tinman togther control cardiac svp expression Genomic imprints, chromatin proteins and Wolbachia. Daisey in the Drosophila dorsal vessel. Kathryn M. Ryan, Richard M. Y. Kent. Mount Allison University, Biology, 63B York St, Sackville, Cripps. Dept Biol, Univ New Mexico, Albuquerque, NM. Canada.

309C 319A Functional analysis of promoter-enhancer interactions at the Quantitative 3D Analysis of Expression Patterns Driven by cis- Drosophila bithorax complex. Ben Schiller, Margaret Ho, Omar Regulatory Modules. Soile V. E. Keränen1, Barret D. Pfeiffer1, Akbari, Esther Bae, Robert A. Drewell. Biology Department, Bill Fisher1, Ann Hammonds1, Cris L. Luengo Hendriks1, HarveyMudd College,Claremont, CA, 91711. Charless Fowlkes2, Clara N. Henriquez1, David W. Knowles1, Jitendra Malik3, Michael Eisen1, Mark D. Biggin1, Susan 310A Celniker1. 1) Lawrence Berkeley Natl Lab, Berkeley, CA; 2) Structure, function, and evolution of a Notch- and EGFR- University of CaliforniaIrvine, Irvine, CA; 3) University of California regulated eye enhancer. Christina I. Swanson, Scott Barolo. Berkeley, Berkeley, CA. Cell and Developmental Biology, University of Michigan,Ann Arbor, MI. 320B Post-translational modifications during formation of Da/Sc/E(spl) 311B complexes. Marianthi Kiparaki1,2, Christos Delidakis1,2. 1) In situ detection of Hox protein interactions with DNA regulatory Institute of Molecular Biology and Biotechnology, Foundation elements in single nuclei. Elvira Tour, Adam Paré, William for Research and Technology Hellas, Heraklion, Crete, Greece; McGinnis. Department of Cell & Developmental Biology, 2) Department of Biology, University of Crete, Heraklion, Greece. University of California, San Diego, La Jolla, CA. 321C 312C Regulation of Vasa stability by Gustavus and Wallenstein. Jan- Study of the transcriptional regulation of unpaired expression in M. Kugler1, Jae-Sung Woo2, Byung-Ha Oh2, Paul Lasko1. 1) Drosophila eye development. Chuan-Ju Wang, Ya-Hsin Liu, Y. DBRI, Dept. of Biology, McGill University, Montreal, Canada; 2) Henry Sun. Academia Sinica, Inst Molecular Biology, Taipei, Dept. of Life Science, Pohang University, Pohang, Korea. Taiwan. 322A 313A Computational model of eggshell patterning by the EGFR and Temporal switching of regulation and function on eye gone (eyg) Dpp pathways. Jessica Lembong, Nir Yakoby, Stanislav Y. in Drosophila eye development. Lan-hsin Wang1,2, Y. Henry Shvartsman. Lewis-Sigler Institute for Integrative Genomics, Sun1,2. 1) Institute of Molecular Biology, Academia Sinica, Taipei, Princeton University, Princeton, NJ. Taiwan, Republic of China; 2) Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of 323B China. Targeted Deletions of the slowpoke transcriptional control region through Homologous Recombination. Xiaolei Li, Nigel 314B Atkinson. Section of Neurobiology, The university of Texas at Regulation of the pro-neural gene atonal by selector factors and Austin, Austin, TX. signaling pathways during eye development. Tianyi Zhang, Swati Ranade, Qingxiang Zhou, Francesca Pignoni. Dept Ophthalmology, Harvard Medical Sch/MEEI, Boston, MA. 48 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

324C 332B Medea SUMOylation restricts the signalling range of the Dpp Inhibition of RNA interference by cell death signaling. Weiwu morphogen in the Drosophila embryo. Wayne Miles1, Shugaku Xie, James Birchler. Biological Sci Div, Univ Missouri-Columbia, Takeda1, Ellis Jaffray2, Laura Bayston1, Sanjay Basu1, Laurel Columbia, MO. Raftery3, Ron Hay2, Hilary Ashe1. 1) Faculty Life Sci, Univ Manchester, Manchester, United Kingdom; 2) School of Life 333C Sciences, University of Dundee, Dundee, UK; 3) Cutaneous Disruption of CP190’s C-Terminal domain genetically reduces Biology Research Centre, Massachusetts General Hospital and Gypsy insulator functionality.Omar Akbari, Chi-Yun Pai, Daniel Harvard Medical School, Charlestown. Oliver. Dept Biol, Univ Nevada, Reno, Reno, NV.

325A 334A Identification of functional domains and target genes of the Activity of Gt protein gradient differentially positioning pair-rule Hindsight zinc-finger protein. Liang Ming1,2, Ronit Wilk2, stripes. Luiz P. Andrioli, Ligiane Oliveira, Thiago Caséé. Depto Amanda Pickup2, Howard Lipshitz1,2. 1) Department of Genéética e Biologia Evolutiva, IB, USP, Sãão Paulo, Sãão Molecular Genetics, University of Toronto, Toronto, ON, Canada; Paulo, Brazil. 2) Program of Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada. 335B Characterizing the Role of Self-Association in Transcriptional 326B Repression by Yan. Thomas G. W. Graham1, Maureen P. Determining the in vitro DNA-binding specificities of transcription Cetera1, Pavithra Vivekanand1,2, Ilaria Rebay1. 1) Ben May factors regulating early embryogenesis. Nobuo Ogawa, Stuart Department for Cancer Research, University of Chicago, Davidson, Xiao-Yong Li, Lucy Zeng, Hou-Cheng Chu, Chicago IL; 2) Department of Biology, Franklin & Marshall Michael B. Eisen, Mark D. Biggin. Genome Sci Dept, Lawrence College, Lancaster, PA. Berkeley Natl Lab, Berkeley, CA. 336C 327C The Polycomb group of genes control the repression states of E23 is an ABC transporter that regulates the hormone 20- genes that subdivide compartments in Drosophila. Luis hydroxyecdysone in a cell autonomous manner. Elana A. Gutierrez, Jürg Müller. Gene Expression Unit, European Paladino1, Lauren Besquillo1, Dan Garza2, Andrew Andres1. Molecular Biology Laboratory, Heidelberg, Baden, Germany. 1) School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV; 2) Novartis Institutes for Biomedical Research, 337A Cambridge, MA. Regulatory mechanism of temporal expression of Blimp-1, an important factor for determination of pupation timing. Akagi 328A Kazutaka1, Hitoshi Ueda1,2. 1) The Grad. Sch. of Nat. Sci. and Identification of Broad-regulated genes during leg development Tech., OkayamaUniv., Okayama,Japan; 2) Dept of Biol, Fac of in Drosophila. Elspeth Pearse1, Xiaochen Wang1, Xue-Wen Sci., Okayama Univ.,Okayama, Japan. Chen2, Robert Ward1. 1) Department of Molecular Biosciences, University of Kansas, Lawrence, KS; 2) Department of Electrical 338B Engineering & Computer Science, University of Kansas, Windei is a binding partner of the histone methyltransferase Lawrence, KS. Eggless and is required for efficient trimethylation of histone 3 lysine 9 during oogenesis. Carmen M. Koch1, Diane Egger- 329B Adam2, Andreas Wodarz1. 1) Department of Stem Cell Biology, rasiRNA pathway components participate in posttranscriptional CMPB, Georg-August-University Goettingen; 2) Faculty of silencing of telomeric retrotransposon HeT-A. Sergey G. Shpiz, Biology, University Kostanz. Yuri A. Abramov, Alla I. Kalmykova. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russian 339C Federation. Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci. Eric Lai, David 330C Tyler, Katsutomo Okamura, Wei-Jen Chung, Joshua Hagen. Post-transcriptional Regulation of nanos mRNA During Early Dev Biol, Sloan Kettering Inst,New York, NY. Drosophila Development. Danielle R. Snowflack, Elizabeth R. Gavis. Molecular Biology, Princeton University, Princeton, NJ. 340A Translation of germ cell-less is regulated by Bruno in a BRE- 331A independent manner. Jocelyn Moore, Paul Lasko. DBRI, The Drosophila MRP gene functions as an inducible pesticide Department of Biology, McGill University, Montreal, QC, Canada. transport. Jolene Tarnay-Cogbill1, Flora Szeri2, Andras Varadi2, Steven Robinow3. 1) Cell & Molecular Biology, University of 341B Hawaii, Honolulu, HI; 2) Institute of Enzymology, Hungarian Pumilio-dependent repression of CyclinB mRNA in the Academy of Sciences, Budapest, Hungary; 3) Department of prospective somatic cytoplasm of the embryo. Krystle J. Nomie, Zoology, University of Hawaii, Honolulu, HI. Robin Wharton. Molecular Genetics and Microbiology, Cell Biology, Duke University, HHMI, Durham, NC. POSTER SESSIONS 49 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

342C 351C Lamin interacts with the gypsy insulator complex. Daniel K. Hedgehog-induced phosphorylation regulates Smoothened Oliver. Biology, University of Nevada,Reno, Reno, NV. activity by promoting a conformational switch in its C-terminal cytoplasmic tail. Yun Zhao, Jin Jiang. Department of 343A Developmental Biology, UT SW Medical Ctr, Dallas, TX. dBlimp-1, a developmental timer protein controls pupation timing through its stability. Moustafa Sarhan1, Hitoshi Ueda1,2. 1) Biol. 352A Dept., Fac. of Sci. Okayama Univ., Okayama, Japan; 2) The A structure-function analysis of the Drosophila STAT Stat92E. Grad. Sch. of Nat. Sci. and Tech., Okayama Univ.Okayama, Laura Ekas, Timothy Cardozo, Foster Gonsalves, Erika Bach. Japan. Dept Pharmacology, New York Univ Sch Medicine, New York, NY. 344B Post-transcriptional control of cyclin B during Drosophila 353B spermatogenesis. Alicia Shields, Byung Soo Gim, Margaret The function of StIP in the JAK/STAT pathway. Linzhu Han, T. Fuller. Department of Genetics, Stanford UniversitySchool of Douglas A. Harrison. Biol Dept, Univ Kentucky, Lexington, KY. Medicine, Stanford, CA. 354C 345C A Role for Ipk2 Kinase Activity in Regulating Cell Proliferation The role of oligomerization and histone deacetylation in Groucho- and Apoptosis of Imaginal Disc Tissue During D. melanogaster mediated silencing during wing development. Clint Winkler, Development. Man-kin Marco Tsui, Andrew Seeds, John York. Wiam Turki-Judeh, Alberto Ponce, Albert Courey. Howard Hughes Medical Institute, Department of Pharmacology Deptartment of Chemistry & Biochemistry, University of and Cancer Biology, DukeUniversity, Durham, NC 27710. California,Los Angeles. 355A 346A Molecular and genetic characterization of upd, upd3 and os. Mechanisms of metabolic suppression in D. melanogaster Liqun Wang, Douglas Harrison. Dept Biology, Univ 1,2 1,2 surviving extremely low O2 environments. Dan Zhou , Jin Xue , Kentucky,Lexington, KY 40506. Orit Gavrialov1,2, Gabriel Haddad1,2. 1) Departments of Pediatrics and Neuroscience,University of California, San Diego, 356B La Jolla, CA 92093-0735; 2) The Rady Children's Hospital - San Fucose modification is essential for Delta- but not Serrate- Diego, San Diego, California. dependent activation of Notch signaling in Drosophila. Tomonori Ayukawa1, Hiroyuki O. Ishikawa2, Kenji Matsuno1,2. 1) Dept Biol Sci, Tokyo Univ of Science; 2) Genome and DrugResearch Signal Transduction Center,Tokyo University of Science. 347B 357C The Ca+2-dependent protease Calpain A modulates embryonic Mechanism of Bearded family protein activity during Notch- dorsal-ventral patterning. HelenaAraujo1, Katia Carneiro1, mediated lateral inhibition in Drosophila. Joseph R. Fontana, Marcio Fontenele1, Rodrigo Agrellos1, Adriana Oliveira- James W. Posakony. Division of Biological Sciences/CDB, Silva1, Ethan Bier2. 1) Dept Histology & Embriology, Fed Univ University of California San Diego, La Jolla, CA 92093. Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil; 2) Section on Cell and Developmental Biology, University of California at 358A San Diego. The DEAD-box RNA helicase Belle differentially regulates Notch signaling during Drosophila development. John Poulton, Wu- 348C Min Deng. Dept. of Biological Science, Florida StateUniversity, Fine Dissection of the BMP-dependent Regulation of brinker. Tallahassee, FL. Enrica Charbonnier, George Pyrowolakis. Developmental Biology Unit, Institute for Biology, University of Freiburg, 359B Germany. Role of Lqf in Notch signaling. Xuanhua Xie. Dept MCDB, Univ Texas at Austin, Austin, TX. 349A TGF-b signaling in the ring gland. Scott Gesualdi, Theodor 360C Haerry. Dept Biol, Florida Atlantic Univ, Boca Raton, FL. Roles of Drosophila Deltex in Suppressor of Hairless- independent Notch signaling. Kenta Yamada, Kazuya Hori, 350B Takashi J. Fuwa, Kenji Matsuno. Dept. Biol. Sci. / Tec., Tokyo Fused-Costal2 regulates Hedgehog-induced Smo Univ.Sci., Japan. phosphorylation and cell-surface accumulation. Yajuan Liu1, Xuesong Cao1, Jin Jiang2, Jianhang Jia1. 1) Sealy Center for 361A Cancer Cell Biology and Department of Biochemistry and Regulation of Neuroblast Self-renewal in Drosophila by aPKC Molecular Biology, University of Texas Medical Branch, and Numb. Xiofei Chen, Krista Golden, Sahar Rahmani, Galveston, TX 77555; 2) Department of Developmental Biology, Cheng-Yu Lee. Center for Stem Cell Biology, Life Sciences University of Texas Southwestern Medical Center, Dallas, Texas Institute, University ofMichigan, Ann Arbor, MI 48109. 75390. 50 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

362B 371B PDE1c and PDE11 and their role in Drosophila male fertility. Identification of Ddok-interacting proteins involved in Dorsal Rachel A. Clemens-Grisham, Anke Vermehren, David B. Closure. Soumit Roy, Y. G. Yeung, E. Richard Stanley. Morton. Integrative Biosciences, Oregon Health and Science Developmental & Molecular Biology, Albert EinsteinCollege of University,Portland, OR. Medicine, Bronx, NY.

363C 372C A novel ecdysone (20E) signaling pathway is used in the mid- Localization of the Toll-7 protein in Development of D. third instar to turn on glue genes. Benjamin Costantino1, Daniel melanogaster. Natalie A. Sandoval, Elizabeth Eldon. Biological Bricker1, Kate Shen1, John Merriam2, J. Callender3, Vincent Sciences, CaliforniaState UniversityLong Beach, Long Beach, Henrich3, Andrew Andres1. 1) University of Nevada-Las CA. Vegas,School of Life Sciences, 4505 Maryland Parkway, Las Vegas, NV 89154-4004; 2) Department of Molecular and Cellular 373A Biology, University of California-Los Angeles, 405 Hilgard Functional analysis of the proprotein convertase amon: Does Avenue, Los Angeles, CA 90024-1606; 3) Department of Biology, amon regulate energy metabolism? Kate R. Small, Michael University of North Carolina-Greensboro, 312 Eberhart Ave, Bender. Department of Genetics, University of Georgia,Athens, North Carolina 27412-5001. GA.

364A 374B Regulation of Drosophila Phototransduction by Ceramide Kinase. Beta-arrestin Kurtz is an endocytic adaptor that functions in early Ujjaini Dasgupta, Usha Acharya. Program in Gene Function embryonic patterning. Marla Tipping, Alexey Veraksa. Biology, and Expression, University of Massachusetts, Worcester, MA. University of Massachusetts Boston, Dorchester, MA.

365B 375C Investigating Dscam Signaling. Maria-Luise Erfurth1,2, Dietmar Identification of substrates and regulators of the Eya Schmucker1,2. 1) Department of Cancer Biology, Dana-Farber phosphatase. Carolyn Wrobel, Justin Cassidy, Ilaria Rebay. Cancer Institute, Boston, MA; 2) Department of Neurobiology, Ben May Department for Cancer Research, University of Harvard Medical School, Boston, MA. Chicago,Chicago, IL.

366C 376A Examining the Activation of Slipper, a Drosophila JNKKK. An in vivo UAS-RNAi based pilot screen for wound healing genes Rebecca Gonda, Beth Stronach. Dept Biological Sci, Univ in Drosophila larvae. Yujane Wu, Yan Wang, Michael Galko. Pittsburgh, Pittsburgh, PA. Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, Houston, TX. 367A Receptor Mediated Endocytosis of rat-Neu in Drosophila: A 377B model of Receptor Tyrosine Kinase down regulation. Noor A screen for dominant modifiers of PDZ-GEF/Dizzy, a Rap1- Hossain, Nasrine Yacoub, Leena Patel, Roger Jacobs. Biology specific guanine nucleotide exchange factor. Zhongchun Zhang, Department, McMasterUniversity, Hamilton, Ontario, Canada. Marc Therrien. Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada. 368B Metal sensing mechanism of metal-responsive transcription 378C factor 1 (MTF-1). Haiqing Hua1, Xiaohua Chen3, Kuppusamy Spatial control of MAPK activation and signaling in the early Balamurugan2, Dominik Staiger1, Alisa Davis1, Viola embryo. Yoosik Kim1,2, Matthieu Coppey1,2, Gerardo Jiménez3, Günther1, Oleg Georgive1, David Giedroc3, Walter Schaffner1. Stanislav Shvartsman1,2. 1) Dept of Chemical Engineering, 1) Inst of Molecular Biology, Zurich, Switzerland; 2) Inst of Princeton University, Princeton, NJ; 2) Lewis-Sigler Institute for Physiology, Zurich, Switzerland; 3) Department of Chemistry, Integrative Genomics, Princeton University, NJ; 3) Institut de Indiana University, Bloomington, IN. Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain. 369C Warts pathway signalling requires merlin, but not expanded, for 379A R8 photoreceptor specification. David Jukam, Desplan Claude. PLC-g regulates differentiation and growth in D. melanogaster. Dept Biol, 1009 Main Bldg, New York Univ, New York, NY. Juan Manuel Murillo-Maldonado1, Justin Thackeray2, Juan Riesgo-Escovar1. 1) Dept Developmental Neurobiol, Inst 370A Neurobiologia, UNAM, Queretaro, Queretaro, Mexico; 2) Role of the Eyes Absent retinal determination protein in Deparment of Biology, Clark University, 950 Main Street, phosphotyrosine signaling networks. SantiagoA. Morillo1, Worcester MA 01610. Wenjun Xiong2, Andrea Roche3, Ilaria Rebay1,4. 1) Department of Molecular Genetics and Cell Biology; 2) Committee on Cancer 380B Biology; 3) Committee on Developmental Biology; 4) Ben May Genetic Dissection of Signaling from a Single Phospho-Tyrosine Institute for Cancer Research, The University of Chicago, of an Oncogenic EGF Receptor in Drosophila. Leena Patel, J. Chicago, IL. Roger Jacobs. Biology Department, McMaster University, Hamilton, Ontario, Canada. POSTER SESSIONS 51 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

381C 390C Regulated secretion of the EGFR ligand Spitz via palmitoylation The retromer complex is involved in Wingless/Wnt secretion by and proteolysis. Josefa Steinhauer, Jessica Treisman. controlling retrograde transport of Wntless from endosomes to Developmental Genetics, Skirball Institute, NYU Medical Center, the trans Golgi network. Xinhua Lin1,2, Tatyana Y. Belenkaya1, New York, NY. Yihui Wu1, Xiaofang Tang1,2, Bo Zhou1,2, Longqiu Cheng1, Yagya V. Sharma3, Dong Yan1,2, Erica M. Selva3. 1) Division of 382A Developmental Biology, Cincinnati Children's Hospital Medical closca, a new element of the Torso tyrosine kinase receptor Center, Cincinnati,OH 45229; 2) The Graduate Program in pathway. Gemma Ventura1, Rui Gonçalo Martinho2, Ruth Molecular and Developmental Biology, Cincinnati Children's Lehmann3, Jordi Casanova1, Marc Furriols1. 1) IRBB-IBMB- Hospital Medical Center, University of Cincinnati College of CSIC, Barcelona, Spain; 2) Instituto Gulbenkian de Ciencia, Medicine, Cincinnati, OH 45229; 3) Department of Biological Oeiras, Portugal; 3) Skirball Institute, New York University Medical Sciences, University of Delaware, Newark, DE 19716. Center, NY. 391A 383B Functional Genomic Analysis of the Wnt-Wingless Signaling A genetic screen for G protein-coupled receptors involved in Pathway. Raluca Pancratov, Emily R. Olson, Binita Rho1 signaling during leg imaginal disc morphogenesis. Nikette Changkakoty, Ramanuj DasGupta. New York University School Benjamin, Rachel Morgan, Laurence von Kalm. Department of Medicine - Cancer Institute, Department of Pharmacology, of Biology, University of Central Florida, Orlando, FL. Smilow 1111, New York, NY 10016.

384C 392B The proteolytic domain of the Stubble type II transmembrane Coop is a co-repressor of Pangolin that negatively regulates the serine protease is essential for proper regulation of apical cell Wingless pathway. Haiyun Song, Sandra Götze, Chloé shape change during epithelial morphogenesis. Rachel Morgan, Spichiger, Konrad Basler. Institute of Molecular Biology, Zurich, Laurence von Kalm. Department of Biology, University of Zurich, Switzerland. Central Florida,Orlando, FL. 393C 385A Unexpectedly robust assembly of the Axin destruction complex Investigating regulation of Armadillo protein stability in regulates Wg signaling as revealed by analysis in vivo. Marcel Drosophila. Kelly Alexandre, David Roberts, Daniel Wehrli, Naz Erdeniz, Wynne Peterson-Nedry, Susan Kremer, Schneider, Greg Rogers, Robert Duronio, Mark Peifer. Jessica Yu, Shahana Baig-Lewis. Cell & Dev Biol/L215, Oregon Biology, University of North Carolina, Chapel Hill, NC. Health & Sci Univ, Portland, OR.

386B Identification of novel mediators of the Wg signaling cascade. Pattern Formation Tina Buechling, Thomas Horn, Michael Boutros. Signaling 394A and Functional Genomics, German Cancer Research Center, The role of the JAK/STAT pathway in proximo-distal (P-D) Heidelberg, Germany. patterning in the wing. Aidee Ayala, Erika Bach. Pharmacology Dept., New YorkUniv School of Medicine, New York, NY. 387C The Wnt signaling pathway influences the activity of aPKC in 395B polarity and adhesion.Pamela Colosimo, Nicholas Tolwinski. Ectopic germ plasm assembly in the Drosophila oocyte. Agata Developmental Biology Program, Sloan Kettering Inst, New York, N. Becalska, Elizabeth R. Gavis. Dept Molecular Biology, NY. Princeton Univ, Princeton, NJ. 388A 396C Regulation of Dishevelled in Fz/planar cell polarity and Wnt/b- Generating the brinker repressor gradient in the wing imaginal Catenin signaling. Andreas Jenny1, Ekatherina Serysheva2, disc. Gerard Campbell, Melissa Moser. Department of Hebist Berhane1, Michael Boutros3, Marek Mlodzik2. 1) Dept Biological Sciences, University of Pittsburgh, Pittsburgh, PA. Mol and Dev Biol, Albert Einstein College of Medicine, Bronx, NY; 2) Dept. of Developmental and Regenerative Biology Mount 397A Sinai School of Medicine, New York, NY; 3) Signaling and The Hippo pathway promotes Notch signaling in regulation of Functional Genomics, German Cancer Research Center, cell differentiation, proliferation, and oocyte polarity. Wu-Min Heidelberg, Germany. Deng, Jianzhong Yu, John Poulton. Dept Biological Sci, Florida State Univ, Tallahassee, FL. 389B Negative regulation of Wingless signaling by the microRNA miR- 8. Jennifer A. Kennell, Kenneth M. Cadigan. Dept of Molec, Cell & Dev Biol, Univ of Michigan, Ann Arbor, MI. 52 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

398B 408C Doctor no and Ken and Barbie are involved in the left-right Functionalization of Duplicate Genes - A Study of Teashirt and asymmetrical development of the Drosophila embryonic gut. Reo Tiptop During Eye Development. Rhea Datta, Justin Kumar. Maeda1, Shunya Hozumi1, Kiichiro Taniguchi1, Takeshi Dept Biol, Indiana Univ, Bloomington, IN. Sasamura1, Toshiro Aigaki2, Ryutaro Murakami3, Kenji Matsuno1. 1) Dept. Biol. Sci./Tech., Tokyo Univ. of Science, 409A Japan; 2) Dept. Biol. Sci., Tokyo Met. Univ., Japan; 3) Dept. Phy. Eye Development and the Six Family of Homeobox Transcription Biol. Inf., Yamaguchi Univ., Japan. Factors. Abigail Henderson, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. 399C Nonmuscle Myosin II heavy chain, Zipper is required for the left- 410B right asymmetric morphology of the proventriculus and midgut A mosaic screen for X-linked mutations affecting photoreceptor in Drosophila embryo. Takashi Okumura, Hiroo Fujiwara, differentiation identifies potentially novel components of the Kiichiro Taniguchi, Reo Maeda, Syunya Hozumi, Kenji Wingless and EGFR pathways. Kevin Legent, Josefa Matsuno. Dept Biol Sci/Tec, Tokyo Univ Science, Chiba, Japan. Steinhauer, Magali Richard, Jessica Treisman. Skirball Institute, New York University Medical Center, New York, NY. 400A Distribution of the potential morphogen Upd during oogenesis. 411C Travis Sexton, Douglas Harrison. Dept Biol, Univ Kentucky, A genetic screen to identify autosomal genes that interact with Lexington, KY. the Drosophila pro-neural gene atonal. Daniel R. Marenda1, Arpit Shah1, Ginnene Middleton1, Andrew J. Gangemi1, Evan 401B Cooke1, Alysia D. Vrailas-Mortimer2, David Melicharek1. 1) Activation and structure-function analysis of Snake, a serine Dept of Biological Sciences, University of the Sciences in protease playing an important role in Drosophila embryonic Philadelphia, Philadelphia, PA; 2) Dept of Cell Biology, Emory patterning. Sufang Tian, Ellen LeMosy. Dept Cell Biol & University School of Medicine, Atlanta, GA. Anatomy, Medical Col Georgia, Augusta, GA. 412A 402C Epitaxial patterning in a discrete reaction-diffusion system Tramtrack69 controls cell fate, morphogenesis, and Notch representing signaling in the Drosophila eye. Matthew W. signaling during Dorsal Appendage formation. Michael J. Pennington1, David K. Lubensky2. 1) Biophysics, University of Boyle1,2, Celeste A. Berg1,2. 1) Molecular & Cellular Biology Michigan, Ann Arbor, MI; 2) Physics, University of Michigan, Ann Program, University of Washington, Seattle, WA; 2) Department Arbor, MI. of Genome Sciences, University of Washington, Seattle, WA. 413B 403A Ventral eye margin is defined by opposing interactions between Cell behaviour during mesoderm spreading in the gastrula. Ivan homothorax and Notch pathway genes Lobe and Serrate. Amit Clark, H.-Arno Müller. College of Life Sciences, University of Singh1,2, Meghana Tare1, Wonseok Son3, Krishanthan Dundee, Dundee, United Kingdom. Vigneswaran3, Kwang-Wook Choi3,4,5. 1) Dept of Biology, Univ Dayton, Dayton, OH; 2) Tissue Regeneration and Engineering 404B Center at Dayton (TREND), University of Dayton, Dayton, OH; A role for the Small GTPase Rap1 in eye development. Eduardo 3) Dept Molecular & Cell Biol, Baylor Col Medicine, Houston, Gonzalez, Layne Dylla, Stacey Lambeth, Jennifer Curtiss. TX; 4) Dept of Ophthalmology, Baylor Col Medicine, Houston; 5) Biology, NMSU, Las Cruces, NM. Developmental Biology Programme, Baylor Col Medicine, Houston, TX. 405C Function of FGF8-like1 and FGF8-like2 in mesoderm movements 414C during gastrulation. Anna Klingseisen1, Tanja Gryzik2, H.-Arno optomotor-blind inhibits cell proliferation, morphogenetic furrow Müller1. 1) Division of Cell & Developmental Biology, College of initiation, and retinal differentiation during Drosophila eye Life Sciences, University of Dundee; 2) Institute of Genetics, development. Yu-Chen Tsai1, Stefan Grimm2, Ju-Lan Chao3, Heinrich Heine University Düsseldorf. Jih-Guang Yao3, Kerstin Hofmeyer2, Jie Shen4, Y. Henry Sun3, Gert O. Pflugfelder2,4. 1) Dept Life Sci, Tunghai Univ, Taichung, 406A Taiwan; 2) Theodor-Boveri-Institut, Biozentrum, Lehrstuhl für Identification of Eve and Runt target genes required for germband Genetik und Neurobiologie, Universität Würzburg, Am Hubland, extension. Athea Vichas, Jennifer Zallen. Developmental Würzburg, Germany; 3) Inst. of Mol. Biol., Academia Sinica, Biology Program, Sloan-Kettering Institute, New York, NY. Taipei, Taiwan; 4) Institut für Genetik, Universität Mainz, Mainz, Germany. 407B Search for New Retinal Determination Genes. Jason Anderson, 415A Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. Deconstructing Pax6 in the Retina. Bonnie Weasner, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. POSTER SESSIONS 53 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

416B 425B Conservation of Retinal Function Across Evolution - The Six Regulatory interaction between Eyeless and Cut in the Family of Transcription Factors.Brandon Weasner, Justin separation of eye-antennal identity.Cheng-wei Wang1,2, Y. Henry Kumar. Dept Biol,Indiana Univ, Bloomington, IN. Sun1,2. 1) Dept Academia Sinica, Inst Molecular Biology, Taipei, Taiwan; 2) Department of Life Sciences and Institute of Genome 417C Sciences, National Yang-Ming University, Taipei, Taiwan, Republic Protein-Protein Interactions Between Homeodomains. of China. Yoshitsugu Adachi1, Frederic Prince1, Serge Plaza2, Walter Gehring1. 1) Dept Cell Biol, Biozentrum, Basel, Switzerland; 2) 426C Centre de Biologie du Développement, Université Paul Sabatier, Molecular fluctuations and interpreting spatial gradients, applied Toulouse, France. to Hunchback pattern formation. David M. Holloway1, Alexander V. Spirov2, Francisco J. P. Lopes2. 1) Mathematics, British 418A Columbia Institute of Technology, Burnaby; Biology, Univ. Victoria, PermanentUltrabithorax repression triggered by high Ubx protein BC, Canada; 2) Applied Mathematics, and Developmental levels. Daniel L. Garaulet, Ernesto Sánchez-Herrero. Centro Genetics, Stony Brook University, NY. de Biología Molecular Severo Ochoa (CBMSO) CSIC-UAM, Madrid, Spain. 427A Cis-regulatory control of sharpening and positioning in the 419B Drosophila hunchback expression pattern. Francisco Lopes1, Analysis of Drosophila Hox complex miRNAs and Hox gene David Holloway2, Alexander Spirov1. 1) Dept. of Applied expression. Derek Lemons, William McGinnis. Biology, UCSD, Mathematics and Center for Developmental Genetics, Stony San Diego, CA. Brook Univ., Stony Brook, NY; 2) Mathematics, British Columbia Institute of Technology, Burnaby; Biology, Univ. of Victoria, B.C. 420C Canada. Investigating the acquisition of segmentation function of fushi tarazu during arthropod evolution. Alison M. Heffer1,2, Leslie 428B Pick1,2. 1) Molecular and Cell Biology; 2) Dept.Entomology Complex movements of segmentation gene expression domains University of Maryland, College Park, MD 20742. in Drosophila wild type embryos and Kr- mutants. Svetlana Surkova1, Maria Samsonova1, John Reinitz2. 1) State 421A Polytechnical University, St. Petersburg, Russia; 2) Stony Brook Regulation of spider segmentation by Wnt8. Alistair P. University, Stony Brook, NY. McGregor1, Matthias Pechmann1,2, Natália M. Feitosa1, Wim G. M. Damen1. 1) Institute for Genetics, University of Cologne, 429C Cologne, Germany; 2) Georg-August-Universität Göttingen, Positional information and the Hunchback repression gradient Johann-Friedrich-Blumenbach-Institut für Zoologie und in Drosophila. Danyang Yu, Stephen Small. Department of Anthropologie GZMB, Abteilung für Entwicklungsbiologie, Biology, New YorkUniv, New York, NY. Göttingen, Germany. 430A 422B The Ancestral morphogen otd can replace many bcd patterning Apical localization of RhoGEF2 and adherens junctions during functions in the early embryo. Gozde Yucel, Na-Eun Yoo, gastrulation. Verena Koelsch1,2, Maria Leptin1. 1) Institute for Stephen Small. Biology, New York Univ, New York, NY. Genetics, University of Cologne, Cologne, Germany; 2) present address: University of California, San Diego, Section of Cell and 431B Developmental Biology, La Jolla, CA. A screen for modifiers of Hedgehog signaling in Drosophila identifies swm and mts. David J. Casso1, Songmei Liu1, D. 423C David Iwaki1, Stacey K. Ogden2, Thomas B. Kornberg1. 1) The K50 homeodomain transcription factor Orthodenticle Dept Biochem/Biophysics, Univ California, San Francisco, San differentially regulates nervous system patterning through Francisco, CA; 2) Department of Pharmacology and Toxicology separable functional domains. ElizabethMcDonald1, Michael Dartmouth Medical School Hanover, NH. Workman1, Joachim Reischl2, Kerstin Meier2, Ernst Wimmer2, Tiffany Cook1. 1) Developmental Biology/Pediatric 432C Ophthalmology, CCHMC, Cincinnati,OH; 2) Department of The roles of knirps and abrupt in wing vein development. Orna Developmental Biology, Georg-August-University Goettingen, Cook1, Hanh Nguyen1, Koen J. T. Venken2, Hugo J. Bellen2, Germany. Ethan Bier1. 1) Dept. of Biology, UCSD, La Jolla CA; 2) Program in Developmental Biology, Baylor College of Medicine, Houston, 424A TX. Larval tracheoblasts as a model for cell migration. Chrysoula Pitsouli1, Norbert Perrimon1,2. 1) Genetics Department, Harvard 433A MedicalSchool, Boston, MA; 2) Howard Hughes Medical Institute. Wing membrane topography is determined by the dorsal wing epithelium. Kristy Doyle, Simon Collier. Dept Biological Sci, Marshall Univ, Huntington, WV. 54 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

434B 443B Coregulation of a neighboring EST with crossveinless-c (cv-c). Identification and characterization of Fkh targets in Drosophila Tia Hughes, Jeffrey Marcus. Dept Biol, 11080,Western salivary gland. Rika Maruyama, Elizabeth Grevengoed, Kentucky Univ, Bowling Green, KY. Deborah J. Andrew. Department of Cell Biology, Johns HopkinsUniversity School of Medicine, Baltimore, MD. 435C The Glucose Transporter (GLUT4) Enhancer Factor is Required 444C for Normal Wing Positioning in Drosophila. Jonathan Terman, From regulatory networks to cell morphology: control of Umar Yazdani, Zhiyu Huang. Departments of Neuroscience epidermis differentiation. Francois Payre1, Jennifer Zanet1, and Pharmacology, The University of Texas Southwestern Philippe Valenti1, Serge Plaza1, Alistair McGregor2, David Medical Ctr, Dallas, TX. Stern2. 1) Biologie du Development, University ToulouseIII CNRS, Toulouse,France; 2) Department of Ecology and 436A Evolutionary Biology, Princeton University. Impact of the sulfation state of heparan sulfate proteoglycans on Hedgehog signaling. Alexandre Wojcinski1, Takuya 445A Akiyama2, Cathy Soula1, Hiroshi Nakato2, Bruno Glise1. 1) Identification and Characterization of Genes Involved in Centre de Biologie du Développement, CNRS, Université Paul Subcellular Lumen Formation. Oscar E. Ruiz, Mark M. Sabatier, UMR-5547, Toulouse, France; 2) Department of Metzstein. Department of Human Genetics, University of Genetics, Cell Biology and Development, The University of Utah,Salt Lake City, UT. Minnesota, Minneaopolis, USA. 446B Genetic control of imaginal hindgut development in Drosophila. Organogenesis and Gametogenesis Shigeo Takashima, Marianna Mkrtchyan, Amelia Younossi- Hartenstein, John Merriam, Volker Hartenstein. Univ 437B California,Los Angeles, Los Angeles, CA. Obstructor, a novel Megatrachea interactor protein, is essential for tracheal tube maturation. Matthias Behr, Birgit Stümpges, 447C Tina Radtke, Christian Wingen, Michael Hoch. Molecular Multiple requirements for Rho1 GTPase during salivary gland Developmental Biology, LIMES Institute, University of Bonn, development. Na Xu1, Benison Keung2, Monn Monn Myat1. 1) Germany. Dept Cell & Developmental Biol, Weill Medical Col of Cornell, New York, NY; 2)New York MedicalCollege, Valhalla, NY 10595. 438C The transcription factor SoxNeuro directs trichome formation in 448A the ventral embryonic epidermis upstream of and in parallel to Multipotent Drosophila intestinal stem cells specify daughter cell Shavenbaby. Marita Buescher, William Chia. Drosophila fates by differential Notch signaling. Benjamin Ohlstein. development, Temasek Lifesciences Laboratories, 1 Research Department of Genetics and Development, Columbia University Link, Singapore 117604. HHSC 1130, New York, NY 10032. 439A 449B Serrano (Sano) is a novel planar cell polarity regulator that Identification of the ov and v subloci and the analysis of controls tube length in Drosophila trachea. Se-Yeon Chung1, complementing nonsense mutants in the dumpy gene. Amber Melissa Vining1, Chih-Chiang Chan2, Pamela Bradley1, Keith Carmon, Ross MacIntyre. Dept Molec Biol & Genetics, Cornell Wharton2, Deborah J. Andrew1. 1) Dept Cell Biol, Johns Univ, Ithaca, NY. Hopkins Univ, Baltimore, MD; 2) Dept Pathol and Mol Biol, UT Southwestern Medical Center, Dallas, TX. 450C Characterization of slowmotion, a novel modulator of cell 440B adhesion in D. melanogaster.Eliezer Gilsohn, Talila Volk. Making and shaping subcellular tracheal tubes. Amin Ghabrial1, Molecular Genetics department, Weizmann Institute of Science, Boaz Levi2, Mark Krasnow2. 1) Dept Cell/Developmental Biol, Rehovot, Israel. Univ Pennsylvania, Philadelphia,PA; 2) Dept of Biochemistry/ HHMI, Stanford University School of Medicine, Stanford, CA. 451A Dead man walking encodes a conserved cathepsin B-like ECM 441C protein that interacts genetically with PS3 integrin. Ellen LeMosy, The role of VHL in tracheal development. Anita Hsouna, Tien Michael Dinkins. Dept Cell Biol & Anatomy, Medical Col Hsu. Hollings Cancer Ctr, Medical Univ South Carolina, Georgia, Augusta, GA. Charleston, SC. 452B 442A cut and slow border cells act antagonistically to regulate Plexin-Semaphorin signaling is necessary for proper salivary adherens junction polarity during follicle cell migration. Benjamin gland migration. Afshan Ismat1, Melissa S. Vining1, Alex Levine, Truesdale Angela, Bergen Andrew, Dobens Leonard. Kolodkin2, Deborah J. Andrew1. 1) Department of Cell Biology, Dept Molecular Biol, Univ MissouriKansas City, Kansas City, Johns Hopkins University School of Medicine, Baltimore, MD; MO. 2) Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD. POSTER SESSIONS 55 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

453C 462C Control of apical cell shape: role of Zona Pellucida proteins in Fly Muscle LIM Protein, Mlp84B, cooperates with Sallimus to epidermal cell morphogenesis. Francois Payre1, Isabelle maintian muscle structural integrity. Kathleen Clark1,2, Mary C. Fernandes1, Helene Chanut-Delalande1,2, Pierre Ferrer1, Beckerle1,2. 1) Huntsman Cancer Inst, Univ Utah, Salt Lake City, Serge Plaza1. 1) Biologie du Development, University ToulouseIII UT; 2) Dept. of Biology, Univ Utah, Salt Lake City, UT. CNRS Toulouse, France; 2) Bizentrum der Universitat Basel, Basel, Switzerland. 463A The role of Myocyte enhancer factor-2 in controlling adult skeletal 454A muscle development. Richard Cripps, TyAnna Lovato, Melanie The role of Tissue Inhibitor of Metalloproteases (Timp) in Adams, Phill Baker. Dept Biol, Univ New Mexico, Albuquerque, Drosophila oogenesis. John Pearson, Acaimo Gonzalez- NM. Reyes. Centro Andaluz de Biologia del Desarrollo (CABD),CSIC Universidad Pablo Olavide, Seville, Spain. 464B Requirement for the Zn-finger transcription factor CF2 in larval 455B blood and indirect flight muscle. Kathleen M. Gajewski1, Richard Cohesive migration of the embryonic salivary gland. Carolyn P. Sorrentino2, Tien Hsu3, Robert A. Schulz4. 1) Dept Pirraglia, Monn Monn Myat. Cell & Developmental Biology, Biochemistry & Molec Biol, MD Anderson Cancer Ctr, Houston, WeillMedical College of Cornell University, New York, NY. TX; 2) Department of Biology, University of Guyana, Georgetown, Guyana; 3) Laboratory of Cancer Genomics, Medical University 456C of South Carolina, Charleston SC; 4) Dept of Biological Sciences, Slit and Roundabout regulate E-Cadherin-mediated cell University of Notre Dame, South Bend IN. adhesion required for Drosophila heart tube lumen formation. Edgardo Santiago-Martínez1,2, Nadine H. Soplop1,2, Rajesh 465C Patel1, Sunita G. Kramer1,2. 1) Dept of Pathology & Laboratory Hemangioblast Differentiation: Pathways Interacting with the Medicine, UMDNJ-Robert Wood Johnson Medical School, Notch/Delta Signal. Melina Grigorian, Lolitika Mandal, Volker Piscataway, NJ; 2) Prog in Cell & Developmental Biology, Rutgers Hartenstein. Dept MCDB, Univ California, Los Angeles, Los University & UMDNJ-Graduate School of Biomedical Sciences, Angeles, CA. Piscataway, NJ. 466A 457A The cytokinesis protein Tumbleweed/RacGAP50C plays an Guidance Molecules in Epithelial Tube Maintenance: Slit essential role in postmitotic myotube guidance. Colleen M. Signaling in the Drosophila Hindgut. Nadine Soplop1,2, Edgardo Guerin1,2, Robert Connacher1, Sunita G. Kramer1,2. 1) Dept Santiago-Martínez1,2, Sunita G. Kramer1,2. 1) Department of Pathology, RWJMS, Piscataway, NJ; 2) Cell & Dev Bio, GSBS- Pathology and Laboratory Medicine; 2) Cell and Developmental UMDNJ & Rutgers, Piscataway, NJ. Biology Program, Graduate School of Biomedical Sciences; Rutgers University and University of Medicine and Dentistry of 467B New Jersey at Robert Wood Johnson Medical School, Live imaging reveals that myoblast fusion requires dynamic Piscataway, NJ. remodeling of the actin cytoskeleton by SCAR/WAVE and Arp2/ 3. Brian Richardson1, Mary Baylies1,2. 1) Program in 458B Biochemistry, Cell and Molecular Biology, Weill Cornell Graduate Shaping the "ball and socket" joints. Reiko Tajiri, Shigeo School of Medical Sciences, New York, NY; 2) Developmental Hayashi. RIKEN Center for Developmental Biology, Kobe, Biology Program, Sloan-Kettering Institute, New York, NY. Japan. 468C 459C Expression and functional analysis of a novel Fusion Competent Highly conserved cysteines in the vitelline membrane domain Myoblast specific GAL4 driver. Kate Rochlin1, Karen Beckett1, of the sV23 eggshell protein are functionally distinct. Tianyi Wu, Hong Duan2, Hanh Nguyen2, Mary Baylies1. 1) Dept Gail Waring. Dept Biological Sci, Marquette Univ, Milwaukee, Developmental Biol, Sloan-Kettering Inst, New York, NY; 2) Dept WI. of Medicine, Developmental and Molecular Biol, Albert Enstein School of Medicine, Bronx, NY. 460A An O-glycosyltransferase is required for proper cell adhesion in 469A Drosophila. Liping Zhang, Ying Zhang, Kelly G. Ten Hagen. SNS and DUF mediate cell interactions in the development of NIDCR/NIH, Bethesda, MD. the garland cell cluster in the Drosophila embryo. Shufei Zhuang, Huanjie Shao, Jeffrey McDermott, Susan Abmayr. Stowers 461B Institute for Medical Research, Kansas City, MO. Fusion Regulates Myoblast Proliferation and Fiber Numbers during Adult Myogenesis in Drosophila. Krishan Badrinath. Dept 470B Zoology, Miami Univ, Oxford, OH. Structure and function of ring canals in somatic cells. Stephanie Airoldi, Lynn Cooley. Genetics, Yale University, New Haven, CT. 56 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

471C 480C Clonal analysis using a follicle cell dependent dominant female Drosophila Pxt: a cyclooxygenase-like facilitator of follicle sterile mutation. PhoenixBouchard Kerr, Aliaa Eleiche, Laura maturation. Tina L. Tootle, Allan C. Spradling. Embryology, Nilson. Department of Biology, McGill University, Montréal, QC, Carnegie Institution, Baltimore, MD. Canada. 481A 472A Determining the Role of 18-Wheeler in Morphogenesis of the Studies on Tis11 in D. melanogaster. Robert Fedic1, Perry J. Ovarian Follicle Cell Epithelium. Erika Vielmas, Crissy Blackshear2, James M. Mason1. 1) Lab Molecular Genetics, Danusastro, Elizabeth D. Eldon. Biological Sciences, California NIEHS/NIH, Res Triangle Park, NC; 2) Laboratory of State UniversityLong Beach, Long Beach, CA. Neurobiology, NIEHS/NIH, Res Triangle Park, NC. 482B 473B The Rho-GTPase Cdc42 is required for Drosophila heart Ecdysone Receptor and Ultraspiracle are Required for Chorion morphogenesis. Georg Vogler1, Li Qian2, Jiandong Liu3, Rolf Gene Amplification. Jennifer Hackney, Leonard Dobens. Dept Bodmer1. 1) Burnham Institute for Medical Research, San Diego, Molecular Biol & Biochem, Univ Missouri, Kansas City, Kansas CA; 2) Gladstone Institute of Cardiovascular Disease, San City, MO. Francisco, CA; 3) University of California, San Francisco, CA.

474C 483C Making inroads into the Drosophila female reproductive system. The variable nurse cells gene encodes Ard1, an N-terminal Yael Heifetz1, Anat Kapelnikov1, Einat Zelinger1, Vidya acetyltransferase subunit implicated in sister chromatid cohesion Nagalakshmi1, Paul Mack2, Michael Bender3, Kahn and cell cycle progression. Ying Wang, Tolga Turan, Michelle Rhrissorrakrai4, Kristin C. Gunsalus4, Patricia Rivlin5, Ronald Mijares, Megan Gall, Kevin Manage, Anna Javier, Rahul Hoy5. 1) Dept. of Entomology, The Hebrew Univ, Rehovot, Israel; Warrior. Dept of Developmental & Cell Biol, Univ California, 2) Dept. of Sciences and Mathematics, Mississippi Univ. for Irvine, Irvine, CA. Women, Columbus, MS; 3) Dept. of Genetics, Georgia Univ., Athens, GA; 4) Center for Comparative Functional Genomics, 484A Dept. of Biology, New York Univ., NY; 5) Dept. of Neurobiology Arginine methylation of SmB is needed for its localization in the and Behavior, Cornell Univ., Ithaca, NY. pole plasm and formation of germ cells. Joel Anne, Bernard M. Mechler. Dept Developmental Genetics, DKFZ, Heidelberg, 475A Germany. Juvenile hormone controls entry to metamorphosis through Methoprene-tolerant and Broad-complex. Marek Jindra, 485B Barbora Konopova. Biology Center ASCR, Ceske Budejovice, The Regulation of Germ Cell Sex Determination in Drosophila. Czech Republic. Abbie Casper, Mark Van Doren. Dept Biol, Johns Hopkins Univ, Baltimore, MD. 476B Oviduct under construction: Post-mating changes in Drosophila 486C reproductive tract. Anat Kapelnikov1, Patricia Rivlin2, Ronald DSXM and DSXF expression in subsets of Drosophila somatic Hoy2, Yael Heifetz1. 1) Dept. of Entomology, The Hebrew Univ., gonad cells. Leonie Hempel, Brian Oliver. LCDB/NIDDK/NIH, Rehovot, Israel; 2) Dept. of Neurobiology and Behavior, Cornell Bethesda, MD. Univ., Ithaca, NY. 487A 477C Mjl expression depends on somatic and germline signals. Rasika A combinatorial enhancer recognized by Mad, TCF and Brinker Kalamegham, Brian Oliver. LCDB, NIDDK/NIH, Bethesda, MD. first activates then represses dpp expression in the posterior spiracles. Stuart Newfeld1, Denis Bulanin2, Aaron Johnson1, 488B Teresa Orenic2, Norma Takaesu1. 1) Sch Life Sci, Arizona State JAK/STAT regulation of germ cell sex determination. Gretchen Univ, Tempe, AZ; 2) Dept Biol, Univ Illinois,Chicago. H. McConnell, Carla M. Wood, Mattew J. Wawersik. Biology, The College of William and Mary, Williamsburg, VA. 478A Antagonistic role of Notch and Ecdysone signaling in regulating 489C the endocycle/gene amplification switch in Drosophila follicle Identification of targets of the germline sex determination cells. Jianjun Sun, Alexander Armento, Wu-Min Deng. Dept pathway. John Smith, Brian Oliver. Lab. of Cellular & Biological Sci, FloridaState Univ, Tallahassee, FL. Developmental Biology, National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda, MD. 479B Identification of Genes Required for Adult Muscle Development 490A in Drosophila. Chrisna V. Thomas, Richard Cripps. Biology, The octopamine receptor OAMB regulates ovulation through University of New Mexico, Albuquerque, NM. Ca2+/Calmodulin-dependent protein kinase II. Hyun-Gwan Lee1, Kyung-An Han1,2. 1) The Huck Institute Genetics Graduate Program; 2) Department of Biology, Pennsylvania State University, University Park, PA 16802. POSTER SESSIONS 57 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

491B 500B Proteomics of Drosophila reproductive system 1: Profiling of Mutants in the Drosophila SUN gene dspag uncouple Drosophila seminal fluid proteins by two-dimensional gel individualization complex progression from membrane electrophoresis. Nobuaki Takemori1, Masaki Yamada2, Takashi investment in gametogenesis. Martin Kracklauer1, Heather Ohsako1, Masa-Toshi Yamamoto1. 1) Drosophila Genetic Wiora1, Xin Chen2, Janice Fischer1. 1) Dept MCD Biol, Univ Resource Center, Kyoto Institute of Technology, Kyoto, Japan; Texas, Austin, Austin, TX; 2) Dept Dev Biol, Stanford U School 2) Life Science Laboratory, Shimadzu Corporation 1, Kyoto, of Medicine, Stanford, CA. Japan. 501C 492C Developmental control of testis-TAF expression and male germ Mat89Bb is required for proper coupling of centrosomes/basal cell differentiation. Chenggang Lu, Xin Chen, Margaret Fuller. body to the nucleus during spermatogenesis. Michael Department of Developmental Biology, Stanford University Anderson, Laura Lee. Dept Cell & Developmental Biol, School of Medicine, Stanford , CA. Vanderbilt Univ, Nashville, TN. 502A 493A Suppressors and Enhancers of Segregation Distorter. Janna Translational control of boule in spermatogenesis. Catherine McLean, Reid McLean, David Sisneros, Hollie Vigil. Dept of Baker, Margaret Fuller. Developmental Biology, Biol, Colorado State Univ-Pueblo, Pueblo, CO. StanfordUniversity School of Medicine, Stanford, CA. 503B 494B Di-(2-ethylhexyl) phthalate (DEHP) affects D. melanogaster yuri gagarin is required for actin, tubulin and basal body functions development possibly through meiotic defects during in spermatogenesis. Kathleen M. Beckingham, Michael J. spermatogenesis. Janet Rollins1, Kwesi Blackman3, Penel Texada, Ravi P. Munjaal, Rebecca A. Simonette, Cassidy B. Joseph3, Thomas Onorato2. 1) Division of Science, College of Johnson, William J. Deery. Biochemistry and Cell Biology, Mount St. Vincent, Riverdale, NY; 2) Department of Natural RiceUniversity, Houston, TX. Applied Sciences, LaGuardia Community College, Long Island City, NY; 3) Department of Biology, Kingsborough Community 495C College, Brooklyn, NY. Studying of the expression and protein-protein interactions of Stellate protein in testes of D. melanogaster. Ksenia Egorova, 504C Ludmila Olenina, Oxana Olenkina, Vladimir Gvozdev. A role for basigin (bsg) in late-stage Drosophila spermatogenesis. Department of Molecular Genetics of Cell, Institute of Molecular Kathryn Vecomnskie, Meaghan Crook, James Fabrizio. Genetics RAS, Moscow, Russian Federation. BiologyDepartment College of Mount Saint Vincent 6301 Riverdale Ave. Bronx, NY 10471. 496A Characterization of the hypomorphic male sterile nmdry4 allele 505A and analysis of the nmd paralog CG4701’s putative role in Cloning and characterization of mitoshell, a gene required for spermatid mitochondrial shaping. Bevin C. English, Sarah D. normal mitochondrial aggregation during Drosophila Durnbaugh, Kara M. Koehrn, Sara H. Holmberg, Sheena E. spermatogenesis. Kathleen H. Wood, Laura M. Bergner, Favors, Karen G. Hales. Department of Biology, Davidson Francis E. Hickman, Michael C. Beaucaire, Amanda C. College, Davidson, NC. Aldridge, Sheena E. Favors, Karen G. Hales. Department of Biology, Davidson College, Davidson, NC. 497B Separation of Nebenkern anchoring and mitochondrial 506B elongation phenotypes in germ line clones of a milton allele. The Ubiquitin Specific Protease Scrawny is Required in Diverse Sheena E. Favors, Karen G. Hales. Department of Biology, Drosophila Stem Cells. Michael Buszczak1,2, Shelley Paterno2, Davidson College,Davidson, NC. Allan C. Spradling2. 1) Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX; 2) Department of 498C Embryology, Carnegie Institution, Baltimore, MD. Condensin II catalyzes chromosome individualization to enable meiosis I segregation and separation of polytene chromosomes. 507C Tom Hartl, Helen Smith, Giovanni Bosco. Dept Molec & Sex-lethal is required for asymmetric fate specification of ovarian Cellular Biol, Univ Arizona, Tucson, AZ. germ cells. Johnnie Chau, Laura Shapiro Kulnane, Helen K. Salz. Department of Genetics, Case Western ReserveUniversity, 499A Cleveland, OH. Theped gene and delayed translation of dhod RNA during spermatogenesis. David Keesling, Jianyuan Luo, John Rawls. 508A Department of Biology, University of Kentucky, Lexington, KY The role of NURF301 in the Drosophila testis stem cell niche. 40506. Christopher Cherry, Erika Matunis. Department of Cell Biology, JohnsHopkins University School of Medicine, Baltimore, MD. 58 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

509B 518B A circuit discriminating somatic stem cells from niche cells in Regulation of somatic stem cells in the Drosophila ovary through the testis. Stephen DiNardo, Natalie Terry, Sarah Freilich, multiple signaling pathways. Cynthia Vied, Daniel Kalderon. Colin Palmer. Dept Cell & Developmental Biol, Univ Dept Biological Sci, Columbia Univ, New York, NY. Pennsylvania Sch Medicine, Philadelphia, PA. 519C 510C Somatic stem cells contribute to the apical hub in the Drosophila Brat is required for correct differentiation of Drosophila ovarian testis. Justin Voog, Leanne Jones. Laboratory of Genetics, The stem cells. Robin Harris, Hilary Ashe. Faculty of Life Sciences, Salk Institute, La Jolla, CA. University of Manchester, Manchester,Lancashire, United Kingdom. 520A Genetic interactions suggest a role for chromatin modification 511A in maintenance of germline stem cells. Lin Yu1, Yan Song2, Socs36E mediated JAK/STAT signal attenuation regulates the Tammy Lee1, Robin Wharton1. 1) Department of Molecular balance of germline and somatic stem cells in the Drosophila Genetics and Microbiology/Cell Biology, Duke University/HHMI, testis niche. Melanie Issigonis1, Natalia Tulina2, Margaret de Durham, NC; 2) Department of Pathology, Stanford University Cuevas1, Crista Brawley1, Laurel Mellinger1, Erika Matunis1. School of Medicine, Stanford, CA. 1) Cell Biology Department, Johns Hopkins University School of Medicine, Baltimore, MD; 2) Genetics and Gene Regulation Program, University of Pennsylvania Medical School, Chromatin and Gene Expression Philadelphia, PA. 521B Role of Su(var)3-9 in preserving genome integrity. Irene Chiolo, 512B Jamy Peng, Gary Karpen. Genome Biology, LBNL, Berkeley, CA. GFP-NRE sensor directly monitors Pumilio activity in germline stem cells. Sung Yun Kim, Jiyoung Kim, Changsoo Kim. 522C School of Biological Sciences and Technology, Chonnam HIRA-dependent chromatin assembly during Drosophila National University, Gwangju-Si, Korea. development. Benjamin Loppin, Cécile Doyen, Tzvetina Brumbarova, Guillermo Orsi, Pierre Couble. Centre de 513C Genetique Moleculaire et Cellulaire, CNRS, University of Lyon, Dietary Regulation of Normal and Tumorous Germline Stem Cells Lyon, France. Occurs Via both Insulin-Dependent and -Independent Mechanisms. Leesa M. LaFever, Hwei-Jan Hsu, Daniela 523A Drummond-Barbosa. Cell and Developmental Biology, An inducible over-expression screen identifies new factors that Vanderbilt University Medical Center,Nashville, TN. alter heterochromatic gene silencing. Jonathan I. Schneiderman, Kami Ahmad. BCMP Dept, Harvard Medical 514A School,Boston, MA. Coordination between two stem cell populations in the Drosophila ovary. Lucy Morris, Allan C. Spradling. Carnegie Institution of 524B Washington, Baltimore, MD. The role of Scaffold Attachment Factors in nuclear organization and gene expression. Catalina Alfonso, Keith Maggert. Dept 515B Biochemistry/Biophysics, Texas A&M Univ, College Station, TX. Elucidating the Role of the Notch Pathway in the Drosophila Testis Stem Cell Niche. Tishina C. Okegbe, Natalie Terry, Tim 525C Kelliher, Steve Dinardo. Cell and Developmental Biology, Heterochromatin Comes Unhinged: Studies of HP1. Diane E. UPenn, Philadelphia, PA. Cryderman, Karrie A. Hines, Andrew J. Petersen, Luka N. Zirbel, Beatrice Curio-Penny, Lori L. Wallrath. Biochemistry, 516C University of Iowa, Iowa City, IA. Germline stem cells are resilient during Drosophila development. Halyna R. Shcherbata, Ellen J. Ward, Karin A. Fisher, Jenn- 526A Yah Yu, Hannele Ruohola-Baker. Biochemistry, University of Genetic interactions between RNA silencing components and Washington, Seattle, WA. RNA polymerase II. Harsh Kavi, James Birchler. Dept Biological Sci, Univ Missouri,Columbia, Columbia, MO. 517A integrin-dependent anchoring of a stem cell niche. Guy 527B Tanentzapf1,2, Danelle Devenport2,4, Dorothea Godt3, Nicholas Epigenetic blocking of an enhancer region controls irradiation- H. Brown2. 1) Cellular and Physiological Sciences, University of induced pro-apoptotic gene expression in Drosophila embryos. British Columbia, Vancouver, BC, Canada; 2) The Gurdon Nianwei Lin1, Yanping Zhang1, Gina Chan1,2, Rong Yuan1,2, Institute and Department of Physiology, Development, and Bing Yao2, Samuel Wu3, Pamela M. Carroll4. 1) Department of Neuroscience, University of Cambridge, Cambridge, UK; 3) The Molecular Genetics & Microbiology, Univ Florida, Gainesville, Department of Cell & Systems Biology University of Toronto FL; 2) UF Shands Cancer Center, Univ Florida, Gainesville, FL; Toronto, ON, Canada; 4) Laboratory of Mammalian Genetics 3) Department of Statistics, College of Medicine,Univ Florida, and Development, Rockefeller University, New York. Gainesville, FL; 4) Department of Applied Genomics, Bristol- Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. POSTER SESSIONS 59 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

528C 538A Studying the role of rDNA in gene expression. Silvana Paredes, Tissue-specific contributions of the Drosophila LEM domain Keith Maggert. Dept Biol, Texas A&M Univ, College Station, protein dMAN1 in the nuclear lamina.Belinda Pinto1, Shameika TX. Wilmington2, Lori L. Wallrath1,2, Pamela Geyer1,2. 1) Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA; 529A 2) Department of Biochemistry, University of Iowa, Iowa City, IA. Chromatin organization of the fourth chromosome of D. melanogaster. Nicole Riddle, Wilson Leung, Kathryn 539B Huisinga, Brent Brower-Toland, Sarah Elgin. Department of Epigenetic Regulation of Gene Expression by Drosophila Myb Biology, Washington University, St. Louis, MO. and E2F2-RBF via the Myb-MuvB/dREAM Complex. Hong Wen1, Laura Andrejka1, Jonathan Ashton1, Roger Karess2, Joseph 530B S. Lipsick1. 1) Dept Pathology & Genetics, Stanford Univ, Stanford, Studies on the function of the MSL complex. James Birchler, CA; 2) CNRS, Centre de Génétique Moléculaire, France. Harvey Fernandez, Xiaoping Sun, Lin Sun. Biological Sciences, University of Missouri, Columbia, MO. 540C Genetic analyses of bocksbeutel and otefin: genes encoding 531C nuclear lamina LEM domain proteins. Shameika Wilmington1, High-Resolution Mapping of histone modifications in Drosophila Emma Hornick1, Belinda Pinto2, Lori L. Wallrath1,2, Pamela Stage 5 embryos. Sasha Langley, Gary Karpen. Dept MCB, Geyer1,2. 1) Department of Biochemistry, University of Iowa, Iowa Univ California Berkeley/LBNL, Berkeley, CA. City, IA; 2) Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA. 532A Comparison of chromatin structure in wild type and BEAF 541A (Boundary Element-Associated Factor) knockout larvae. Defining the requirements for Polycomb group response Matthew Gilbert, Craig Hart. Dept Biological Sciences, elements (PREs) at the engrailed locus. Melissa Durant, Judy Louisiana State Univ, Baton Rouge, LA. Kassis. NICHD, NIH, Bethesda, MD.

533B 542B tRNA genes: a potential role as boundary elements in D. Functional analysis of the Polycomb repressive complex 2 melanogaster. Paola Guerrero, Keith Maggert. Paola Guerrero, (PRC2) histone methyltransferase.P. Joshi1, N. Jahren1, K. Keith Maggert. Department of Biology, TexasA & M Hines1, J. Wang1, C. Ketel1, D. Cryderman2, E. Miller1, E. University,College Station TX 77843. Carrington3, R. S. Jones3, L. L. Wallrath2, J. A. Simon1. 1) Genetics, Cell Biology and Development, University of 534C Minnesota,Minneapolis, MN; 2) University of Iowa; 3) Southern Genome-wide identification of BEAF binding sites in Drosophila. Methodist University. NanJiang1, Brenda Winbery2, Craig Hart1. 1) Dept Biological Sciences, Louisiana State Univ, Baton Rouge, LA; 2) Louisiana 543C Scholar's College, Northwestern State Univ, Natchitoches, LA. Recruitment of Drosophila Polycomb-group proteins by a novel Polycomblike containing complex. Urmi Savla, Judith Benes, 535A Richard Jones. Dept Biological Sci, Southern Methodist Univ, Does the Boundary Element-Associated Factor (BEAF) play a Dallas, TX. broad role in maintaining global patterns of gene expression? Swarnava Roy, Craig Hart. Dept Biological Sci, Louisiana State 544A Univ, Baton Rouge, LA. Dissecting the functions of the CHD1 chromatin remodeling factor. Jennifer Armstrong, Ivy McDaniel, Kimia Raafat, 536B Michael Dauer. Joint Science Dept, Claremont Gene expression analysis of D. melanogaster after acute and Colleges,Claremont, CA. short intermittent and constant hypoxia treatment. Priti Azad1, Gabriel Haddad1,2. 1) Departments of Pediatrics (Section of 545B Respiratory Medicine) and Neuroscience, University of Blimp-1 Regulates Pupal Eye and Head Development. Gerald California-San Diego, La Jolla, CA 92093; 2) The Rady Children's Call1, Joy Wu2, Chris Bui2, Stacy Chan2, Jingwen Tan2, Amrita Hospital, San Diego, CA 92123. Cheema2, Jiong Chen2, Utpal Banerjee2. 1) Dept of Pharmacology, Midwestern University, Glendale, AZ; 2) 537C Molecular, Cellular and Developmental Biology, University of An investigation into the molecular function of the hybrid incompatibility California, Los Angeles, Los Angeles, CA. gene, Lhr. Shamoni Maheshwari, Daniel A. Barbash. Dept Molecular Biol & Genetics, Cornell Univ, Ithaca, NY. 546C Functional cooperation between the Brahma chromatin remodeling complex and histone demethylase enzymes. Brenda J. Curtis1, Daniel R. Marenda2, Claudia B. Zraly1,3, Andrew K. Dingwall1,3,4. 1) Biochemistry Program, Loyola University Chicago, SSOM, Maywood, IL; 2) Dept Biological Sciences, Univ Sciences-Philadelphia; 3) Oncology Institute; 4) Dept Pathology. 60 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

Neurogenetics and Neural Development 556A CycE regulated cell fate specification in the embryonic CNS. 547A Christian Berger1, Ramakrishnan Kannan2, L. S. The divergent TGF-b ligand Dawdle utilizes an activin pathway Shashidhara2, Gerd M. Technau1. 1) Inst Genetics, Johannes- to influence neuronal function in Drosophila. Kavita Arora1, Gutenberg Univ, Mainz, Germany; 2) Centre for Cellular and Jeremy Ellis1, Minh Nguyen1,2, Louise Parker1,3. 1) Dept Molecular Biology, Hyderabad, India. Developmental & Cell Biol, Univ of California,Irvine, CA; 2) NIH/ NIDCR Bethesda,MD; 3) Univ of California,Berkeley, CA. 557B Development of the adult abdominal ganglion during 548B metamorphosis: formation of the terminal nerve trunk. Meredith Role of the Liprin family of proteins in axon targeting and synapse Dorr, Camilo Molina, Kathleen Broomall, Rakesh formation. Sergio Astigarraga, Kerstin Hofmeyer, Reza Rachamaduggu, Joyce Fernandes. Dept Zoology, Miami Univ, Farajian, Jessica Treisman. Developmental Genetics, Skirball Oxford, OH. Institute, New YorkUniversity School of Medicine, New York, NY. 558C 549C Role Of ZFH-2 in Drosophila gene insulation and apoptosis. Dissecting Dscam Localization and Signaling. Rachel Bortnick1, Ananya Guntur, Martha Lundell. Dept Biol, Univ Texas, San Hyoje Ryu1, Dan Dascenco1, Maria-Luise Erfurth1, Akhila Antonio, San Antonio, TX. Parthasarthy1, Michael Hughes2, Dietmar Schmucker1. 1) Department of Cancer Biology, Dana Farber Cancer Institute; 559A Program in Neuroscience, Harvard Medical School, Boston, MA; Histone Deacetylase 1 (Rpd3) in Dendrite Targeting of Drosophila 2) Department of Pharmacology, Institute for Translational Olfactory Projection Neurons. Takahiro Chihara1,3,4, Joy S. Medicine and Therapeutics, University of Pennsylvania School Wu1,2,4, Liqun Luo1,2. 1) HHMI and Dept Biol; 2) Neuroscience of Medicine, Philadelphia, PA. program, Stanford University. Stanford,CA, USA; 3) Dept Gen, Grad Sch Pharmaceut, University of Tokyo, Tokyo, Japan; 4) 550A These authors contributed equally to this work. Transcriptional Regulation of Commissureless. Casey Jowdy, Mark Seeger. Dept. of Molecular Genetics and Center for 560B Molecular Neurobiology, The Ohio State University, Columbus, Protein stability of Glide/GCM regulated by F-box proteins Slimb OH. and Archipelago during Drosophila glial development. Margaret Ho1, Hungwen Chen2, Angela Giangrande3, Cheng-Ting 551B Chien1. 1) Dept. Molecular Biol., Academia Sinica, Taipei, Taiwan; Vav, a Rho GTPase activator, controls axon guidance during 2) Dept. Biological Chemistry, Academia Sinica, Taipei, Taiwan; Drosophila embryonic and larval development. Marianne B. Y. 3) Institut de Génétique et de Biologie Moléculaire et Cellulaire, Malartre, Maria D. Martin Bermudo. Centro Andaluz de Biologia Strasbourg, France. del Desarollo, Seville, Spain. 561C 552C Degeneration of optic lamina caused by defective endocytic Analysis of the Drosophila crmp gene to determine the role of function in glial cells. Yuan-Ming Lee1,2, Y. Henry Sun1,2. 1) Inst the CRMP protein in neurogenesis. Deanna Morris, John Molecular Biology, Academia Sinica, Taipei, Taiwan; 2) Inst of Rawls. Department of Biology, University of Kentucky, Lexington, Genomic Science, National Yang Ming University, Taipei, Taiwan. KY. 562A 553A Identity, origin, and migration of peripheral glial cells in the Dissecting the Frazzled cytoplasmic domain in-vivo and a genetic Drosophila embryo. Christian M. von Hilchen, Ruth screen for novel regulators of axon guidance at the Drosophila Beckervordersandforth, Christof Rickert, Benjamin midline. Mike P. O'Donnell, David S. Garbe, Greg J. Bashaw. Altenhein, Gerhard Technau. Institute of Genetics, Department of Neuroscience, University of Pennsylvania, JohannesGutenberg University,Mainz, Germany. Philadelphia, PA. 563B 554B Cultured Primary Mushroom Body Neurons Reveal a Notch- Helmsman is Expressed in Trachea and Retina: inactivation alters Deficient Phenotype. Randy Boyles1, Kate Shen1, Robert tracheal morphology and visually guided behavior. John Kraft2, Linda L. Restifo2, Andrew Andres1. 1) School of Life Pollock1, James McKay2, Barbara Nightingale1. 1) Dept Sciences, Univ Nevada - Las Vegas, Las Vegas, NV; 2) Arizona Biological Sci, Duquesne Univ, Pittsburgh, PA; 2) Department Research Laboratories, Division of Neurobiology, University of of Molecular Biology, University of Texas Southwestern Medical Arizona, Tucson, Arizona 85721. Center Dallas, TX. 564C 555C Heterochronic microRNAs are required for appropriate cell Remodeling the larval motor system during metamorphosis: the division control and neuromuscular junction formation during fate of motor neuron subsets.Soumya Banerjee, Holly Drosophila metamorphosis. Elizabeth E. Caygill, Laura A. Rataiczak, Meredith Dorr, Badrinath Krishan, Joyce Johnston. Department of Genetics & Development, College of Fernandes. Zoology, Miami University, Oxford, OH. Physicians & Surgeons, Columbia University, New York, NY. POSTER SESSIONS 61 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

565A 574A The role of Crk-associate substrate (Cas) in neural development. Microarray analysis of sensory neurogenesis in the embryo using Guang-Chao Chen1, Ai-Pei Chi2. 1) Inst Biological Chemistry, FACS. Andrew Jarman, Sebastián Cachero, Petra zur Lage, Academia Sinica, Taipei, Taiwan; 2) Graduate Institute of Ian Simpson, Lina Ma, Fay Newton, Douglas Armstrong, Molecular and Cellular Biology, NationalTaiwan University,Taipei, Lynn Powell, Sadie Kemp. Centre for Integrative Physiology, Taiwan. University of Edinburgh,Edinburgh, United Kingdom.

566B 575B Characterization of aaquetzalli (aqz), a gene required for Insights into the mechanism controlling stochastic spineless development of the nervous system during D. melanogaster expression required for the color vision retinal mosaic. Robert embryogenesis. Miguel Mendoza-Ortiz, Juan Riesgo-Escovar. Johnston, Claude Desplan. Dept Biol, New York Univ, New Dept Developmental Biol, Inst Neurobiologia, UNAM, Queretaro, York, NY. Qro, Mexico. 576C 567C Analysis of a CK2 phosphorylation-specific variant of E(spl)M8 Interstitial branching at glial boundaries determines the leads to a reinterpretation of the mechanism of E(spl)D. Bhaskar organization of the adult Drosophila brain. Shana R. Spindler, Kahali, Anasua Bose, Clifton Bishop, Ashok Bidwai. Dept Wayne Pereanu, David Nguyen, Volker Hartenstein. Biol, West Virginia Univ, Morgantown, WV. Molecular, Cell, Developmental Biology, University of California, Los Angeles, Los Angeles, CA. 577A Anteroposterior control of stem cell identity: specification of 568A neuroblast 5-6 by overlapping action of Hox factors and Hox co- JAK/STAT signal regulates proneural wave progression in the factors. Daniel Karlsson, Magnus Baumgardt, Stefan Thor. optic lobe development. Tetsuo Yasugi, Daiki Umetsu, Satoshi Dept. of Clinical and Experimental Medicine, Linköping Murakami, Makoto Sato, Tetsuya Tabata. Lab Morphogenesis, University, Linköping, Sweden. IMCB, Univ Tokyo, Tokyo, Japan. 578B 569B The C-terminal domain (CtD) of Enhancer of split M8, regulates Understanding the function of Sanpodo during asymmetric cell repression in a CK2 phosphorylation-dependent manner. Jee- divisions in the Drosophila CNS.Burcu Babaoglan, Hemi Eun Kim, Bhaskar Kahali, Umesh Karandikar, Clifton Bishop, Mistry, Kate O'Connor, Adam Schickedanz, Beth Wilson, Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. James Skeath. Dept Genetics, Washington Univ. in St Louis,St Louis, MO. 579C Characterization of Echinoid and Friend of Echinoid Binding. 570C Woongki Kim, Susan Spencer. Dept Biol, St Louis Univ, St extramacrochaetae (emc), a Notch target gene, is required for Louis, MO. R7 and cone cell development. Abhishek Bhattacharya, Nicholas E. Baker. Dept Molecular Genetics, AECOM, Bronx, 580A NY. Potential Antagonistic Roles of CK2 and PP2A in Notch Signaling in Drosophila. Ezgi Kunttas, Anasua Bose, Clifton Bishop, 571A Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. Analysis of the interactions between CK2, PP2A and E(spl) during neurogenesis. Anasua Bose, Ezgi Kunttas-Tatli, 581B Bhaskar Kahali, Clifton Bishop, Ashok Bidwai. Dept Biol, West Dbx represses Eve in the ventral nerve cord of Drosophila Virginia Univ, Morgantown, WV. embryos. Haluk Lacin1, Yi Zhu1, Beth Wilson1, Heather Broihier2, James Skeath1. 1) Dept. Genetics, Washington Univ, 572B St Louis, St Louis, MO; 2) Dept. of Neurosciences Case Western Expression levels of the ontrols neural vs support cell fate Reserve University, Cleveland, OH. determination in the Drosophila eye. Mark A. Charlton-Perkins, S. Leigh Whitaker, Tiffany Cook. Developmental Biology/ 582C Pediatric Opthamology, Cincinnati Children's Hospital Research Functional studies of Optix in Drosophila. Yumei Li1,2, Kristi Foundation, Cincinnati, OH. Hoffman2, Abanti Chattopadhyay2, Umesh Karandikar3, Keqing Wang1, Graeme Mardon2,3, Rui Chen1,2. 1) HGSC, 573C Baylor College of Medicine, Houston, TX; 2) Dept Molecular & Genome wide dissection of eyeless function during retinal cell Human Gen, Baylor College of Medicine, Houston, TX; 3) fate determination in Drosophila. Rui Chen1,2, Yumei Li1, Jianlan Department of Pathology, Baylor College of Medicine, Houston, Peng1, Erin Haase1, Richard Gibbs1, Graeme Mardon1,2,3. 1) TX. HGSC, Molec & Human Genetics, Baylor Col Medicine, Houston, TX; 2) Program in Developmental Biology, Baylor College of 583A Medicine, Houston, TX; 3) Department of Pathology, Baylor The Notch ligands Delta and Serrate are necessary in the R1 College of Medicine, Houston, TX. and the R6 photoreceptors to prevent R7 fate via autonomous cis-inhibition of Notch activation.Adam Miller, Tory Herman. Institute of Molecular Biology, University of Oregon,Eugene, OR. 62 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

584B 593B Identifying interactors of Sanpodo essential for Notch-mediated A mosaic screen for genes required for postembryonic neural asymmetric divisions in Drosophila nervous system. Rajalaxmi proliferation. Jennifer Grant, Cedric Maurange, Louise Cheng, Natarajan, James Skeath. Dept of Genetics, Washington Julia Pendred, Alex Gould. Developmental Neurobiology, University School of Medicine, St. Louis, MO. NIMR, London, London, United Kingdom.

585C 594C Examining Echinoid’s Role in the Endocytosis of EGFR, Notch, Characterization of the torp4a gene, a Drosophila homolog of and Delta. Grant Simmons, Susan Spencer. Saint Louis human DYT1 (Torsin A) associated with early-onset dystonia. University, Saint Louis, MO. Noriko Wakabayashi-Ito1, Minoru Yamanishi2, Hideaki Moriyama3, James F. Gusella1, Naoto Ito1. 1) Center for Human 586A Genetic Research, Massachusetts General Hospital, Boston, Changing sensory specificity in functional terminally MA; 2) School of Biological Science; 3) Department of Chemistry, differentiated photoreceptors by switching Rhodopsin expression. University of Nebraska-Lincoln, Lincoln, NE. Simon Sprecher, Claude Desplan. Dept Biol, New York Univ, New York, NY. 595A A role for Hox genes in regulating apoptosis during Drosophila 587B embryonic central nervous system development. Ana Rogulja- A Genetic Screen For Genes Controlling Tv Neuron Identity And Ortmann, Gerd M. Technau. Institute of Genetics, University of FMRFamide Expression.Carina Ulvklo, Patrik Nilsson, Anna Mainz, Mainz, Germany. Angel, Fredrik Fransson, Stefan Thor. Molecular Genetics, Clinical and Experimental Medicine, Linköping, Sweden. 596B Characterization of Aedes aegypti rhodopsins in transgenic 588C Drosophila. James H. England, Joseph Real, Xiaobang Hu, Senseless functions as a molecular switch for color Zachary Lemmon, Aaron Lani, Jennifer Tung, Michelle A. photoreceptor differentiation in Drosohila.Baotong Xie, Mark Whaley, Joseph E. O'Tousa. Biological Sciences, University of Charlton-Perkins, Elizabeth McDonald, Brian Gebelein, Notre Dame, Notre Dame, IN. Tiffany Cook. Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children’s Hospital Medical 597C Center, Cincinnati, OH. Gene-expression profiling of sensory organ precursor (SOP) cells from wing and leg imaginal discs. Mariano A. Loza Coll, 589A James W. Posakony. Division of Biological Sciences, University Drosophila dCtBP is required for adult peripheral nervous system of California at San Diego, La Jolla, CA. development and sharpens a proneural transcriptional activity of Pnr. Inna Biryukova, Pascal Heitzler. Institut de Génétique 598A et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Retinophilin (CG10233) localizes to rhabdomeres of Drosophila Strasbourg, BP 10142. photoreceptor cells. Kirk L. Mecklenburg1, Benjamin Currie2, Joseph E. O'Tousa2. 1) Department of Biology, Indiana 590B University South Bend, South Bend, IN, 46634; 2) Department Understanding the function of twin of eyegone (toe) in eye of Biological Sciences, University of Notre Dame, Notre Dame, development in D. melanogaster.Abanti Chattopadhyay1, Indiana 46556. Abuduaini Abulimiti3, Keqing Wang3, Rui Chen1,2,3. 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) 599B Program in Developmental Biology, Baylor Col Medicine, Degradation of proneural proteins controls the timing of neural Houston, TX; 3) Human Genome Sequencing Center, Baylor precursor division. Hai-Wei Pi1, Pao-Ju Chang1, Yun-Ling Col Medicine, Houston, TX. Hsiao1, An-Chi Tien2, Yi-Ju Li1. 1) Department of Life Science, Chang-Gung University, Tao-Yuan, Taiwan; 2) Program in 591C Developmental Biology, Baylor College of Medicine, One Baylor Genetic interaction of dorsoventral patterning genes during Plaza, Houston, Texas 77030. embryonic development of the Drosophila brain. Janina Seibert, Dagmar Volland, Gerhard Technau, Rolf Urbach. Institute of 600C Genetics, JohannesGutenberg University,Mainz, Germany. Feedback from Rhodopsin 6 protein is required to maintain pR8 identity through inhibition of Rh5 expression. Daniel 592A Vasiliauskas1, Esteban O. Mazzoni2, Claude Desplan1. 1) Over-expression of the Notch Intracellaur Domain in Neural Cells Department of Biology, New York University, New York, NY; 2) Potentiates Long-Term Survival of Drosophila in Hypoxia. Pathology Department, Columbia University, New York, NY. DeeAnn W. Visk1, Dan Zhou2, Gabriel Haddad2. 1) Biology, University of California, San Diego, La Jolla, CA; 2) School of 601A Medicine, University of California, San Diego, La Jolla, CA. Identification of cofactors that participate in Orthodenticle- dependent development of the retinal mosaic. Michael Workman, Elizabeth McDonald, Tiffany Cook. Developmental Biology/Pediatric Opthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH. POSTER SESSIONS 63 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

602B 610A Drosophila EHBP-1 is Required for Notch Signaling during The role of Rac GTPase activity in synaptic structural and External Sensory Organ Development. Shinya Yamamoto1, functional plasticity at the Drosophila neuromuscular junction. Nikolaos Giagtzoglou2, Hillary K. Andrews1, Hao Wang3, Maude Warren-Paquin, Kazuya Tsurudome, Pejmun Karen L. Schulze2, Hugo J. Bellen1,2,4. 1) Program in Haghighi. Department of Physiology, McGill University, Montreal, Developmental Biology; 2) Howard Hughes Medical Institute; 3) Canada. Summer Science for Seniors Program; 4) Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX. Neurophysiology and Behavior 611B 603C Involvement of the circadian clock in xenobiotic responses of D. The role of cato, an atonal-related gene, during Peripheral melanogaster. Shawn M. Butcher1, Henry Priest2, Louisa Nervous System development. Petra zur Lage, Andrew Hooven1, Todd Mockler2, Jaga Giebultowicz1. 1) Zoology, Jarman. SBMS, University of Edinburgh, Edinburgh, United Oregon State University, Corvallis, OR; 2) Botany and Plant Kingdom. Pathology, Oregon State University,Corvallis, OR. 604A 612C Fragile X protein functions in neural stem cells. Matthew A. What's Lov got to do with it? A role for Jim Lovell in Drosophila Callan, Daniela C. Zarnescu. Molecular and Cellular Biology, courtship. Sonia Bjorum, Kate Beckingham. Dept Biochem & University of Arizona, Tucson, AZ. Cell Biol, Rice Univ, Houston, TX. 605B 613A The Tumor Suppressor Gene insensitive Encodes a Nuclear Alogjam in the neural circuits controlling oviposition behavior. Protein That Controls Asymmetric Cell Division by Regulating Ginger Carney, Kara Boltz, Stephanie Grady. Dept of Biology, Expression of lethal (2) giant larvae. Jamian D. Reed, Nick Texas A&M Univ, College Station, TX. Reeves, James Posakony. Division of Biological Sciences, Section of Cell and Developmental Biology, UC San Diego, La 614B Jolla, CA, 92093. Fitting it all together: how the courtship- and mating-responsive fit gene affects male reproductive behaviors. Lisa L. Ellis, Ginger 606C E. Carney. Dept Biol, Texas A&M Univ, College Station, TX. Falafel is a novel regulator of asymmetric segregation of the Miranda complex in Drosophila neuroblasts. Rita Sousa- 615C Nunes1,2, William Chia1,2, W. Gregory Somers1,2. 1) MRC Centre Takeout family members play a role in courtship in non-neuronal for Developmental Neurobiology, King's College London, New tissues. Valbona Hoxha1, Hyeeun Kim1, Paul E. Hardin2, Gregg Hunt's House, Guy's Campus, London SE1 1UL, UK; 2) Temasek Roman1, Brigitte Dauwalder1. 1) Department of Biology and Lifesciences Laboratory, 1 Research Link, National University Biochemistry, University of Houston, Houston,TX; 2) Department of Singapore, 117604 Singapore. of Biology, Texas A&MUniversity, College Station, TX. 607A 616A Co-regulators of programmed cell death in neuroblasts. Wei Non-transitive sex and violence between genotypes in D. Tang1, Megumu Mabuchi1, Susan Pierre2, Reena Patel1, Barret melanogaster. Sergey V. Nuzhdin, Larry Cabral, Brad Foley. Pfeiffer3, Kristin White1. 1) Cutaneous Biology Research Center, Molecular Computation Biology, University of Southern Massachusetts General Hospital, Charlestown, MA; 2) Flybase, California, Los Angeles, CA. Harvard University, Cambridge MA; 3) Janelia Farm Research Campus, HHMI, Ashburn, VA. 617B Social decision-making in D. melanogaster. Julia Saltz. 608B Population Biology Graduate Group, UC Davis, Davis, CA. Dfezl encodes a novel regulator of neural stem cell self-renewal in Drosophila. Mo Weng1,2, Shufen Situ2, Caitlin Gamble5, 618C Cheng-Yu Lee1,2,3,4. 1) Department of Cell and Developmental An ecdysone signal lost: How is loss of EcR influencing adult Biology, University of Michigan, Ann Arbor, MI; 2) Life Science behavior? Christoph C. Schwedes, Ginger E. Carney. Biology, Institute, University of Michigan, Ann Arbor, MI; 3) Molecular TexasA&M University , College Station, TX. Medicine and Genetics, Unversity of Michigan, Ann Arbor; 4) Center for Stem Cell Biology, Unviersity of Michigan, Ann Arbor; 619A 5) Department of Biology, Univeristy of Oregon, Eugene, OR. Neuronal Mechanisms of anesthetic tolerance. Yazan M. Al- Hasan, Harish Krishnan, Alfredo Ghezzi, Yan Wang, Nigel 609C Atkinson. Neurobiology, Neuroscience, Austin, TX. RhoGAP100F is Required for R7 Photoreceptor Axon Targeting. Scott Holbrook, Tory Herman. Institute of Molecular Biology, 620B University of Oregon, Eugene, OR 97403. An Epistatic Analysis between Catecholamines up and a- synuclein in Drosophila. Faiza Ferdousy, Hakeem Lawal, Carrie Williams, Janis O'Donnell. Department of Biological Sciences, Univ Alabama, Tuscaloosa, AL 35401. 64 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

621C 629B Site-directed mutagenesis of the Drosophila NMDA-R1 using Second-Order Learning in D. melanogaster. Christopher homologous recombination. Moon Draper, Mariah Dedrick, Tabone, J. Steven de Belle. School of Life Sciences, University Adron Harris, Nigel Atkinson. Dept Neurobiology C0920, Univ of Nevada, Las Vegas, Las Vegas, NV. Texas, Austin, TX. 630C 622A Induction of the Stress Response Mitigates Heat Disruption of straightjacket, a Drosophila calcium channel a2d subunit, is Mushroom Body Development and Gene Expression in D. required for the proper localization of synaptic voltage gated melanogaster. Xia Wang1, Lisa L. Strobel2, J. Steven de Belle1, 1 2,3 1 calcium channel a1 subunits. Cindy Ly , Chi-Kuang Yao , Patrik Stephen P. Roberts . 1) School of Life Sciences, University of Verstreken2,3,5, Tomoko Ohyama2, Hugo Bellen1,2,3,4. 1) Nevada, Las Vegas, Las Vegas, NV; 2) Lehrstuhl für Genetik Department of Neuroscience, Baylor College of Medicine, und Neurobiologie, Universität Würzburg, Am Hubland, Houston, TX; 2) Department of Molecular and Human Genetics, Würzburg, Germany. Baylor College of Medicine, Houston, TX; 3) Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX; 4) 631A Program in Developmental Biology, Baylor College of Medicine, Creating Anastasia Yemelyanova, Christopher J. Jones. Houston, TX; 5) VIB Department of Developmental Genetics, Department of Biological Sciences, Moravian College, K.U. Leuven Center for Human Genetics, Leuven, Belgium. Bethlehem, PA.

623B 632B The development of a semi-automated phototaxis assay to PKA activity differentially regulates multiple processes required identify genes responsible for ethanol tolerance. Kapil V. for wing expansion in bursicon-secreting neurons. Fengqiu Diao, Ramachandran, Rosie B. Ramazani, Nigel S. Atkinson. Nathan C. Peabody, Benjamin H. White. Laboratory of Section of Neurobiology, University of Texas at Austin, Austin, Molecular Biology, NIMH/NIH, Bethesda, MD. TX. 633C 624C Neprilysin 4: An essential endopeptidase is expressed in eve- Elucidating the role of the evolutionarily conserved elements in positive pericardial cells and in the CNS of D. melanogaster. the promoter region of the BK-type Ca2+-activated K+ channel, Mareike Panz, Eva Hab-Cordes, Achim Paululat, Heiko Meyer. slowpoke, in the induction of tolerance to alcohol. Maureen R. University of Osnabrueck, Department of Biology, Section of Scholl, Xiaolei Li, Nigel S. Atkinson. Section of Neurobiology, Zoology. University of Texas at Austin, Austin, TX. 634A 625A Genetic analysis of the proprotein convertase amontillado JAK/STAT signaling is required for long term memory in (amon): amon is required for growth control and glucose Drosophila. Tijana Copf, Thomas Preat. Genes et Dynamique homeostasis. Jeanne Rhea1, Lowell Rayburn1, Christian des Systemes de Memoire, CNRS-ESPCI, Paris, France. Wegener2, Michael Bender1. 1) Dept Genetics, Univ Georgia, Athens, GA; 2) Department of Biology, PhillipsUniversity, 626B Marburg, Germany. Impact of reduced numbers of Kenyon cell classes on odor memory in Drosophila. Brian Dunkelberger, Christine Serway, 635B

Nicole Nolan, J. Steven de Belle. University of Nevada, Las The 5-HT7Dro Serotonin Receptor: Expression in the CNS and Vegas, School of Life Sciences, Las Vegas, NV. Function. Jaime Becnel, Oralee Johnson, Charles D. Nichols. Department of Pharmacology and Experimental Therapeutics, 627C LSU Health Sciences Center, New Orleans, LA. Distinctive Neuronal Networks and Biochemical Pathways in Appetitive and Aversive Memory in Drosophila Larvae. Ken 636C Honjo, Asami Tanaka, Katsuo Furukubo-Tokunaga. Graduate Fly Tracker as a novel system for analyzing movement behaviors School of Life and Enviromental Sciences, University of Tsukuba, in D. melanogaster. David J. Moore, Young-Cho Kim, Jeong- Tsukuba, Japan. Hye Min, Kyung-An Han. The Huck Institutes Neuroscience Graduate Program, The Pennsylvania State University, University 628A Park, PA. A critical component of the nuclear pore complex is implicated in learning and mushroom body development in Drosophila; 637A mushroom body miniature B is Pendulin, the Drosphila Importin Mutations of the Drosophila Vesicular Monoamine Transporter alpha 2. Christine N. Serway1, Nicole W. C. Nolan1,2, Stephanie decrease larval locomotion and the adult response to cocaine. Freer1,3, J. Steven de Belle1,4. 1) School of Life Sciences, Anne Simon1, Richard Daniels2, Rafael Romero-Calderon1, University of Nevada, Las Vegas, Las Vegas, NV; 2) Creighton Grygoruk Anna1, Wu Mark3, Amita Sehgal3, Larry Ackerson1, University Medical School, Omaha, NE; 3) The University of Nigel Maidment1, Aaron DiAntonio2, David E. Krantz1. 1) North Carolina at Chapel Hill, Chapel Hill, NC; 4) The National Semel Institute for Neurosciences and Human Behavior, UCLA, Science Foundation, Arlington, VA. Los Angeles, CA; 2) Dept of Mol. Biol. and Pharma., Washington University, School of Medicine, St. Louis, Mo; 3) Howard Hughes Medical Institute, Dept of Neurosc., School of Medicine, University of Pennsylvania, Philadelphia, PA. POSTER SESSIONS 65 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

638B 649A Zinc and Dopamine: A functional relationship? O'Neil Wright, Characterization of Drosophila bitter taste receptor. Seok Jun Janis O'Donnell. Dept Biological Sci, Univ Alabama, Moon, Craig Montell. Department of Biological Chemistry The Tuscaloosa, AL. JohnsHopkins University,Baltimore, MD.

639C 650B The effect of individual Acps on sperm competition and uterine Atypical soluble guanylyl cyclases in Drosophila are expressed muscle contraction. Frank Avila. Dept. of Molecular Biology and in taste chemosensilla and are involved in taste preference Genetics, CornellUniversity, Ithaca, NY. behaviors. Anke Vermehren, Judith Stewart, Wendy Timmermans, David B. Morton. Integrative Biosciences, 640A OHSU, Portland, OR. A Cluster of Autonomic Neurons regulates peristolsis Drosophila larval midgut. Dennis LaJeunesse, Brooke Johnson. Department 651C of Biology, University of North Carolina, Greensboro, NC. Bitter taste in Drosophila. Linnea A. Weiss, Anupama Dahanukar, Jae Young Kwon, John R. Carlson. Molec., Cell. 641B & Dev. Biology, Yale University, New Haven, CT. Dissecting patterns of aggressive behavior established by fruitless. Steven Nilsen1, Dylan Nelson2. 1) Biology, Oxford 652A College of Emory University, Oxford, GA; 2) Neurobiology, Phosphatidyl inositol 4,5 bisphosphate signaling determines Harvard Medical School, Boston, MA. synaptic morphology of the Drosophila neuromuscular junction. Ron L. P. Habets1,2, Thang M. Khuong1,2, Patrik Verstreken1,2. 642C 1) Laboratory of Neuronal Communication, K.U. Leuven, Center Drosophila CG16801/fdx modulates eclosion, wing expansion for Human Genetics, Belgium; 2) VIB, Department of Molecular behaviors, and possibly fertility. Steven Robinow, Qing Chang, and Developmental Genetics, Belgium. Laura Wong, Elizabeth Nguyen, Nelson Lazaga, Carl Sung. Dept Zoology, Univ Hawaii,Honolulu, HI. Evolution and Quantitative Genetics 643A 653B Development of Assays Used to Study Tolerance to Drugs of Variability of the Dras1 gene in D. virilis sibling species. Anna Abuse in Drosophila. Nyssa A. Sherazee, Jascha B. Pohl, Chekunova1, Vyacheslav Sergienko1, Helen Zelentsova2, Kevin Bieri, Tanzeen Yusuff, Nigel S. Atkinson. Department Larisa Gauze1, George Bakhtojarov1, Alex Kulikov1, Vladimir of Neurobiology, University of Texas at Austin. Mitrofanov1. 1) Dept Genetics, Koltsov Institute of Developmental Biology RAS, Moscow, Russian Federation; 2) 644B Dept of Molecular Mechanisms of Biological Adaptation, Genetic mapping of the bas mutation in Drosophila. Simon Engelhardt Institute of Molecular Biology Russian Academy of Tabchi, Nathaniel Tussey, Christopher J. Jones. Department Sciences, Moscow, Russian Federation. of Biological Sciences, Moravian College,Bethlehem, PA. 654C 645C Testing the functional consequences of adaptive protein evolution Mapping the bss mutation in Drosophila. Nathaniel Tussey, at the bag of marbles gene for Drosophila germline stem cell Simon Tabchi, Christopher J. Jones. Department of Biological differentiation. Heather A. Flores, Charles F. Aquadro, Daniel Sciences, Moravian College,Bethlehem, PA. A. Barbash. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY. 646A Pain sensitization induced by tissue damage in Drosophila larvae. 655A Daniel T. Babcock1, Christian Landry2, Michael J. Galko1. 1) Evolution of Hox gene expression and function and the effect on Department of Biochemistry & Molecular Biology, University of limb specification in arthropods. Cheryl Hsia1, Adam Paré1, Texas MD Anderson Cancer Center, Houston, TX; 2) ProDev Matthew Ronshaugen2, William McGinnis1. 1) Division of Engineering, Houston, TX. Biology, University of California, San Diego, La Jolla, CA; 2) Faculty of Life Sciences, University of Manchester, Manchester, 647B UK. aguesic is a gustatory-related DEG/ENaC ion channel. Yehuda Ben-Shahar1, Michael Welsh1,2. 1) HHMI; 2) Internal Medicine, 656B University of Iowa College of Medicine, Iowa City, IA. Distinct modifications of EGF receptor signaling in homoplastic evolution of eggshell morphology in genus Drosophila. Tatsuo 648C Kagesawa, Yukio Nakamura, Minori Nishikawa, Kenji john glenn (jog), a gene implicated in gravitaxis, may be involved Matsuno. Dept. Biol. Sci. / Tec., Tokyo Univ. Sci., Japan. in axon outgrowth and function downstream of Drosophila APP (APPL). Cassidy B. Johnson1, Vanaja Konduri1, Sven 657C Huelsmann2, Nick Brown2, Kathleen M. Beckingham1. 1) Dept. Transkingdom sex: viral mediated cytoplasmic incompatibility in Biochemistry & Cell Biol, Rice Univ, Houston, TX; 2) Gurdon the Wolbachia/Drosophila symbiosis system. Timothy Karr, Ben Institute and Dept. of Physiology, Development and Heath. Dept Biol & Biochemistry, Univ Bath, Bath, United Neuroscience, University of Cambridge, UK. Kingdom. 66 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

658A 667A Various evolutionary rates in the Drosophila virilis species group. Measuring spontaneous mutation rate at locus dumpy in D. Alex Kulikov, Oleg Lazebny, Vladimir Mitrofanov. Dept melanogaster. Olga G. Grushko1, Christopher Duchesneau1, Genetics, Koltsov Institute of Developmental Biology, RAS, Amber Carmon2, Ross J. MacIntyre2, Alexey S. Kondrashov1. Moscow, Russian Federation. 1) Life Sciences Institute, University of Michigan, Ann Arbor, MI; 2) Department of Molecular Biology and Genetics, Cornell 659B University, Ithaca, NY. Interspecies variability and genetic analysis of phallus hairiness in sibling species of the Drosophila virilis species group. Alex 668B Kulikov, Nick Gornostaev, Oleg Lazebny, Vladimir Mitrofanov. Effect of X-linkage on rates of evolution of sex-biased genes in Dept Genetics, Koltsov Institute of Developmental Biology, RAS, Drosophila. Tatiana Gurbich, Doris Bachtrog. Division of Biological Moscow, Russian Federation. Sciences, University of CaliforniaSan Diego, La Jolla, CA.

660C 669C Evolution of head development and axis specification in higher Molecular Evolution of Glutathione S-Transferases in the Genus, flies. Steffen Lemke, Matteen Rafiqi, Urs Schmidt-Ott. Drosophila. W. Y. Low1, H. L. Ng2, C. J. Morton2, M. W. Parker2, Department of Organismal Biology and Anatomy, University of P. Batterham1, C. Robin1. 1) Bio21 Molecular Science and Chicago, Chicago, IL. Biotechnology Institute, University of Melbourne, Vic 3010, Australia; 2) Biota Structural Biology Laboratory and the ACRF 661A Rational Drug Discovery Facility, St. Vincent's Institute of Medical Genetic analysis of between-species differences in embryonic Research, Melbourne, Vic 3065, Australia. pattern formation. Susan E. Lott1, Michael Z. Ludwig2, Martin Kreitman1,2. 1) Committee on Genetics, Univ Chicago, 670A Chicago,IL; 2) Department of Ecology and Evolution, Univ Radiation sensitivity and progeny production in D. melanogaster Chicago, Chicago, IL. under selective pressure. Lucy McNamara, John Jenkins, Nicole Boyle, Valerie Vassor. Biology Department, Swarthmore 662B College, Swarthmore, PA. Components of protein complexes linked to cognition are conserved and co-expressed in the Drosophila brain. Bilal R. 671B Malik1, Andrew J. Pocklington1, Alex Bayes2, Richard Emes3, Recurrent recruitment of retrogenes involved in nuclear transport. Seth G. N. Grant2, J. Douglas Armstrong1. 1) ANC, School of Mansi Motiwale, Charles Tracy, Xavier Río, Esther Betrán. Informatics, University of Edinburgh; 2) Wellcome Trust Sanger Biology, University of Texas at Arlington,Arlington, TX. Institute, Hinxton, Cambridge, CB10 1SA, UK; 3) Institute for Science and Technology in Medicine, School of Medicine, Keele 672C University, Staffordshire ST5 5BG, United Kingdom. Selective Constraints Suggest Most of Drosophila Genome Comprised of Non-coding RNAs and cis-Regulatory Elements. 663C Daniel Pollard1, Daniel Halligan2, Casey Bergman3, Peter Cell number and distribution in cycle 14 Drosophila embryos Keightley2, Michael Eisen1. 1) Dept Genome Sci, LBNL, selected for large and small egg size. Cecelia Miles, Martin Berkeley, CA; 2) Inst of Evol Bio, Univ of Edinburgh, UK; 3) Life Kreitman, Michael Ludwig. Dept Ecology & Evolution, Sci, Univ of Manchester, UK. University ofChicago, Chicago, IL. 673A 664A Evolution of genic content in the innate immune system of Comparative genomics reveals that the TGF-beta and Wnt Drosophila. Timothy Sackton1,2, Andrew Clark2. 1) Field of signaling pathways have distinct patterns of developmental Ecology & Evolutionary Biol, Cornell Univ, Ithaca, NY; 2) Dept evolution. Stuart Newfeld1, Charlotte Konikoff1, Michael of Molecular Biology and Genetics, Cornell Univ, Ithaca, NY. Stinchfield1, Sudhir Kumar1, Robert Wisotzkey2. 1) Sch Life Sci, Arizona State Univ, Tempe, AZ; 2) Dept. Biol, California State 674B Univ East Bay, Hayward, CA. Genomic Rearrangements Inferred from Gene Order Data Collected from Ten Species of Drosophila. Stephen Schaeffer1, 665B Arjun Bhutkar2,3, Mu Xu1, Susan Russo2,4, Temple Smith3, How different are the mitochondria during spermatogenesis? William Gelbart2,4. 1) Dept Biol, Pennsylvania State Univ, Chitra Chandrasekaran1, Esther Betrán2. 1) Biology, Texas University Park, PA; 2) Dept Mol and Cell Biol, Harvard Univ, Wesleyan University, Fort Worth, TX; 2) Biology, University of Cambridge, MA; 3) BioMolecular Engineering Research Center, Texas Arlington, Arlington, TX. Boston Univ, Boston, MA; 4) FlyBase, The Biological Laboratories, Harvard Univ, Cambridge, MA. 666C Mining Odorant Receptor Genes from 12 Drosophila and Other 675C Insect genomes. Seong-il Eyun1, Stephen O. Opiyo1, Pooja Contrasting features of sex and autosome chromosomal K. Strope1, Etsuko N. Moriyama1,2. 1) School of Biological evolution in malaria mosquitoes. Igor Sharakhov1, Ai Xia1, Maria Sciences; 2) Plant Science Initiative, University of Nebraska- Sharakhova1,2, Zhijian Tu2, Yogesh Shouche3. 1) Department Lincoln, Lincoln, NE 68588. of Entomology, Virginia Tech, Blacksburg, VA; 2) Department of Biochemistry, Virginia Tech, Blacksburg, VA; 3) National Centre for Cell Science, Ganeshkhind, Pune 411 007, India. POSTER SESSIONS 67 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

676A 686B Fine-scale recombination rate estimation in D. melanogaster. Molecular evolution and population genetics of two D. mettleri Nadia D. Singh, Charles F. Aquadro, Andrew G. Clark. cytochrome P450 genes involved in host plant adaptation. Jeremy Molecular Biology and Genetics, Cornell University, Ithaca, NY. M. Bono, Luciano M. Matzkin, Therese A. Markow. Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ. 677B Study of testes-specific proteasome retrogenes. Mehran 687C Sorourian, Esther Betrán. Biology, University of Texas at Natural selection shapes genome wide patterns of copy number Arlington, Arlington, TX. polymorphism in D. melanogaster. Margarida Cardoso- Moreira1,2,3, J. J. Emerson1, Justin O. Borevitz1, Manyuan 678C Long1. 1) Dept Ecology & Evolution, Univ Chicago, Chicago, IL; Demasculinization of X chromosomes in the Drosophila genus. 2) Graduate Program in Areas of Basic and Applied Biology, David Sturgill1, Yu Zhang1, Michael Parisi2, Brian Oliver1. 1) Universidade do Porto, Portugal; 3) Faculdade de Ciências, Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Universidade do Porto, Portugal. Bethesda,MD; 2) Department of Biology, University of Pennsylvania, Philadelphia, PA. 688A Reduced nucleotide diversity on neo-Y chromosome of D. 679A albomicans. Hsiao-Han Chang1, Chau-Ti Ting2, Hwei-yu Chromosome movement of retrogenes and X inactivation during Chang3. 1) Institute of Biotechnology, National Tsing Hua Drosophila spermatogenesis. Maria D. Vibranovski1, Hedibert University, Hsinchu, Taiwan 300, ROC; 2) Department of Life F. Lopes2, Timothy L. Karr3, Manyuan Long1. 1) Department Science, Institute of Ecology and Evolutionary Biology, & Institute of Ecology and Evolution, The University of Chicago, Chicago, of Zoology, National Taiwan University, Taipei, Taiwan 106, ROC; IL; 2) Graduated School of Business, The University of Chicago, 3) Department of Entomology, National Taiwan University, Taipei, Chicago, IL; 3) Department of Biology and Biochemistry, Taiwan 106, ROC. University of Bath, Bath, UK. 689B 680B Evolution of a Female Reproductive Protease Gene Family in Constraint and turnover in sex-biased gene expression in the Cactophilic Drosophila. Erin Kelleher, Therese Markow. Dept genus Drosophila. Yu Zhang1, David Sturgill1, Michael Parisi1,3, EEB, Univ Arizona, Tucson, AZ. Sudhir Kumar2, Brian Oliver1. 1) LCDB/NIDDK/NIH, Bethesda, MD; 2) Center for Evolutionary Functional Genomics, Biodesign 690C Institute, Arizona State University, Tempe AZ; 3) Department of Factors impacting Y chromosome evolution in D. melanogaster. Biology, University of Pennsylvania, PA. Amanda Larracuente, Andrew Clark. Dept Molecular Biol & Genetics, Cornell University, Ithaca, NY. 681C Bacteria associated with natural populations of the cactophilic 691A species D. aldrichi and D. arizonae. Vanessa Corby-Harris, Jorj Population genetics in the Drosophila simulans sibling, D. Wagner, Therese A. Markow. Dept. of Ecology and Evolution, sechellia. New insights into its evolutionary history based on University of Arizona, Tucson, AZ. selected and neutral polymorphisms. Delphine Legrand, Da Lage Jean-Luc, Lachaise Daniel, Cariou Marie-Louise. 682A Laboratoire Evolution Génomes et Spéciation, CNRS, Gif-sur- Post-mating gene expression in tissues of the lower female Yvette, France. reproductive tract in D. pseudoobscura. Dean A. Croshaw, Dalziel Dominguez, Carlos A. Machado.University of 692B Arizona,Tucson, AZ. Molecular variation in the Lim3 locus controlling neuron development is associated with D. melanogaster lifespan. Olga 683B Y. Rybina, Elena Pasyukova. Institute of Molecular Genetics Identification and expression analysis of putative mRNA-like non- of RAS, Moscow, Russian Federation. coding RNAs (mlncRNAs) in D. pseudoobscura . Zifeng Jiang, Carlos Machado. Department of Ecology and Evolutionary 693C Biology, University of Arizona, Tucson, AZ. Detecting selective sweeps using Hidden Markov Models on sequence data. Christian Schloetterer1, Simon Boitard2, 684C Andreas Futschik2. 1) Inst Tierzucht, VMU Wien, Wien, Austria; Evolutionary relationships, timescales, and selective pressures 2) Dept. of Statistics, University of Vienna. in the 12 fruit fly genomes.Sudhir Kumar1,2, Sonja Prohaska1,3, Alan Filipski1,2. 1) Center for Evolutionary Functional Genomics, 694A Biodesign Institute, Arizona State Univ, Tempe, AZ; 2) School of Understanding the effects of cis-polymorphisms on gene Life Sciences, Arizona State Univ, Tempe, AZ; 3) Department of expression variation. Aaron Tarone1, Matthew Hahn2, Anne Biomedical Informatics, Arizona State Univ, Tempe, AZ. Genissel3, Joseph Dunham1, Sergey Nuzhdin1. 1) Molecular and Computational Biology Program, University of Southern 685A California, Los Angeles, CA; 2) Department of Biology and Population size is not a major determinant of rates of protein School of Informatics,Indiana University, Bloomington, IN; 3) adaptation in Drosophila. Doris Bachtrog. Div Biological Sci, Section of Evolution and Ecology, University of California at Univ California, San Diego, La Jolla, CA. Davis, Davis, CA. 68 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

695B 705C Genomic analysis of local adaptation in D. melanogaster. Age-Specific Variation in Immune Response and its Correlations Thomas Turner. Center for Population Biol, Univ California, with Lifespan, Reproduction, and Lipid Storage. Adrienne Davis, Davis, CA. Starks, Jeff Leips. Dept Biological Science, UMBC, Baltimore, MD. 696C Developmental and ecological determinants of life-history in D. 706A melanogaster. Alan Bergland, Marc Tatar. Dept Ecology & Investigating the interactions between the hybrid incompatibility Evolution, Brown Univ, Providence, RI. protein LHR and heterochromatic proteins HP1 and HP6. Nicholas Brideau, Daniel A. Barbash. Dept Molecular Biol & 697A Genetics, Cornell Univ, Ithaca, NY. Evolution and development of natural variation in abdominal pigmentation. Ryan D. Bickel1,2, Sergey Nuzhdin1, Artyom 707B Kopp2. 1) Molecular and Computational Biology, University of Functional Studies of the Hybrid Male Sterility Gene, Odysseus Southern California, Los Angeles, CA; 2) Section of Evolution (OdsH), in D. melanogaster. Ya-Jen Cheng1, Niapm H. Patel2, and Ecology, University of California-Davis, Davis, CA. Chau-Ti Ting1,3. 1) Inst. of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC; 2) Dept. 698B of Molecular and Cell Biology, and Dept. of Integrative Biology, Identification of quantitative trait loci function through analysis and HHMI, UC Berkeley, CA; 3) Dept. of Life Science, Inst. of of multiple cuticular hydrocarbons differing between D. simulans Ecology and Evolutionary Biology, and Inst. of Zoology, National and D. sechellia. Jennifer M. Gleason1, R. Andrew James2, Taiwan University, Taipei, Taiwan, ROC. Claude Wicker-Thomas3, Michael G. Ritchie2. 1) Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045; 708C 2) School of Biology, University of St. Andrews, St. Andrews, Symbiotic Bacteria Affect Mating Choice in D. melanogaster. Alex Fife KY16 9TH, Scotland; 3) Laboratoire des Mecanismes de Kulikov1, Alexander Markov2, Oleg Lazebny1, Irina Communication, NAMC UMR-CNRS, Université Paris-XI Sud, Goryacheva3, Maxim Antipin3. 1) Dept Genetics, Koltsov 91405 Orsay, France. Institute of Developmental Biology, RAS, Moscow, Russian Federation; 2) Institute of Paleontology, RAS Moscow, Russian 699C Federation; 3) Vavilov Institute of General Genetics, RAS, Functional Regulatory Divergence of the Innate Immune System Moscow, Russian Federation. in Interspecific Drosophila Hybrids. Erin Hill, Andrew Clark. Department of Molecular Biology & Genetics, Cornell University, 709A Ithaca, NY. Two divergent island populations of the Drosophila simulans sibling, D. mauritiana. The splitting of a species into two? 700A Delphine Legrand, Chenel Thomas, Lachaise Daniel, Cariou Relating among species divergence to mutation and standing Marie-Louise. Laboratoire Evolution Génomes et Spéciation, variation. David Houle, Kim van der Linde. Biological Science, CNRS, Gif-sur-Yvette, France. FloridaState University, Tallahassee, FL. 710B 701B Genome-wide expression studies of phenotypic divergence and The influence of diet on genetically based variation in the cost hybrid dysfunction in species of the D. pseudoobscura group. of reproduction. Mary F. Kaminski, Jeff Leips. Dept Biological Carlos Machado, Zifeng Jiang. Dept Ecology/Evolutionary Biol, Sciences, UMBC, Baltimore, MD. Univ Arizona, Tucson, AZ.

702C 711C Structural equation modeling of pigmentation in Drosophila. High-resolution Genome-wide Dissection of the Two Rules of Brooke A. LaFlamme1, Jason G. Mezey2, ACERT National Speciation in Drosophila. John P. Masly1, Daven C. ESR Center. 1) Molecular Biology and Genetics, Cornell Presgraves2. 1) Department of Molecular and Computational University, Ithaca, NY; 2) Biostatistics and Computational Biology, Biology, University of Southern California, Los Angeles, CA; 2) Cornell University, Ithaca, NY. Department of Biology, University of Rochester, Rochester, NY.

703A 712A Generation of quantitative variation in wing vein position by the Thermotolerance and temperature preference are important Hedgehog and Decapentaplegic signaling pathways. James factors in habitat isolation between D. yakuba and D. santomea. Lorigan, Fangfei Ye, Jason Mezey. Dept Biological Statistics, Daniel Matute, Jerry A. Coyne. Ecology and Evolution, Cornell Univ, Ithaca, NY. University of Chicago, Chicago, IL.

704B 713B Quantitative Trait Loci Affecting the Intraspecific Difference in Intrinsic reproductive isolation barriers between D. yakuba and Male Abdominal Pigmentation in D. malerkotliana. Chen-Siang D. santomea.Daniel R. Matute, Jerry A. Coyne. Ecology and Ng1,2, Andrew Hamilton1, Amanda Frank1, Artyom Kopp1,2. 1) Evolution, University of Chicago,Chicago, IL. Section of Evolution & Ecology, University of California, Davis, CA 95616; 2) Center for Population Biology, University of California, Davis, CA 95616. POSTER SESSIONS 69 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

714C 723C Genomic differentiation between ecologically divergent forms A dual mechanism to promote Pseudomonas aeruginosa of D. melanogaster that coexist in Brazzaville, Congo. Carolyn infectivity: KerB restricts elicitors and enhances suppressors of McBride1, Tom Turner1, Pierre Capy3, Sergey Nuzhdin2. 1) innate immunity. Yiorgos Apidianakis1, Sagi Sapira1, Center for Population Biology, Univ California, Davis, Davis, CA; Guillaume Charriere1, Jianxin He1, Ding Ding An1, Michael 2) Molecular and Computational Biology, Univ Southern Mindrinos2, Regina Baldini1, Laurence Rahme1. 1) California, Los Angeles, CA; 3) Laboratory of Evolution, Massachusetts GeneralHospital, Boston,MA; 2) Genomes, and Speciation, Centre Nacional de la Recherche StanfordUniversity, Palo Alto, CA. Scientifique, Gif-sur-Yvette, France. 724A 715A Characterization of Tep genes during the innate immune Role of polymorphism in putative pheromone binding receptor response Drosophila. Richard Bou Aoun, Nicolas Matt, Jules Gr68b in mate recognition of D. virilis species group. Nikolai Hoffmann, Dominique Ferrandon. UPR9022 IBMC, CNRS, Mugue1, Varvara Vedenina2. 1) Inst Developmental Biology, Strasbourg, Alsace, France. Moscow, Russian Federation; 2) Institute of Information Transmission Problems, Moscow, Russian Federation. 725B Novel virus-like particles from Leptopilina boulardi, a parasite of 716B fruit flies D. melanogaster. Felix Castellanos, Jorge Morales, Role of Gr32a sequence variation in courtship and mate Shubha Govind. Biology Department, The City College of The recognition in D. virilis species group. Nikolai Mugue1, Varvara City University of New York, 138th Street and Convent Avenue, Vedenina2. 1) Inst Developmental Biology, Moscow, Russian New York, NY 10031. Federation; 2) Institute of Information Transmission Problems, Moscow, Russian Federation. 726C "Too much of a good thing": extracellular adenosine effects in 717C flies. Tomas Dolezal, Monika Zuberova, Milena Novakova, Incomplete lineage sorting in the D. simulans clade. Yu-Ping Michaela Fenckova. Faculty of Sciences, University of South Poh1,2, Chau-Ti Ting3, Shun-Chern Tsaur2. 1) Institute of Bohemia,Ceske Budejovice, Czech Republic. Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan; 2) Research Center for Biodiversity, Academia 727A Sinica, Taipei, Taiwan; 3) Department of Life Science, Institute Identification of Rift Valley Fever Virus cellular determinants using of Ecology and Evolutionary Biology, & Institute of Zoology, high throughput screening. Claire Marie Filone1, Robert Doms1, National Taiwan University, Taipei, Taiwan. Sara Cherry1,2. 1) Department of Microbiology, University of Pennsylvania, Philadelphia, PA; 2) Penn Genomics Institute, 718A University of Pennsylvania,Philadelphia, PA. Functional and cytological analysis of the Hybrid male rescue (Hmr) gene. Aruna Satish, Patrick M. Ferree, Daniel A. 728B Barbash. Department of Molecular Biology and Genetics, Undertaker, a Drosophila Junctophilin links Draper and dCed-6 Cornell University, Ithaca, NY. to calcium homeostasis during phagocytosis. Nathalie Franc, Leigh Cuttell, Claire Escaron, Mark Lavine, Emeline Van Goethen, Jean-Pierre Eid, Magali Quirin. MRC Cell Biol Unit, Immune System and Cell Death MRC LMCB UCL, London, United Kingdom. 719B 729C The Role of Caspases in Midgut Cell Death During Drosophila The hidden enemy: Insect endosymbionts and the evasion of Metamorphosis. Donna Denton, Kathryn Mills, Sharad Kumar. host immunity. Harriet Harris1,2, Stephinie Geis1, Lesley Hanson Institute, IMVS, Adelaide, SA, Australia. Brennan1,2. 1) Dept Biol, Concordia Univ Col,Edmonton, AB, Canada; 2) Dept Biological Sci, Univ of Alberta, Edmonton, AB, 720C Canada. Apoptosis independent role of DIAP1-Dronc in promoting cell proliferation. Celia Domingues, Hyung Don Ryoo. Dept Cell 730A Biol, New York Univ, New York, NY. Suppression of innate immune responses by elevated CO2 levels. Tomasz Krupinski1,4, Iiro T. Helenius1,4, Dennis Wang1, Doug 721A Turnbull2, Neal Silverman3, Eric Johnson2, Jacob I. Sznajder1, Imaging analysis of the spatiotemporal pattern of caspase Peter Sporn1, Greg Beitel1. 1) Northwestern Univ.; 2) Univ. of activation during epithelial cell sheet replacement. Yuichiro Oregon; 3) Univ. Massachusetts; 4) co-first authors. Nakajima, Erina Kuranaga, Masayuki Miura. Department of Genetics, Grad. Sch. Pharm. Sci., University of Tokyo, Tokyo, Japan.

722B JNK activity induces oncogenic transformation after stress events. Evgeny Shlevkov, Gines Morata. Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, 28049Madrid, Spain. 70 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

731B 740B Characterization of CG1837 protein as a candidate ligand for klumpfuss regulates expression of genes involved in retinal Draper, a Drosophila phagocytosis receptor. Takayuki Kuraishi1, apoptosis. Jamie Rusconi, Erica Hutchins, Malcolm Takashi Ishimoto2, Naoko Yamamoto2, Koichi Ueda2, Hiroshi Schongalla, Sunil Ganesh. Dept. Biological Sciences, Nakayama3, Masamitsu Yamaguchi4, Takeshi Awasaki5, Yumi University at Albany, Albany, NY. Hashimoto1, Takeshi Moki2, Akiko Shiratsuchi1, Yoshinobu Nakanishi1.1) Grad. Sch. Med. Sci., Kanazawa Univ; 2) Grad. 741C Sch. Nat. Sci. & Tech., Kanazawa Univ; 3) Biomol. Charact. Team, Drosophila Cbl is essential for control of cell death and cell RIKEN; 4) Dept. Applied Biol., Kyoto Inst. Tech; 5) Dept. differentiation during eye development. Yuan Wang1,2, Christian Neurobiol., Univ. Mass. Med. Sch. Werz3, Dongbin Xu1,2, Zhihong Chen1,2, Ying Li1,2, Ernst Hafen3, Andreas Bergmann1,2. 1) The University of Texas M.D. Anderson 732C Cancer Center, Department of Biochemistry & Molecular Biology; Cellular immune response and JAK/STAT signalling pathway. 2) The Genes & Development Graduate Program; 3) ETH. Rami Makki1, Virginie Daburon1, Delphine Pennetier1, Joanna Krzemien1, Marie Meister2, Alain Vincent1, Michèle Crozatier1. 742A 1) Centre de Biologie du Développement, Toulouse,France; 2) Characterization of Mutants from Cell Death Screens for the 3rd Musée de Zoologie, Strasbourg, France. Chromosome in Drosophila. Dongbin Xu, Andreas Bergmann. Dept. of Biochemistry & Molecular Biology, Genes & 733A Development Graduate Program. University of Texas, MD Identification of host factors involved in Poxvirus infection using Anderson Cancer Center,Houston, TX. genome-wide screening approaches in Drosophila. Theresa Moser, Sara Cherry. Department of Microbiology, University of 743B Pennsylvania, Philadelphia, PA. Evolution of the morgue cell death gene in the phylum Arthropoda. Ying Zhou2, John Nambu1,2. 1) Department of 734B Biology, University of Massachusetts, Amherst, MA; 2) Program Melanotic mass formation and lamellocyte differentiation in lam of Neuroscience and Behavior, University of Massachusetts, mutant larvae. Maja Pavlovic1,2, Per Kylsten1, Mitch Dushay1,3. Amherst, MA. 1) Life Sciences, Södertörns högskola, 141 89 Huddinge, Sweden; 2) Biosciences and Nutrition, Karolinska Institute, 744C Stockholm, Sweden; 3) Comparative Physiology, EBC, Uppsala ATP-sensitive potassium channels mediate survival during viral University, Norbyvägen 18A, 752 36 Uppsala, Sweden. infection in Drosophila. Ioannis Eleftherianos, Safia Deddouche, Jules Hoffmann, Jean-Luc Imler. Institut de 735C Biologie Moleculaire et Cellulaire, Strasbourg, Alsace, France. High-Throughput Screening of Sindbis virus in Disparate Hosts to identify novel antiviral targets. Patrick P. Rose1, Rich W. 745A Hardy2, Sara Cherry1. 1) Department of Microbiology, University Examining the Cross-Talk between the Insulin Signaling and of Pennsylvania, Philadelphia, PA. 19104; 2) Department of Immune System Pathways in D. melanogaster. Ingrid Hansen1,2, Biology, IndianaUniversity, Bloomington, IN 47405. Scott Pletcher1,2,3. 1) Huffington Ctr on Aging, Baylor Col Medicine, Houston, TX; 2) Interdepartmental Program in Cell 736A and Molecular Biology, Baylor Col Medicine, Houston, TX; 3) A role for autophagy in innate antiviral immunity: Lessons learned Department of Molecular and Human Genetics, Baylor Col from Drosophila. Spencer Shelly, Nina Lukinova, Allison Medicine, Houston, TX. Berman, Sara Cherry. Microbiology, University of Pennsylvania, Philadelphia, PA. 746B Activation of insect phenoloxidase after injury: endogenous 737B versus foreign elicitors. Thomas Hauling1, Gawa Bidla1, Mitchell E3 ligase and proteasome pathway in phagocytosis of apoptotic Dushay2, Ulrich Theopold1. 1) Molecular Biology & Functional corpses by embryonic macrophages. Elizabeth Silva, Nathalie Genomics, Stockholm University, Stockholm, Sweden; 2) Franc. MRC Laboratory for Molecular Cell Biology and Cell Department of Comparative Physiology, Uppsala University, Biology Unit, UniversityCollege London,London, United Norbyvägen 18A, Uppsala, Sweden. Kingdom. 747C 738C Peroxiredoxin 5 modulates immune response in D. melanogaster. Characterization of Apoptosis Inducing Factor (AIF) in the William Orr, Svetlana Radyuk, Katarzyna Michalak, Vladimir Drosophila visual system. Zachary Lemmon, Joseph O'Tousa. Klichko, Judith Benes. Dept of Biological Sci, Southern Biological Sciences, University of Notre Dame, Notre Dame, IN. Methodist Univ, Dallas, TX.

739A 748A DNaseII acts cell autonomously during nurse cell death in Danger signals in Drosophila Host defence. Jean-Marc oogenesis. Kim McCall, B. Paige Bass, Elizabeth Tanner, Reichhart. UPR 9022 CNRS, IBMC, Strasbourg, France. Daniel Mateos San Martín. Department of Biology, Boston University, Boston, MA. POSTER SESSIONS 71 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

749B 758B The effects of accessory gland proteins and sperm on immune Immunity and Maternal Effects in D. melanogaster. Jodell E. response in female D. melanogaster. Sarah M. Short1, Brian P. Linder, Daniel E. L. Promislow. Dept Genetics, University of Lazzaro2. 1) Field of Genetics and Development, Cornell Georgia, Athens, GA. University , Ithaca, NY; 2) Department of Entomology, Cornell University, Ithaca, NY. 759C Innate immune response activity correlates to diversity of 750C Drosophila susceptibility to bacterial infection. Kiyoshi Okado, Recognition and Signaling in the Drosophila IMD Pathway. Neal Naoaki Shinzawa, Hiroka Aonuma, Shinya Fukumoto, Shin- Silverman, Deniz Erturk-Hasdemir, Nicholas Paquette, ichiro Kawazu, Kanuka Hirotaka. NRCPD, Obihiro University Kamna Aggarwal. Dept. of Medicine, Division of Infectious of Agri. and Vet. Med., Obihiro, Hokkaido, Japan. Diseases, UMass MedicalSchool, Worcester, MA. 760A 751A Deciphering the interactions between D. melanogaster and a Identification of the trafficking receptor of the serpin degradation yeast Candida glabrata.Jessica Quintin, Joelle Asmar, pathway during the innate immune response. Sandra F. Soukup, Dominique Ferrandon. UPR9022 IBMC, CNRS, Strasbourg, David Gubb. Functional Genomics, CICBiogune, Derio, Basque Alsace, France. Country, Spain. 761B 752B Identification of Host Factors Involved in Flock House Virus A serpin that controls the melanization reaction in the tracheal Infection. Leah R. Sabin1, Paul Ahlquist2, Sara Cherry1. 1) system of Drosophila. Huaping Tang1, Zakaria Kambris2, Bruno Microbiology, University of Pennsylvania, Philadelphia, PA; 2) Lemaitre2, Carl Hashimoto3. 1) Department of Molecular, Institute for Molecular Virology, University of Wisconsin-Madison, Cellular, and Developmental Biology, Yale University, New Haven, Madison, WI. CT 06520; 2) Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France; 3) Department of Cell Biology, Yale 762C University School of Medicine, New Haven, CT 06520. Black dots and inflammation: the role of p38 MAPK signalling in a chronic inflammation-like phenotype in Drosophila. Gerhard 753C Seisenbacher, Ernst Hafen, Hugo Stocker. Institute of Regulation of Drosophila Inhibitor of Apoptosis Protein1 in Molecular Systems Biology, ETH Zurich, Switzerland. Developmental Apoptosis. Hui Li, Kenneth Cadigan. Dept MCDB, Univ Michigan, Ann Arbor, MI. 763A Mitochondrial Morphology and Programmed Cell Death in the 754A Drosophila Ovary. Elizabeth Tanner, Todd Blute, Kim McCall. Probing the Diap1-Dronc Interaction in vitro and in vivo. Peter J. Biology, Boston University, Boston, MA. Shapiro, Hyung-Don Ryoo. Cell Biology, NYU Medical Center, New York, NY. 764B Reaper- and Grim-induced cell death is suppressed by Ras/ 755B MAPK signaling in Drosophila developing indirect flight muscles. Dicer-2 mediated inducible antiviral response in Drosophila. Safia Hidenobu Tsujimura, Tomohiro Yoneda, Hiroka Aonuma, Deddouche1, Delphine Galiana-Arnoux2, Stefanie Mueller1, Hideaki Arai, Shinobu Hirai. Dept Developmental Biol, Tokyo Bassam Berry3, Christophe Antoniewski3, Anette Univ Agric & Technology, Tokyo, Japan. Schneeman4, Jules Hoffmann1, Jean-Luc Imler1. 1) UPR9022 CNRS, Institut de Biologie Moléculaire et Cellulaire, 67000 765C Strasbourg, France; 2) UMR5242 CNRS Institut de Genomique Spiroplasma infection in natural populations of Drosophila Fonctionnelle de Lyon ENS, 69000 Lyon, France; 3) URA2578 species. Thomas Watts, Sergio Castrezana, Therese Markow, CNRS, Institut Pasteur, 75015 Paris, France; 4) Department of Nancy Moran. Ecology & Evolutionary Biol, Univ Arizona, Molecular Biology, CB262 The Scripps Research Institute, La Tucson, AZ. Jolla, CA 92037. 766A 756C JAK/STAT signaling controls prohemocyte fates in a dose Trancriptional architecture of age-specific variation in immune dependent manner in Drosophila larval hematopoiesis. Soichi function. T. M. Felix1, G. Wu2, J. Leips1. 1) Dept Biological Tanda, Ying Shen, Catherine Dominguez, William Bell. Dept Sciences, UMBC, Baltimore, MD; 2) Department of Molecular Biological Sci, Ohio Univ, Athens, OH. Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.

757A Identification of host factors and pathways essential for West Nile Virus replication. Sheri Hanna, Robert Doms, Sara Cherry. Dept Microbiology, University of Pennsylvania, Philadelphia, PA. 72 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

Techniques and Genomics 774C Identification of novel transcription initiation and polyadenylation 767B sites in the Hdc gene. Daniel E. Boozer1, Sara Smolinski2, Genome-Wide Mapping of Chromosomal Proteins in Drosophila. Martin G. Burg1,2. 1) Cell and Molecular Biology Program, Grand Akiko Minoda1, Art Alekseyenko2, Nicole Riddle3, Yuri Valley State University, Allendale, MI; 2) Department of Schwartz4, Cameron Kennedy1, Sarah Elgin3, Peter Biomedical Sciences, Grand Valley State University, Allendale, Kharchenko5, Mitzi Kuroda2, Peter Park5, Vincenzo MI. Pirrotta4,Gary Karpen1. 1) Dept. of Genome Biology, Lawrence Berkeley National Lab, Berkeley, CA; 2) Dept. of Genetics, 775A Harvard-Partners Center for Genetics & Genomics, Boston, MA; Genome-Wide Expression-Based Lineage Analysis. John M. 3) Department of Biology, Washington University, St. Louis, MO; Olson1, Cory J. Evans1, Eunha Kim1, Kathy Ngo1, Noemi E. 4) Department of Molecular Biology and Biochemistry, Rutgers Lee1, Edward Kuoy1, Alexander N. Patananan1, Daniel Sitz1, University, Piscataway, NJ; 5) HPCGG, Harvard Medical School, PhuongThao Tran1, Minh-Tu Do1, Kevin Yackle1, Albert Boston, MA. Cespedes1, Gerald B. Call2, UCLA URCFG1, Volker Hartenstein1, Utpal Banerjee1. 1) Molecular, Cell, and 768C Developmental Biology, University of California, Los Angeles, Adaptive image segmentation methods applied to the analysis Los Angeles, CA; 2) Midwestern University, Glendale, AZ. of Snail repressor dosage impact on nascent vnd, sog, rho and brk transcription. William Beaver1, Adam Paré2, David 776B Kosman2, Ethan Bier2, William McGinnis2, Yoav Freund1. 1) An improved Tet-On expression system. Carlos Flores, Jerry Department of Computer Science and Engineering, University C.-P. Yin, William R. Engels. Dept Genetics, Univ Wisconsin, of California, San Diego, La Jolla, CA; 2) Cell and Developmental Madison, WI. Biology Department, University of California, San Diego, La Jolla, CA. 777C Optimizing Ends-Out Gene Targeting In Drosophila. Yang Hong, 769A Juan Huang, Wenke Zhou, Annie Watson. Cell Biology and Mining embryonic expression images reveals novel Physiology, Univ of Pittsburgh Medical School, Pittsburgh, PA. developmental pathway components. Erwin Frise, Amy Beaton, Richard Weiszmann, Ann Hammonds, Susan E. Celniker. 778A Berkeley Drosophila Genome Project, Lawrence Berkeley Nat Development of a Spatial and Temporal Control of Transgene Lab, Berkeley, CA. Expression in Drosophila. Haojiang Luan, Andrew Vreede, Benjamin White. Lab Molecular Biol, NIMH, Bethesda, MD. 770B Drosophila genome resources at NCBI. Terence Murphy, Pavel 779B Bolotov, Stacy Ciufo, Karen Clark, Wratko Hlavina, Yuri A Temperature-Sensitive Protein Switch. Guihong Tan, Ming Kapustin, Boris Kiryutin, Michael Ovetsky, Sergey Chen, Change Tan. Department of Biological Sciences, Bond Resenchuk, Sasha Souvorov, Igor Tolstoy, Paul Kitts, Donna Life Sciences Center, University of Missouri-Columbia. Maglott, Ilene Mizrachi, Tatiana Tatusova, Kim Pruitt. NCBI/ NLM/NIH/DHHS, Bethesda, MD. 780C ProStack, the image analysis software to process and quantify 771C patterns of gene expression in the Drosophila blastoderm. QBLAST: a so far missing but indispensable on-line tool. Erwin Konstantin N. Kozlov1, Pisarev Andrei1, Samsonova Maria1, Seinen, Ody C. M. Sibon. SSCB, UMCG, Groningen,Groningen, Reinitz John2. 1) St. Petersburg State Polytechnical University, Netherlands. St. Petersburg, Russian Federation; 2) Stony Brook University, Stony Brook. 772A FlyExpress: Computational Biology and Bioinformatics for spatial 781A gene expression patterns from Drosophila embryogenesis. Multiphoton Investigation of Myosin-Based Drosophila Muscle Bernard Van Emden1, Christopher Busick1, Hector Ramos1, Degeneration Induced by Proteasome Inhibition. Chiao-Ying Kailah Davis1, Sethuraman Panchanathan1,2, Stuart J. Lin1, Chen-Yuan Dong2, June-Tai Wu3, Chii-Wann Lin1,5, Jyh- Newfeld1,3, Sudhir Kumar1,3. 1) Biodesign Inst., Arizona State Horng Chen1, Sung-Jan Lin4,5. 1) Department of Electrical Univ., Tempe, AZ; 2) School of Computing and Informatics, Engineering, National Taiwan University, Taipei, Taiwan; 2) Arizona State Univ., Tempe, AZ.C; 3) School of Life Sciences, Department of Physics, National Taiwan University, Taipei, Arizona State Univ., Tempe, AZ. Taiwan; 3) Department of Medical Research, National Taiwan University Hospital , Taiwan; 4) Department of Dermatology, 773B National Taiwan University Hospital and College of Medicine, Automatically determining the developmental stage of embryos Taipei, Taiwan; 5) Institute of Biomedical Engineering, College captured in the Gene Expression Pattern Images. Jieping Ye1,3, of Engineering and College of Medicine, National Taiwan Jianhui Chen1,3, Sudhir Kumar2,3. 1) Department of Computer University, Taipei, Taiwan. Science and Engineering, Arizona StateUniversity, Tempe, AZ; 2) School of Life Sciences, Arizona State University, Tempe, AZ; 3) Center for Evolutionary Functional Genomics, The Biodesign Institute, Arizona State University, Tempe, AZ. POSTER SESSIONS 73 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

782B 790A A novel epitope tag for purification of macromolecular complexes Expression profiling by massively parallel sequencing. Christian from invertebrates. Matthias Kroiss1, Katharina Apfel1, Julia Schloetterer1, Tatiana Torres1, Muralidhar Metta1, Birgit Wiesner2, Matthias Grimmler1, Albert Sickmann2, Utz Ottenwaelder2. 1) Inst Tierzucht, VMU Wien, Wien, Austria; 2) Fischer1. 1) Department of Biochemistry, Theodor-Boveri- Eurofins, Medigenomics GmbH, Martinsried, Germany. Institute, Würzburg, Germany; 2) Rudolf-Virchow-Center, Würzburg, Germany. 791B Efficient generation of continuous cell lines from Drosophila 783C embryos by expression of oncogenic Ras. Amanda Simcox, New Drosophila GS-TAP vectors for protein complex purification Ting Chen, Jon Buthchar, Steven Justiniano. Dept Molecular and proteome exploration. Alexey Veraksa, Phillip Kyriakakis, Genetics, Ohio State Univ, Columbus, OH. Marla Tipping, Louka Abed. Department of Biology, University of Massachusetts Boston, Boston, MA. 792C D. melanogaster: a GO reference genome. Susan Tweedie, The 784A FlyBase Consortium and The Gene Ontology Consortium. Expanding the coverage and versatility of the Gene Disruption Department of Genetics, University of Cambridge, Cambridge, Project collection using Minos mutators with swappable United Kingdom. cassettes. Robert W. Levis1, Koen J. T. Venken2,3, Yuchun He2,3, Joseph W. Carlson4, Martha Evans-Holm4, Karen L. 793A Schulze2,3, Roger A. Hoskins4, Allan C. Spradling1,3, Hugo J. Phosphoproteome analysis of D. melanogaster embryos. Bo Bellen2,3. 1) Dept Embryology, Carnegie Inst of Washington, Zhai, Judit Villén, Sean A. Beausoleil, Julian Mintseris, Baltimore, MD; 2) Dept Molecular and Human Genetics, Dept Steven P. Gygi. Department of Cell Biology, Harvard Medical Neuroscience, Baylor College of Medicine, Houston, TX; 3) School, Boston, MA. Howard Hughes Medical Institute; 4) Dept Genome & Computational Biology, Lawrence Berkeley National Laboratory, 794B Berkeley, CA. A Genome-Wide RNAi Screen to Identify New Components of the RAS/MAPK Pathway. Dariel Ashton-Beaucage1, Anne- 785B Sophie Guenier1, Jean Duchaine1, Patrick Gendron1, Marc Generation of FRT lines to enable germ line clonal analysis of Therrien1,2. 1) Institue for Research in Immunology and Cancer, pre-existing FRT collections. Ernesto Lujan1, Douglas Montreal, Canada; 2) Département de Pathologie et Biologie Bornemann1, Carmen Rottig2, Ernst Hafen2, Rahul Warrior1. Cellulaire, Université de Montréal, Montréal, Canada. 1) Dept of Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Zoologisches Institut, Universitat Zurich, Switzerland. Drosophila Models of Human Diseases 795C 786C Function of Dorsal and Dif in multistep hematopoietic microtumor Innovations in a one-pot system of genome walking and formation. Marta Kalamarz, Indira Paddibhatla, Christina mutational mapping. Kyl Myrick, William Gelbart. Dept Molecular Nadar, Shubha Govind. Department of Biology, The City College & Cellular Biology, Harvard University, Cambridge, MA. of New York, CUNY, New York 10031. 787A 796A Systematic characterization of Drosophila transcription factor Enthoprotin regulates hematopoiesis in Drosophila. Wei-Ru Li1,2, specificities via a bacterial one-hybrid system. Michael Brodsky1, Yung-Heng Chang1,3, Y. Henry Sun1,2. 1) Inst Molecular Biology, Marcus Noyes1, Xiangdong Meng1, Atsuya Wakabayashi1, Taipei, Taiwan; 2) Inst of Genomic Science, National Yang Ming Saurabh Sinha2, Scot Wolfe1. 1) Program in Gene Function & University, Taipei, Taiwan; 3) Graduate Institute of Life Sciences, Expression, University of Massachusetts Medical School, National Defense Medical Center, Taipei, Taiwan. Worcester, MA; 2) Department of Computer Science, University of Illinois at Urbana-Champaign, Urbana, IL. 797B Inhibition of RAS-Mediated Transformation and Tumorigenesis 788B by Targeting the Downstream E3 Ubiquitin Ligase SIAH. Amy Incorporation of piggyBac-based insertional mutagenesis and Tang1,2, Rebecca Schmidt2, Cheol Park1, Atique Ahmed1, GAL4 system vectors in ten species of Drosophila with Justin Gundelach1, Nanette Reed1, Shen Cheng1, Bruce sequenced genomes. Stacy Holtzman1, David F. B. Miller1, Knudsen1, Amy Tang1,2. 1) Department of Surgery, Mayo Clinic Teruyuki Niimi2, Thomas C. Kaufman1. 1) Biology Department, College of Medicine, Rochester, MN; 2) Department of Indiana University, Bloomington, IN; 2) Lab of Sericulture and Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Entomoresources, Graduate School of Bioagricultural Sciences, Mayo Clinic College of Medicine, Rochester, MN. Nagoya University, Nagoya, Japan. 798C 789C The effects of Bcr-Abl on Ena-associated proteins in D. Genetic information in FlyBase: what goes in must come out. melanogaster . Kaitlyn M. Vernier, Traci L. Stevens. Biology, Peter McQuilton, The FlyBase Consortium. FlyBase- Randolph-Macon College, Ashland , VA. Cambridge, Dept Genetics, University of Cambridge, Cambridge, United Kingdom. 74 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

799A 808A TheHelicobacter pylori virulence factor CagA can function as Binding partners of dFMRP suggest a regulatory role in an adaptor protein in receptor tyrosine kinase pathways. Anica translational initiation. Ophelia Papoulas1, Kate Monzo1, Greg M. Wandler1, Crystal M. Botham2, Karen Guillemin1. 1) Institute Cantin2, Cristian Ruse2, John Yates III2, John C. Sisson1. 1) of Molecular Biology, University of Oregon, Eugene, OR; 2) The Section of MCD Biology and The I.C.M.B., The University Department of Pediatrics, Stanford University, Stanford, CA. of Texas at Austin, Austin, TX; 2) Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA. 800B Drosophila model for Kif1A related hypoplastic left heart 809B syndrome. Takeshi Akasaka1, Grant Hogg1, Karen Ocorr1, Rolf Lamin processing and Progeria: studies in Drosophila. Sandra Bodmer1, Paul Grossfeld2. 1) Burnham Inst, La Jolla, CA; 2) R. Schulze1, Beatrice Curio-Penny2, Melissa Petersen1, Lori Department of Pediatrics, UCSD, La Jolla, CA. L. Wallrath2. 1) Dept Biol, Western Washington Univ, Bellingham, WA; 2) Dept Biochemistry, University of Iowa, Iowa City, IA. 801C A wildtype Drosophila model of age-dependent heart failure. Rolf 810C Bodmer1, Karen Ocorr1, Tim Crawley1, Greg Gibson2. 1) The role of alg10 in regulating N-glycosylation during Drosophila Burnham Inst, La Jolla, CA; 2) University of Queensland, development: a novel model for Congenital Disorders of Brisbane, Australia. Glycosylation. Erica M. Selva, Carly Dominica, Evan Lebois. Dept Biological Sci, Univ Delaware, Newark, DE. 802A Deletion screen in adult Drosophila identifies candidate genes 811A for dilated cardiomyopathy. Michelle E. Casad1, Il-Man Kim2, Dube3a expression in neurons increases synaptic bouton Matthew J. Wolf2, Howard A. Rockman1,2,3. 1) Department of number through regulation of Pbl and downstream Rac targets. Cell Biology, Duke University Medical Center, Durham, NC; 2) Kyle Summers1, Hemachand Tummala2, Priti Azad1, Department of Medicine, Duke University Medical Center, Lawrence Reiter1. 1) Neurology, University of Tennessee Health Durham, NC; 3) Department of Molecular Genetics, Duke Science Center, Memphis, TN; 2) Biology, University of Memphis, University Medical Center, Durham, NC. Memphis, TN.

803B 812B Drosophila model of muscular dystrophy. Jeffery Goldstein1, Characterization of the Drosophila mitochondrial elongation Michael Allikian2, Gira Bhabha2, Patrick Dospoy2, Ahlke factor iconoclast: a model system for Combined Oxidative Heydemann2, Pearl Ryder2, Judy Earley2, Matthew Wolf4, Phosphorylation Deficiency. Catherine Trivigno, Theodor E. Howard Rockman5, Elizabeth McNally2,3. 1) Departments of Haerry. Dept. of Biology, Florida Atlantic Univ, Boca Raton, FL. Pathology; 2) Medicine; 3) and Human Genetics, University of Chicago, Chicago, IL; 4) Departments of Medicine; 5) and Cell 813C Biology, Duke University, Durham, NC. Characterization of a new tracheal gene required in tube elongation. Erika Tång Hallbäck, Anne Uv. Biomedicine, 804C Medical genetics, Göteborg, Göteborg, Sweden. Using Drosophila heart to uncover the genes contributing to Down syndrome congenital heart disease. Tamar R. Grossman1, Amir 814A Gamliel2, Robert J. Wessells3, Ouarda Taghli-Lamallem4, Identification and characterization of a new class of proteins Kristen Jepsen2, Julie R. Korenberg5, Rolf Bodmer4, Ethan required for epithelial barrier formation. Fariba Zare, Anna Bier1. 1) Div. Biol. Sci; 2) HHMI, Dept. Med., UCSD, La Jolla, CA; Tonning, Anne Uv. Department of Clinical and Medical Genetics, 3) Univ Michigan, Ann Arbor, MI; 4) Burnham Institute Med Res, Institution of Biomedicine, Gothenburg, Sweden. La Jolla, CA; 5) Cedars-Sinai Medical Center, Los Angeles, CA. 815B 805A Functional characterization of dPGC, a member of the PPARg Characterizing how mGluR-activated G-coupled pathways are Coactivator 1 family. Gretchen V. Gee, Jennifer T. Paul, Marc misregulated in the absence of dFMR1. Balpreet Bhogal, Tatar. Brown University, Providence, RI. Department of Ecology Thomas Jongens. Dept Genetics, University of Pennsylvania, and Evolutionary Biology. Philadelphia, PA. 816C 806B Drosophila as a model for metabolic syndrome. Laura Reed, A role for the steroid hormone ecdsyone in embryonic tracheal Stephanie Williams, Mastafa Springston, Julie Brown, Greg development. Tina Chavoshi, Anne Uv. Department of Medical Gibson. Dept Genetics, North Carolina State Univ, Raleigh, NC. and Clinical Genetics, Biomedicine, Gothenburg, Gothenburg, Sweden. 817A Regulation of energy homeostasis and obesity in D. 807C melanogaster. Tânia Reis, Iswar Hariharan. MCB, Univ Requirement for O-mannosylation in Drosophila development. California, Berkeley, Berkeley, CA. Dmitry Lyalin, Naosuke Nakamura, Stacey Whitman, Kate Koles, Vlad Panin. Dept Biochemistry & Biophysics, Texas A&M Univ, College Station, TX. POSTER SESSIONS 75 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

818B 825C Stress Protection Pathway - New Ways of Treating Disease. Genetic screen of human neurological disease genes for defects Qiong Wang, Wesley Dobbs, Adriana Villella, Gerasimos in synaptic function and development. Dominik M. Haddad1,2, Sykiotis, Dan Garza, Marc Hild. DMP / Model Organisms, Maarten Leyssen1,2, Patrik Verstreken1,2. 1) Laboratory of NIBRI, Cambridge, MA. Neuronal Communication, K.U. Leuven, Center for Human Genetics, Belgium; 2) VIB, Department of Molecular and 819C Developmental Genetics, Belgium. Genetic suppression of neurodegeneration and neurotransmitter release abnormalities caused by expanded full-length huntingtin 826A accumulating in the cytoplasm. Guang-Ho Cha1, Romero Circadian Rhythms as Model Systems to Study the Effects of Eliana1, Patrik Verstreken1,2,6, Cindy Ly3, Robert Hughes4, Transcriptional Dysregulation in MJD-afflicted Drosophila. Amy Hugo Bellen1,2,3,5, Juan Botas1,7. 1) Dept Molecular Human Gen, B. Hart, John M. Warrick. Department of Biology, University of Baylor Col Medicine, Houston, TX 77030; 2) Howard Hughes Richmond, Richmond, VA. Medical Institute; 3) Department of Neuroscience, Baylor Col Medicine, Houston, TX 77030; 4) Buck Institute 8001 Redwood 827B Blvd, Novato, CA 94945; 5) Program in Developmental Biology, A Drosophila Model for neuroinflammatory response in Baylor Col Medicine, Houston, TX 77030; 6) VIB Department of neurodegenerative disease. Arati Inamdar1, Anathbandhu Molecular and Developmental Genetics K.U.Leuven Department Chaudhuri2, Hakeem Lawal1, Faiza Ferdousy1, Janis of Human Genetics Herestraat 49, bus 602 B3000 Leuven, O'Donnell1. 1) Dept Biological Sci, Univ Alabama, Tuscaloosa, Belgium; 7) Department of Molecular and Cellular Biology, Baylor AL, 35487; 2) Department of Pharmacology and Experimental Col Medicine, Houston, TX 77030. Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198. 820A Protein nuclear transport and polyglutamine toxicity. Wing Man 828C Chan1, Frankie Ho Tsoi2, Pang Chui Shaw1,2, Ho Yin Edwin Overexpression and RNAi silencing the Drosophila ortholog of Chan1,2. 1) Molecular Biotechnology Programme, CUHK, UBQLN1, dUbqln, decreases anoxia tolerance. A. Li1, K. M. HKSAR; 2) Department of Biochemistry, CUHK, HKSAR. McKay1, Y. Dong1,2, Z. Xie1,2, R. E. Tanzi1. 1) Genetics & Aging Research Unit, Department of Neurology; 2) Department of 821B Anesthesia and Critical Care, Massachusetts General Hospital Regulation of Tau Phosphorylation and Toxicity : Insights from a and Harvard Medical School, Charlestown, MA. Drosophila Model. Shreyasi Chatterjee1, Tzu Kang Sang2, George Jackson1. 1) Department of Neurology and Center for 829A Neurobehavioral Genetics. David Geffen School of Medicine at modeling the role of human ApoD in neuropathologies. Julien University of California, Los Angeles. CA; 2) Department of Muffat, Seymour Benzer. Dept Biol, CalTech, Pasadena, CA. Neurology, National Tsing Hua University, Taiwan, Republic of China. 830B Neurotrophic factors GDNF and BDNF as approach for 822C genotherapy of neurodegenerative disorders. Ekatherina A. Characterizing the function of the Drosophila LRRK2 homolog, Nikitina, Elena V. Savvateeva-Popova. Neurogenetics, Pavlov lrrk. Mark W. Dodson1, Changan Jiang2, Ming Guo1,2. 1) Institute of Physiology, St-Petersburg, St-Petersburg, Russian Molecular Biology Institute, University of California, Los Angeles, Federation. Los Angeles, CA; 2) Departments of Neurology and Pharmacology, David Geffen School of Medicine, UCLA. 831C Degradation of functional TPI protein underlies sugarkill 823A pathology. Michael Palladino1,2, Jacquelyn Seigle1,2, Alicia Cell and Organism Toxicity by Atrophins. Manolis Fanto1, Celotto1,2. 1) Pharmacology Dept, Univ Pittsburgh, School of Bernard Charroux2, Ilaria Nisoli1, Elise Peyre2, Francesco Medicine Pittsburgh, PA. 15261; 2) Pittsburgh Institute for Napoletano1. 1) Dulbecco Telethon Institute, DIBIT-HSR, Milan, Neurodegenerative Diseases University of Pittsburgh School of Italy; 2) IBDML, Campus de Luminy Case 907, F-13288 Marseille Medicine Pittsburgh, PA 15260. Cedex 9, France. 832A 824B A Drosophila model for motor neuron disease caused by a Soluble Oligomers of a-Synuclein in the Neurodegeneration of mutation in VAPB/DVAP33A: Evidence for a dominant negative Parkinson Disease. Madhu Gajula Balija1, D. P. Karpinar1, F. mechanism. Anuradha Ratnaparkhi, George Lawless, Felix Opazo3, B. Falkenburger3, D. Riedel2, A. Herzig1, H. Jaeckle1, Schweizer, Peyman Golshani, George Jackson. University of S. Eimer2, J. B. Schulz3, C. Griesinger1, M. Zweckstetter1. 1) California, Los Angeles, Los Angeles, CA. Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; 2) European Neuroscience Institute, Goettingen, 833B Germany; 3) Center for Molecular Physiology of Brain, Developmental functions of two acyl-CoA synthetases, Goettingen, Germany. Bubblegum and Double Bubble, in Drosophila. Anna Sivatchenko, Anthea Letsou. Dept Human Genetics, Univ Utah, Salt Lake City, UT. 76 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

834C 843C A Genetic Screen in Drosophila for Modifiers of Ab42-induced Stimulation of the mitochondrial replicase by deletion mutants lifespan. Ho-Juhn Song1, Neda Hoseinovic1, Thomas Neufeld2, of mitochondrial single-stranded DNA-binding protein. Marcos Mary Konsolaki3, Daniel Curtis1, Dan Garza1. 1) Dept Dev T. Oliveira, Laurie S. Kaguni. Graduate Program in Genetics Molec Pathway, Novartis, Cambridge, MA; 2) Department of and Department of Biochemistry and Molecular Biology, Genetics, Cell Biology, and Development, University of Michigan State University, East Lansing, MI. Minnesota, Minneapolis, MN; 3) Department of Genetics, Rutgers University, Piscataway, NJ. 844A Dominant modifier screens reveal components that interact with 835A the Drosophila Dg-Dys complex. Mario Pantoja1, Mariya Modified co-activator levels in Spinocerbellar Ataxia 3 Drosophila Kucherenko1,2, Andriy Yatsenko1,2, Karin Fischer1, Halyna model. Naoum Tavantzis, John Warrick. University of Shcherbata1, Hannele Ruohola-Baker1. 1) Department of Richmond, Richmond, VA. Biochemistry, University of Washington, Seattle, WA; 2) Ivan Franko National University in Lviv, Lviv, Ukraine. 836B Genetic evidence for the involvement of the 26S proteasome in 845B age-related neurodegenerative diseases. Ayako Tonoki1, Erina D. melanogaster as model system to study mitochondrial Kuranaga1,2, Takeyasu Tomioka1, Jun Hamazaki3, Shigeo respiratory chain diseases. A. Sanchez-Martinez1, R. Murata3, Keiji Tanaka3, Masayuki Miura1,2. 1) Department of Hernández-Sierra1, M. Calleja1, S. Peralta1, V. Domingo1, R. Genetics, Grad. Sch. Pharm. Sci., University of Tokyo; 2) JST, Garesse1, N. Luo2, C. Farr2, Y. Matsushima2, L. S. Kaguni2. 1) CREST; 3) Laboratory of Frontier Science, Core Technology and Departamento de Bioquimica. Instituto de Investigaciones Research Center, Tokyo Metropolitan Institute of Medical Science. Biomédicas "Alberto Sols" CSIC-UAM. CIBERER ISCIII, Facultad de Medicina, Universidad Autónoma de Madrid, Spain; 837C 2) Department of Biochemistry and Molecular Biology, Michigan Roles of E3 ubiquitin ligases in polyglutamine diseases. Azaria State University,East Lansing, Michigan 48824-1319. K. Y. Wong1,2, Alan S. L. Wong1,2, C. M. Chan1,3, H. Y. Edwin Chan1,2,3. 1) Laboratory of Drosophila Research; 2) Molecular 846C Biotechnology Programme; 3) Department of Biochemistry, The Laboratory selection for hyperoxia-tolerance in D. melanogaster: Chinese University of Hong Kong, Hong Kong SAR, China. role of single genes in tolerance. Huiwen W. Zhao1, Dan Zhou1, Gabriel G. Haddad1,2,3. 1) Departments of Pediatrics; 2) 838A Departments of Neuroscience, University of California San Analysis of the de novo purine synthesis gene CG3590 encoding Diego, La Jolla, CA 92093; 3) The Rady Children’s Hospital, adenylosuccinate lyase. Denise V. Clark, Bethany R. Herrmann. San Diego, CA 92123. Dept Biol, Univ New Brunswick, Fredericton, NB, Canada.

839B Physiology and Aging Low chlorophylin concentrations increase genetic damage 847A induced by gamma rays in D. melanogaster. Martha P. Cruces1,2, Beneficial role of ad libitum water on lifespan in D. melanogaster. Emilio Pimentel1. 1) Instituto Nacional de Investigaciones William W. Ja1, Ted Brummel2, Seymour Benzer1. 1) Division Nucleares, Ocoyoacac, Mexico, Mexico; 2) Posgrado en Ciencias of Biology, California Institute of Technology, Pasadena, CA; 2) Biologicas, UNAM. Department of Biology, Long Island University, Brookville, NY. 840C 848B Effects of tissue-specific expression of mutant forms of Lamin Dietary Restriction Increases Lifespan by Targeting Anti-oxidative C: Drosophila as a model for Emery-Dreifuss muscular dystrophy. Defense Systems. Hadise Kabil1,2, Lawrence Harshman1, Scott Diane E. Cryderman1, George Dialynas1, Sandra R. Schulze2, Pletcher2. 1) School of Biological Sciences,University of Beatrice Curio-Penny1, Pamela K. Geyer1, Lori L. Wallrath1. Nebraska-incoln, Lincoln, NE; 2) Huffington Ctr on Aging, Baylor 1) Biochemistry, University of Iowa, Iowa City, IA; 2) Biology, Col Medicine, Houston, TX. Western Washington University, Bellingham, WA. 849C 841A Juvenile hormone as a regulator of the trade-off between Genetic screens uncover new genes potentially involved in the reproduction and lifespan in Drosophila. Thomas Flatt1,2, pathogenesis of Myotonic Dystrophy Type 1. Maria de Haro1, Tadeusz Kawecki2,3. 1) Brown University Division of Biology Ismael Al-Ramahi1, Thomas Cooper2, Juan Botas1. 1) Dept and Medicine Department of Ecology and Evolutionary Biology Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) Box G-W, 80 Waterman Street Providence, 02912 Rhode Island; Dept Pathology, Baylor Col Medicine, Houston, TX. 2) University of Fribourg Unit of Ecology and Evolution Chemin du Musee 10 CH-1700 Fribourg Switzerland; 3) Department of 842B Ecology and Evolution University of Lausanne Biophore CH- The H. pylori virulence factor, CagA, interacts with Rho signaling 1015 Lausanne Switzerland. to disrupt epithelia.Jonathan B. Muyskens, Crystal M. Botham, J. T. Neal, Anica Wandler, Lucy Cho, David Reid, Karen Guillemin. Institute of Molecular Biology, University of Oregon, Eugene, OR. POSTER SESSIONS 77 Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

850A 858C Couch potato aging in Drosophila. Paul Schmidt1, Mariska A Drosophila DEG/ENaC gene expressed in the fat body may Batavia1, C. T. Zhu2, Walter Eanes2. 1) Dept Biol, Univ play a role in lipid metabolism. Yishan Sun1,2, Lei Liu2, Yehuda Pennsylvania, Philadelphia, PA; 2) Department of Ecology and Ben-Shahar4, Michael J. Welsh1,2,3,4. 1) Neuroscience Graduate Evolution, SUNY Stony Brook. Program, Univ of Iowa, Iowa City, IA; 2) Dept of Internal Medicine, Univ of Iowa, Iowa City, IA; 3) Dept of Physiology and Biophysics, 851B Univ of Iowa, Iowa City, IA; 4) HHMI, Iowa City, IA. Sumoylation is necessary for the metamorphosis of D. melanogaster. Ana Talamillo1, Jonatan Sánchez1, Coralia 859A Pérez1, David Martín2, Rafael Cantera3,4, Rosa Barrio1. 1) Mutation in Drosophila adenosine deaminase disturbs the Functional Genomics, CIC bioGUNE, Derio, Bizkaia, Spain; 2) glycogen metabolism. Monika Zuberova, Tomas Dolezal. Institut de Biologia Molecular de Barcelona-CSIC, Barcelona, University of South Bohemia, Faculty of Science, Ceske Spain; 3) Stockholm University, Stockholm, Sweden; 4) IIBCE, Budejovice, Czech Republic. Montevideo, Uruguay. 860B 852C Mechanisms of life-span extension in Or83b mutant flies. Peter Neverland, a conserved Rieske-type family of proteins essential Poon, Daniel Harmon, Xiaowen Chu, Scott Pletcher. for ecdysone synthesis and cholesterol metabolism in the Huffington Center on Aging, Baylor College of Medicine, prothoracic gland. Takuji Yoshiyama1, Ryusuke Niwa2, Hiroshi Houston, TX. Kataoka1. 1) Department of Integrated Biosciences, The University of Tokyo, Kahiwa, Chiba, Japan; 2) Department of 861C Molecular, Cellular and Developmental Biology, Yale University, Effects of mutation of lot’s wife and starvation on crop motility in New Haven, CT. Drosophila.Elizabeth M. Bacon, Edward M. Blumenthal. Biological Sciences, Marquette University, Milwaukee, WI. 853A Characterization of ponchik, a novel gene regulating appetite, 862A adiposity, and lifespan inD. melanogaster. Tammy P. Chan1,2, Alterations in triglyceride levels and feeding behavior by a Sergiy Libert1,3, Emmeline Peng1, Jessica E. Zwiener1, mutation in lot's wife.Cassandra R. Peller, Edward M. Danielle A. Skorupa1,3, Scott D. Pletcher1,2,3,4. 1) Huffington Blumenthal. Biological Sciences, Marquette University, Center on Aging, Baylor College of Medicine, Houston, TX; 2) Milwaukee, WI. Program in Developmental Biology, Baylor College of Medicine, Houston, TX; 3) Interdepartmental Program in Cell and Molecular 863B Biology, Baylor College of Medicine, Houston, TX; 4) Department Effect of different temperature in the spontaneous somatic of Molecular and Human Genetics, Baylor College of Medicine, mutation accumulation in Drosophila. Anamaria Garcia1, Rita Houston, TX. Busuttil2, Cody Ginn1, Brent Calder2, Jan Vijg2, Martha Lundell1. 1) Dept Biol, Univ Texas, San Antonio, San Antonio, 854B TX; 2) Buck Institute for Age Research, Novato, CA. Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic and computational 864C systems analysis. Laurence Coquin1, Jacob Feala2, Andrew Changes in gene expression profiles related to mortality over McCulloch2, Giovanni Paternostro1,2. 1) The Burnham Institute the life span of large caged populations of D. melanogaster. Kylee for Medical Research, 10901 N. Torrey Pines road, La Jolla, CA; M. Gardner1, Kimberly Carlson1, Anjeza Pashaj1, Darby 2) Department of Bioengineering, University of California, San Carlson1, Lawrence Harshman2. 1) Biology, University of Diego, 9500 Gilman Drive, 0412, La Jolla, CA 92093-0412. Nebraska at Kearney, Kearney, NE; 2) School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588. 855C Metabolic control under hypoxia. Christian Frei1, Nan Chen1, 865A Olivier Rinner2, Matthias Gstaiger2. 1) CC-SPMD and Institute Identification of Genes that Impact Age-Related Locomotor for Cell Biology, ETH Zurich, Switzerland; 2) Institute for Impairment. Melanie Jones, Michael Grotewiel. Dept Human Molecular Systems Biology, ETH Zurich, Switzerland. Genetics, VCU, Richmond, VA.

856A 866B Molecular characterization of mutations in the enzymes of the a The shuttle craft locus controlling motoneuron axon guidance glycerophosphate cycle. Ross MacIntyre1, Amber Carmon1, proves to affect D. melanogaster lifespan. Natalia V. Roshina, Jeff Chien1, David Sullivan2. 1) Dept Molec Biol & Genetics, Alexandr V. Symonenko, Eugenia A. Tcybulko, Eugenia V. Cornell Univ, Ithaca, NY; 2) Emeritus Professor, Syracuse Ershova, Elena G. Pasyukova. Institute of Molecular Genetics University, Syracuse, NY. of RAS, Moscow, Russian Federation.

857B 867C Roles for the DHR38 nuclear receptor in carbohydrate Mitochondrial superoxide dismutase or SOD2 is more essential metabolism. Anne-Françoise Ruaud, Carl Thummel. for adult life span. Renee Forde, Subhas Mukherjee, Atanu Department of Human Genetics, University of Utah School of Duttaroy. Howard Univ, Washington, DC. Medicine, Salt Lake City, UT. 78 POSTER SESSIONS Poster board is in bold above the title. See page 13 for presentation schedule. The first author is the presenter. Abstracts begin on page 81.

868A RNA Biology Is the circadian clock system involved in temporal co-ordination of protein homeostasis during oxidative stress in D. 877A melanogaster? Natraj Krishnan, Andrew Davis, Jadwiga A genome-wide screen for genes involved in microRNA Giebultowicz. Department of Zoology, Oregon State University, biogenesis and activity. Caroline Jacquier, Josette Pidoux, Corvallis, OR 97331. Hélène Thomassin, Christophe Antoniewski. Lab of Drosophila Genetics and Epigenetics, Institut Pasteur, 25 rue 869B du Dr Roux, 75015 Paris, France. Factors influencing aging of male germline stem cells in Drosophila. Matthew Wallenfang, Tarnima Ahamed, 878B Khadeejah Bari, Christine Chang, Saira Siddiqui, Ayelet Regulation of Drosophila germline stem cells by a Dcr-1 Spitzer. Dept Biological Sciences, Barnard College, New York, mediated miRNA pathway. Zhigang Jin, Ting Xie. Stowers Inst, NY 10027. Kansas City, MO.

870C 879C A novel muscle-spike waveform in the giant fiber response in Identification of New miRNA Pathway Components. Arthur old flies. Jeff Engel. Dept Biological Sciences, Western Illinois Luhur, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. Univ, Macomb, IL. 880A 871A A sensitive reporter system for testing miRNA targeting applied Initiation and formation of adult type 2 peritrophic matrix. Gae E. to the regulation of Hox protein transcript and protein levels by Kovalick. Biology, University of Texas Permian Basin, Odessa, Hox cluster-encoded microRNAs. Adam Paré, Derek Lemons, TX. William McGinnis. Dept Cell & Developmental Biol, Univ California, San Diego, La Jolla, CA. 872B Fatty Acid Transporters Regulate Obesity, Cardiac Performance 881B and Lifespan in Drosophila. Samantha Morley, Nicole Piazza, MicroRNA Mediated Translational Repression in the Ovary. John Mike Hayes, Robert J. Wessells. Dept Intnl Med/Geriatrics, Univ C. Reich, Mark J. Snee, Paul M. Macdonald. Molecular Cell Michigan, Ann Arbor, MI. and Developmental Biology, University of Texas, Austin, US.

873C 882C Changes in GST gene expression in large caged populations of Analysis of the role of roX1 RNA 5' sequences in X chromosome D. melanogaster.Anjeza Pashaj1, Kimberly Carlson1, Kylee targeting. Ying Kong, Victoria H. Meller. Department of Gardner1, Darby Carlson1, Lawrence Harshman2. 1) Biology, Biological Sciences, Wayne State University, Detroit, MI. University of Nebraska at Kearney, Kearney, NE; 2) School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 883A 68588. In vivo analysis of nonsense mediated mRNA decay. Kimberly A. Frizzell, Shawn G. Rynearson, Mark M. Metzstein. 874A Department of Human Genetics, University of Utah, Salt Lake Antigenotoxicity of some traditional medicinal phytoextracts used City, UT. for the control of diabetes mellitus type 2. Rosario Rodriguez- Arnaiz, America Nitxin Castañeda Sortibrán, Maria 884B Guadalupe Ordaz Tellez. Departamento de Biologia Celular, Drosophila PTB/hnRNPI promotes formation of high-order oskar Facultad de Ciencias, Universidad Nacional Autónoma de RNP complexes and represses oskar translation. Florence México, Mexico, DF, Distrito Federal, Mexico. Besse1,2, Sonia Lopez de Quinto1,2, Anne Ephrussi1. 1) Developmental Biology, EMBL, Heidelberg, Germany; 2) 875B contributed equally to the work. IRES dependent translational regulation of dFoxo. Eugenia Villa- Cuesta, Brian T. Sage, Marc Tatar. Department of Ecology and 885C Evolutionary Biology, Brown University, Providence, RI. Identifying tissue specific regulatory proteins associated with the early spliceosome. Thomas Carr, Alexis Nagengast. Dept 876C Biochemistry, Widener Univ, Chester, PA. Theribose-5-phosphate isomerase mutant extends lifespan in D. melanogaster. Yi-Yun Wang, Jing-Zi Wang, Hsun Li, Horng- 886A Dar Wang. Institute of Biotechnology, Department of Life Bicaudal-C regulates nos expression during oogenesis. Chiara Science, National Tsing Hua University, Hsinchu 30013, Taiwan, Gamberi, Paul Lasko. Biology, McGill University, Montreal, R.O.C. Quebec, Canada.

888C 897C A novel role for klumpfuss as an RNA-binding protein. Erica J. Expression profiling of rasiRNA-affecting mutants. Alina Korbut, Hutchins, Barbara J. Zaffo, Jamie C. Rusconi. Department of Sergey Lavrov, Vladimir Gvozdev. Dep. of Molecular Genetics Biological Sciences, University at Albany, Albany, NY. of the Cell, Institute of Molecular Genetics, Moscow, Moscow, Russian Federation. 889A In vitro selection and characterization of the RNA binding sites 898A for the RNA binding protein Bruno. Brad Reveal, Paul A Genome-wide RNAi Screen Identifies Novel Factors Required Macdonald. Molecular Cell and Developmental Biology, for Histone pre-mRNA Processing. Eric J. Wagner, Brandon University of Texas, Austin, TX. Burch, Ashly Godfrey, Harmony Salzler, William Marzluff, Robert J. Duronio. Biochemistry and Biophysics, University of 890B North Carolina at Chapel Hill, Chapel Hill, NC. RNA localization through RNP transport particles in Drosophila ovaries. Yiyin Ho, Elizabeth Gavis. Dept of Molecular Biology, 899B Princeton University, Princeton, NJ. Identifying tissue specific alternative splicing events. Tim Rudolph1, Neha Sirohi2, Alexis Nagengast1. 1) Dept 891C Biochemistry, Widener Univ, Chester, PA; 2) Dept Biology, The changing dynamics of localized fluorescently labeled gurken Widener Univ, Chester, PA. mRNA in Drosophila. Angie Jaramillo1,2, Timothy Weil2, Elizabeth Gavis2, Trudi Schüpbach1,2. 1) HHMI; 2) Department of Molecular Biology, Princeton University, Princeton, NJ.

892A Understanding the Role of Symplekin in Histone pre-mRNA Processing. Deirdre C. Tatomer1, Mindy Steiniger3, Eric J. Wagner3, Sarah Kennedy5, Matthew R. Redinbo3,5, William F. Marzluff1,2,3,4, Robert J. Duronio1,2,4. 1) Department of Biology, UNC-Chapel Hill, Chapel Hill, NC; 2) Curriculum in Genetics and Molecular Biology, UNC-Chapel Hill, Chapel Hill, NC; 3) Department of Biochemistry and Biophysics, UNC-Chapel Hill, Chapel Hill, NC; 4) Program in Molecular Biology and Biotechnology, UNC-Chapel Hill, Chapel Hill, NC; 5) Department of Chemistry, UNC-Chapel Hill, Chapel Hill, NC.

893B Purification and characterization of mRNA localization complexes. James Wilhelm, Risa Maruyama, Elena Monfort- Prieto. Dept Biological Sciences, Univ California San Diego, La Jolla, CA.

896B The role of Drosophila fmr1 gene in rasiRNA silencing. Mikhail Klenov. Dept Animal Molecular Gen, Inst Molecular Genetics, Moscow.

ABSTRACTS 81 Introduction

Abstracts are divided into two sections: platform and poster presentations. In each section, the number located above the title identifies the abstract in all other listings in this book.

Platform Presentations Abstracts for platform presentations appear first and are organized chronologically by day and presentation. These abstracts are numbered consecutively from 1 through 156. Topic Abstract #s Beginning page # Immune System and Cell Death 1-8 83 Neurophysiology and Behavior 9-16 86 Organogenesis 17-24 89 Regulation of Gene Expression 25-38 92 Evolution and Quantitative Genetics 39-52 97 Cytoskeleton and Cell Biology 53-66 102 Gametogenesis 67-74 107 Signal Transduction 75-82 110 Pattern Formation 83-90 113 Drosophila Models of Human Diseases 91-104 116 RNA Biology 105-111 121 Genome and Chromosome Structure 112-118 124 Techniques and Functional Genomics 119-125 127 Chromatin and Gene Expression 126-132 130 Physiology and Aging 133-140 133 Neurogenetics and Neural Development 141-148 136 Cell Division and Growth Control 149-156 139

Poster Presentations All posters will be displayed from Wednesday, April 2, through Saturday, April 5, in the Grand Exhibit Hall of the Town and Country Resort & Conference Center. Authors are expected to be at their boards according to the following schedule: Thursday: 2:00 pm–3:00 pm: even-numbered posters 3:00 pm–4:00 pm: odd-numbered posters 8:00 pm–9:00 pm: “A” posters 9:00 pm–10:00 pm: “B” posters 10:00 pm–11:00 pm: “C” posters Friday: 8:00 pm–9:00 pm: “C” posters 9:00 pm–10:00 pm: “B” posters 10:00 pm–11:00 pm: “A” posters Saturday: 1:30 pm–2:30 pm: odd-numbered posters 2:30 pm–3:30 pm: even-numbered posters 7:00 pm–10:00 pm: open poster viewing (Authors not required to be present) Abstracts consecutively numbered from 157A through 899B for poster presentations are organized as follows: Topic Abstract #s Beginning page # Cell Division and Growth Control 157A-212B 142 Cytoskeleton and Cellular Biology 213C-266B 161 Genome and Chromosome Structure 267C-281B 179 Regulation of Gene Expression 282C-346A 184 Signal Transduction 347B-393C 206 Pattern Formation 394A-436A 222 Organogenesis and Gametogenesis 437B-520A 237 Chromatin and Gene Expression 521B-546C 265 Neurogenetics and Neural Development 547A-610A 274 Neurophysiology and Behavior 611B-652A 296 Evolution and Quantitative Genetics 653B-718A 310 Immune System and Cell Death 719B-766A 332 Techniques and Genomics 767B-794B 348 Drosophila Models of Human Diseases 795C-846C 358 Physiology and Aging 847A-876C 376 RNA Biology 877A-899B 386

Note: Late abstracts will be listed in the Program Addendum and will be available for viewing in the rear portion of the Grand Exhibit Hall.

PLATFORMS: Immune System and Cell Death 83

1 Immune response to tumors in Drosophila. José C. Pastor-Pareja, Ming Wu, Tian Xu. Howard Hughes Medical Institute, Dept. of Genetics, Yale University School of Medicine, New Haven, CT. We use Drosophila to study cancer biology. In our laboratory, several genome-wide screens for mutations that promote tumor progression and metastasis have been performed. Fly tumors mutant for the polarity determinant scribble that express an oncogenic form of Ras at the same time (RasV12/scrib-/-) exhibit malignant behavior similar to that observed in human metastatic cancers. Features of these tumors include accelerated growth, loss of cell adhesion, basement membrane degradation, migration and invasion, as well as secondary tumor formation. Recently, we have discovered that flies are able to mount an immune response to RasV12/scrib-/- tumors. We are investigating this response and have found so far that tumors activate hemocytes and that hemocytes, in turn, adhere to tumors and affect their growth. We are currently adressing the molecular mechanisms underlying these phenomena and will present our initial findings regarding signaling between tumors and hemocytes and how hemocytes recognize tumors. This Drosophila model provides a tool for dissecting the genetic, molecular and cellular mechanisms of tumor-immune system interactions.

2 The bacterial symbiont Wolbachia confers resistance to viruses in Drosophila melanogaster. Luís Teixeira, Álvaro Ferreira, Michael Ashburner. Department of Genetics, University of Cambridge, Cambridge, United Kingdom. Many microorganisms colonize animals and establish interactions that range from mutualism to parasitism. These symbionts frequently influence their host’s physiology. We found that in Drosophila melanogaster the presence of tetracycline-sensitive bacteria confers resistance to Drosophila C virus (DCV). This resistance is maternally transmitted and the bacteria are intracellular. Using molecular markers we identified the bacteria as Wolbachia pipientis. Wolbachia infects a wide range of invertebrates, including several Drosophila species, and may be the most abundant strain of intracellular bacteria. The lethality induced by DCV infection is reduced in Wolbachia infected flies. Resistance is the result of the viral titre being much lower in Wolbachia infected flies than in those either uninfected or cured by tetracycline treatment. We are now investigating whether or not Wolbachia protects Drosophila from other viruses, and what the mechanism of protection is. To our knowledge this is the first report of a bacterial infection inducing resistance to a viral infection.

3 SENSING OF GRAM POSITIVE BACTERIA IN DROSOPHILA IMMUNITY. Lihui Wang, Petros Ligoxygakis. Dept Biochemistry, Univ Oxford, Oxford, United Kingdom. The genetic tractability of Drosophila melangaster has been proved invaluable in our understanding of host and pathogen interaction. Genetic screenings have identified three putative receptors in the defence against Gram positive bacteria: a glucan binding protein GNBP1, and Peptidoglycan Recognition Proteins: -SA and -SD (PGRP-SA and -SD). Nevertheless, the picture of how these three molecules work in sensing Gram positive invasion is largely sketchy. Using purified recombinant GNBP1, PGRP-SA and -SD, an in- depth investigation was performed on their roles in Gram positive peptidoglycan (PG) sensing from a molecular and biochemical perspective. From this study, it was found that GNBP1 and PGRP-SA form an essential protein complex in recognition of certain species of PG. In addition, GNBP1 exhibited an endomuramidase-like activity releasing smaller PG fragments to enable their recognition by PGRP-SA. PGRP-SD, not a potent PG receptor itself, augmented the binding capacity of GNBP1 to an extended family of PG via protein protein interaction. Furthermore, a high molecular weight complex containing all three proteins could be detected in solution. More importantly, addition of a highly purified PG fragment induced the occurrence not only of the ternary complex but also of dimeric formations among the three proteins. Taken together, these data indicate a synergistic mechanism of GNBP1, PGRP-SA and PGRP-SD in PG sensing to efficiently recognise a broad spectrum of Gram positive bacteria. 84 PLATFORMS: Immune System and Cell Death

4 The Infection-Induced Proteolysis of The Receptor PGRP-LC Activates the IMD Pathway and Melanization Cascades in Drosophila. Amy Tang1,2, Rebecca Schmidt2, Francesca Rinaldo1, Shayla Hesse1, Theodore Trejo1, Zachary Ortiz1, Masakazu Hamada2, Timothy Plummer1, Andrea Page-McCaw3, Jeffery Platt1, Amy Tang1,2. 1) Department of Surgery, Mayo Clinic College of Medicine, Rochester, MN 55902; 2) Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN 55902; 3) Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180. The Drosophila immune deficiency (IMD) pathway, homologous to the mammalian tumor necrosis factor (TNFa) signaling pathway, initiates antimicrobial peptide (AMP) production in response to infection by Gram-negative bacteria. A membrane-spanning peptidoglycan recognition protein, PGRP-LC, functions as a receptor for the IMD pathway. This receptor is known to be activated via pattern recognition and binding of monomeric peptidoglycan (DAP-type PGN) through the PGRP ectodomain. Here we report that treatment of Drosophila with proteases activates the IMD pathway and protease-dependent IMD activation requires the receptor PGRP-LC. PGRP-LC expression is down-regulated upon Gram-negative bacterial infection but is not affected by chemically-fixed bacteria or protease-deficient E.coli in S2 cells and in vivo. An ectodomain (PGRP)-deleted PGRP-LC receptor is functional and constitutively activates both AMP production and melanization. Our results suggest a model in which pathogen invasion and tissue damage may be monitored through the structural integrity of sentinel receptors such as PGRP-LC after host and pathogen are engaged by pattern recognition. We propose that the irreversible cleavage or down-regulation of innate immunity receptors/ligands may provide an additional cue for host recognition of pathogenic microbes and activation of multiple innate defense systems in Drosophila, thereby effectively enhancing its combat of bacterial infection and initiating tissue repair.

5 Developmentally-regulated cell death of Drosophila salivary glands utilizes ER stress-linked apoptosis. Robert Farkas1, Lucia Mentelova1,2, Peter Low3, Gabor Juhasz3, Miklos Sass3. 1) Institute of Experimental Endocrinology, Slovak Academy of Sciences, 83306 Bratislava, Slovakia; 2) Department of Genetics, Comenius University, Bratislava, Slovakia; 3) Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary. Developmentally-associated programmed cell death (PCD) is a genetically encoded form of cell suicide that is required to remove superfluous or potentially dangerous cells. The Drosophila salivary glands (SGs) are highly specialized secretory organs, that serve as an useful model to study PCD as they respond to the steroid hormone ecdysone to undergo programmed histolysis during metamorphosis. Here we present data demonstrating that this tissue dies by an apoptosis-prone mechanism in which endoplasmic reticulum (ER) becomes fragmented into vesicles. Although a low level of autophagy can be seen in the SGs, inducing autophagy with rapamycin does not cause nor accelerates the cell death process. Although the genetic removal of Atg1 function or the RNAi silencing of other Atg genes inhibits the development of autophagy, it does not prevent the vesiculation and subsequent death of the SGs. The Drosophila SG cells exhibit externalization of phosphatidylserine and attract phagocytic macrophages, both pivotal hallmarks of apoptosis. We also show that SG apoptosis is linked to ER disintegration via an ER-stress mechanism. Genetic manipulation of ER-resident proteins (Ca-P60, Crc, Cnx99A,), chaperones and co-chaperones (Cct5, Hop, hsc3, P58IPK) resulted in a widespread and fast vesiculation of the ER, typical of that seen during the final apoptotic stage. In contrast, the genetic removal of the SERCA pump prevented tissue apoptosis. Thapsigargin, an inhibitor of endoplasmic reticular Ca2+-ATPase SERCA causing depletion of ER calcium stores, induces very effective vesiculation and death of SG cells almost instantly. The results presented here describe for the first time the importance of cytosolic chaperones in cell death-associated ER stress. Moreover, activation of Xbp1 implicates the involvement of unfolded protein response (UPR) signaling.

6 Wts function in autophagic cell death of salivary glands. Sudeshna Dutta1,2, Eric Baehrecke1. 1) Cancer Biology, University of Massachusetts Medical School, Worcester, MA; 2) Molecular & Cell Biology Program, University of Maryland, College Park, MD 20742. Proper coordination between cell growth, cell proliferation and cell death is crucial to maintain homeostasis in multicellular organisms. Disruption of these processes can cause disorders including cancer. Previous studies have shown that the Hpo signaling pathway regulates both cell proliferation and apoptotic cell death. We are investigating autophagic cell death of salivary glands, and hope to identify novel mechanisms that distinguish this form of cell death from apoptosis.A high-throughput proteomic screen resulted in the identification of many proteins that are expressed in dying salivary glands including Wts, a serine threonine protein kinase in the Hpo signaling pathway. Either RNAi knockdown of Hpo or loss-of-function mutations in wts, prevent degradation of salivary glands. Surprisingly, wts mutant salivary glands possess markers of both caspase activity and autophagy, which are hallmarks of cell death. The only cellular defect observed in these wts mutant salivary gland cells was their failure in growth arrest. These mutant cells have defects in class I PI3K signaling including altered phosphatidylinositol products and phospho-Akt localization. Although previous studies have shown that Hpo signaling pathway influences apoptosis and cell cycle, our study provides a novel mechanistic link between cell growth and the regulation of autophagic cell death during development. PLATFORMS: Immune System and Cell Death 85

7 Physiological apoptosis in the Drosophila ovarian polar cell lineage involves Hid-mediated activation of a Diap1/Dronc/ Drice cascade. Anne-Marie Pret, Asma Khammari, François Agnès, Pierre Gandille, Elisabeth Boissonneau. Centre de Genetique Moleculaire UPR2167, CNRS/Pierre and Marie Curie University Paris VI, Gif-sur-Yvette, France. Organ morphogenesis involves the strict control of cell number that in some cases has been shown to occur via elimination of excess cells by apoptosis. The underlying mechanisms allowing the precise counting of cells are not well understood. During Drosophila ovarian follicle formation, we have shown that selection of pairs of polar cells, specialized follicular cells present at each follicle extremity, occurs via apoptotic elimination among a small group of approximately 6 precursors produced during early oogenesis. The reduction in polar cell number plays a physiological role during oogenesis since prolonged survival of excess polar cells (via expression of the anti-apoptotic p35 protein) disturbs the differentiation pattern of adjacent follicle cells, including border cells which are necessary for formation of the micropyle, the sperm entry point into the oocyte. We have characterized the cell death machinery responsible for apoptosis of excess early polar cells. We show that it involves the functions of the pro-apoptotic regulator, Hid, the initiator caspase Dronc and its adaptor Dark/Apaf-1 and the effector caspase Drice. In addition, survival of the two mature polar cells requires the function of Diap1. Since we found expression of hid specifically in polar cells destined to die, we are presently testing whether general signaling pathways (Notch, EGFR, Jak-Stat) are responsible for regulation of hid. Finally, we are performing an RNAi-based screen using the GAL4/UAS system to identify regulators of the cell death program in polar cells.

8 Different caspases control distinct mechanisms of apoptosis-induced compensatory proliferation. Yun Fan, Andreas Bergmann. Department of Biochemistry & Molecular Biology, UT MD Anderson Cancer Center, Houston, TX. In multi-cellular organisms, apoptotic cells are capable of inducing compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, it has been shown that dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here, by taking advantage of the developing eye disc, we show that distinct mechanisms control apoptosis-induced compensatory proliferation depending on the proliferating vs. differentiating status of the tissue. In response to apoptotic activity, Dpp and Wg signaling is preferentially induced in proliferating eye tissue similar to wing discs. However, in apoptotic differentiating eye tissue, effector caspases DrICE and Dcp-1 are required redundantly to trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell cycle re-entry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent non-apoptotic function. 86 PLATFORMS: Neurophysiology and Behavior

9 Molecular mechanisms of odor receptor expression and function. Anandasankar Ray1, Wynand van der Goes van Naters2, John R Carlson2. 1) Department of Entomology, University of California, Riverside, CA; 2) MCDB, Yale University, New Haven, CT. Olfactory receptor neurons (ORNs) must select, from a large repertoire, which odor receptors to express. This process produces a highly stereotyped receptor-to-neuron map, thereby posing a remarkably complex problem of receptor gene regulation. We have used phylogenetic analysis amongst multiple Drosophila species to identify positive and negative regulatory elements. Mutational analysis shows that this formidable problem is solved via three classes of mechanisms: by elements that specify the expression of Or genes in the correct olfactory organ, by positive elements that activate Or genes in a subset of ORN classes within an organ, and by negative elements that restrict expression to only one ORN class. This highly precise regulatory program is conserved across species, and the odor response spectra of the ORNs have been remarkably well-conserved for several million years. The odor response spectrum of individual neurons depends upon individual Or genes that are expressed in them. Interestingly, receptors that respond to similar odors can share low sequence identity, which makes it a challenge to investigate mechanisms of receptor-odor interactions. Using a powerful bioinformatic method, we have identified two classes of signature amino acid motifs in the odor receptors; one that is enriched in receptors responding to the same odor, and another that is shared by a large number of receptors. Functional analysis of a signature motif that is present in most receptors reveals a role in signal transduction. On the other hand, the odor-specific class of signatures enables us to predict odor responses of receptors not only from Drosophila but a variety of other insects whose genomes have been sequenced.

10 The Drosophila sex peptide receptor mediates the post-mating switch in female reproductive behaviour. Nilay Yapici, Young- Joon Kim, Carlos Ribeiro, Barry J. Dickson. Molecular Biology and Genetics, Institute of Molecular Pathology, Vienna,Austria. Mating in many species induces a dramatic switch in female reproductive behaviour and physiology. In most insects, this switch is triggered by factors present in the male’s seminal fluid. How these factors exert such profound effects in females is unknown. Here, we identify the receptor for the Drosophila melanogaster sex peptide (SP), the primary trigger of the post-mating response in this species. The sex peptide receptor (SPR) is a G-protein coupled receptor that is specifically activated by low nanomolar concentrations of SP. It is expressed in the female’s reproductive tract, and in the brain and ventral nerve cord of both sexes. Females that lack SPR function, either entirely or only in the nervous system, fail to respond to SP. Such females continue to show virgin behaviours even after mating. SPR is highly conserved structurally and functionally across the insect order, opening up the prospect of novel strategies to control the reproductive and host-seeking behaviours of important agricultural pests and human disease vectors.

11 A novel tetraspanin required for synaptic endocytosis. Chi-Kuang Yao1,3, Yong Qi Lin1,3, Tomoko Ohyama1, Cindy Ly2, Patrik Verstreken1, Karen L. Schulze1,3, Hugo J. Bellen1,2,3. 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) Department of Neuroscience, Baylor Col Medicine, Houston, TX; 3) Howard Hughes Medical Institute, Baylor Col Medicine, Houston, TX. Completion of neurotransmission relies on repeated synaptic vesicle release and retrieval. To identify new players in synaptic transmission, we performed an unbiased forward genetic screen using the eyFLP system. We identified a mutation that affects the morphology of the NMJs and causes a sprouty phenotype. We therefore named the gene flower. More importantly, we observe an obvious defect in endocytosis in these mutants as they exhibit a reduction in FM1-43 dye uptake; a rundown of synaptic transmission at 10Hz but not at 1 Hz stimulation; a severe depletion of vesicle number based on electron microscopy, and an significant increase in endocytic intermediates. flower is required in neurons and the protein localizes to vesicles and periactive zones. The protein contains four transmembrane domains, has not been characterized in any species, and is evolutionarily conserved from worms to man. In addition, most of the conserved amino acids map to the transmembrane (TM) domains, including key negatively charged amino acids in the middle of the TMs. The primary structure of the protein suggests that it may be a channel or transporter. However, the phenotype observed in flower mutants shows striking similarities with synapses that have been treated with La3+, a Ca2+ channel blocker (Kuromi et al., 2004). Based on preliminary data we speculate that flower encodes a novel Ca2+ channel. PLATFORMS: Neurophysiology and Behavior 87

12 Genetic Analysis of AMP-Activated Protein Kinase. Jay Brenman1, Nevzat Kazgan1, Paul Medina1, Vincent Mirouse2, Daniel St Johnston2, Linsay Williamson3, Erik Johnson3. 1) UNC Chapel Hill School of medicine, Chapel Hill, NC USA; 2) University of Cambridge, UK; 3) Wake Forest University, NC USA. AMP-activated protein kinase (AMPK) activity is constituted by a serine-threonine kinase catalytic subunit (α) and two regulatory subunits (β and γ). AMPK is proposed to broadly function as a cellular energy sensor inhibiting energy consuming activities while activating energy producing ones. Outside of a handful of predominantly metabolic enzymes, in vivo downstream targets of AMPK are largely uncertain. Interestingly, however, AMPKα is most homologous to kinases with known roles in cell polarity including SAD kinases and Par-1. During a forward screen searching for genes regulating larval neuronal dendrite development we identified the first know mutations in AMPKα. Animals with mutations in AMPKα contain neuronal dendrites with greatly enlarged dendritic swellings that could be rescued by autonomous expression of a wild type transgene. Mutations in AMPKγ produce nearly the same phenotype. One proposed upstream activator of AMPK activity is LKB1 (Par-4), which has a role in epithelial polarity. When we examined the role of AMPK in epithelial polarity, we found an energy-dependent requirement of AMPKα for maintenance of epithelial cell architecture. In the adult brain loss of AMPK function leads to neurodegenerative phenotypes. Surprisingly, the adult eye phenotypes of different molecules implicated in an AMPK signaling pathway suggest that the current vertebrate signaling pathway may not reflect the in vivo situation. Through structure/function analysis we identify previously unnoticed protein motifs required for AMPKα function in vivo and examine motifs required for subcellular localization. We also identify energy-sensitive behavioral phenotypes in flies expressing different AMPKα mutant transgenes.

13 Functional and Genomic Analyses of Genes Regulated by Fruitless in Adult Head and Central Nervous System Tissues. Thomas Goldman, Michelle Arbeitman. Molecular and Compuational Biology, USC, Los Angeles, CA. In Drosophila, all aspects of somatic sexual differentiation, including differences in adult physiology and behavior, are controlled by the sex determination hierarchy, an alternative pre-mRNA splicing cascade which results in the production of sex-specific transcription factors encoded by fruitless (fru). fru specifies the potential for all aspects of the male courtship ritual, an innate behavior. We have identified genes regulated as a consequence of male-specific FRU (FRU-M) using whole-genome microarray expression analyses. Surprisingly, by comparing genes regulated by FRU-M in all tissues of the adult head to just those in the central nervous system tissues, we found that FRU-M regulates gene expression in non-CNS system tissues, such as those of the adult head fat body. We also present molecular-genetic and behavioral analyses of one FRU-regulated gene, defective proboscis extension response (dpr), which encodes a putative cell adhesion molecule. We show that dpr is co-expressed with FRU-M in cells just below the medial region of the antennal lobe of the brain, and in the first thoracic segment of the ventral nerve cord. A strain homozygous for a hypomorphic allele of dpr, or a strain in which fru expression is reduced in the cells which express dpr, showed reduced latency in male courtship initiation and attempted copulation. Based on the expression patterns and behavioral analyses, we propose a model for how dpr functions in the FRU-M circuit to regulate courtship behaviors.

14 Dynamic range compression by feedback inhibition in the olfactory system. Cory M. Root1, Kaoru Masuyama1, Lina Enell2, Dick R. Nässel2, Jing W. Wang1. 1) Neurobiology Section, Div. of Biological Sciences, University of California, San Diego, La Jolla, CA; 2) Department of Zoology, Stockholm University, Svante Arrhenius vag 14S-106 91 STOCKHOLM, Sweden. The dynamic range of environmental cues is much larger than that of olfactory sensory neurons; therefore, a mechanism to alter sensitivity is required. We have investigated signal compression in a simple neural circuit, the Drosophila olfactory system. Olfactory receptor neurons (ORNs) of the same type converge onto a single glomerulus in the antennal lobe where they synapse onto uniglomerular projection neurons (PNs) and multiglomerular interneurons (INs). All olfactory information is delivered to higher brain centers by PNs and ORNs are the main drivers of PN output. Based on the results of this study, we propose that feedback inhibition is a mechanism to expand the olfactory dynamic range in Drosophila.

In mammalian cortex it is well established that activation of the G protein coupled receptor, GABAB, in presynaptic nerve terminals inhibits voltage gated calcium channels via the βγ complex to suppress neurotransmitter release. Here we report that Drosophila

ORNs express the GABAB receptor that mediates feedback inhibition of ORN presynaptic terminals. Using two-photon imaging of calcium and synaptic transmission, we find that GABAB signalling affects presynaptic calcium and synaptic transmission. Using a promoter fusion transgenic line, as well as RT-PCR and immunohistochemistry, we find that ORNs express the GABAB receptor.

Furthermore, blocking GABAB receptors reduces the dynamic range of ORN synaptic transmission that is matched by similar modulation of PN response, suggesting a presynaptic mechanism to alter the dynamic range of a sensory system. Strikingly, we find that different ORN channels have different levels of GABAB expression that is correlated with physiological sensitivity to GABAB modulation. Our findings reveal that different sensory channels have different levels of presynaptic inhibition that may permit heterogeneous modulation of dynamic range for different environmental cues. 88 PLATFORMS: Neurophysiology and Behavior

15 A novel vesicular neurotransmitter transporter expressed in the mushroom bodies and central complex is required for learning and female receptivity. Elizabeth S. Brooks1, Bac T. Nguyen1, Christopher J. Tabone2, J. Steven de Belle2, David E. Krantz1. 1) Semel Institute for Neuroscience, UCLA, Los Angeles, CA; 2) Department of Biological Sciences, UNLV, Las Vegas, NV. The mushroom bodies (MBs) are a distinct central brain structure required for olfactory learning and memory in Drosophila melanogaster. To date, the neurotransmitters used for signaling by the Kenyon cell neurons intrinsic to the MBs have not been clearly identified. However, we postulate that transporters must exist to move these neurotransmitters across cellular membranes. Here we report the identification of a cDNA for a predicted gene (CG10251) that functions as a vesicular neurotransmitter transporter in a subset of Kenyon cells. The CG10251 protein is similar in structure to, but distinct from, other known vesicular transporters. mRNA expression is low during development and high in the adult head. An antiserum raised against the carboxy terminus of the protein detects expression in a few neurons in the larval ventral ganglion, robust expression in the larval MB Kenyon cells as well as a large extrinsic neuron with projections into the MB lobes. In the adult brain, we similarly find robust protein expression in the MBs in a subset of Kenyon cell fibers in the peduncle and all 5 MB lobes. In addition, projections to both the optic ganglia and central complex become visible in the adult. Biochemical fractionation reveals the CG10251 protein localizes to synaptic vesicles, consistent with its proposed role as a vesicular transporter. We have generated a mutant through imprecise excision of a P-element just upstream of CG10251. Behavioral studies indicate this mutant has reduced fertility as well as altered copulatory behavior including reduced female receptivity. Olfactory learning assays reveal a modest but significant learning deficit in these mutants. We suggest that the CG10251 protein may be responsible for the storage of neurotransmitter in the MBs and the central complex and thus critical for both learning and sexual behavior function in D. melanogaster. We propose to rename the CG10251 gene portabella.

16 Regulation of Drosophila male courtship by complex integration of sensory information. Werner Boll1, Dimitrije Krstic1,2, Markus Noll1. 1) Institute of Molecular Biology, University of Zürich, Zürich, Switzerland; 2) Ph.D. Program in Molecular Life Sciences, Zürich, Switzerland. Courtship in Drosophila melanogaster is performed as a stereotyped and robust sequence of innate behavioral steps. A male perceives the presence of a potential mate through his visual, olfactory, and gustatory senses that direct him to initiate courtship, which in turn elicits a response from the courted animal. These different senses control courtship behavior and provide to courter and courtee a plethora of information about gender, conspecificity, receptivity, and sexual fitness. Understanding how the various sensory cues are interpreted by a male encountering a potential mate appears essential to comprehend the logic of the neuronal network that directs this complex behavior. To this aim, we utilized three mutations that inactivate different sensory modalities, either separately or in combinations, without affecting the functions of the CNS. Our results do not only describe the interplay and relative importance of the different sensory modalities in the male during courtship, but also show that the neural circuitry is reconfigured in the dark to compensate for the absence of visual cues. Analysis of male-male courtship reveals that the integration of visual, gustatory, and behavioral, but not olfactory cues determines the sexual orientation of the courting male. Based on these studies, we propose a model illustrating how the brain of a naïve Drosophila male integrates the various sensory stimuli during courtship. PLATFORMS: Organogenesis 89

17 Control of stem cell self-renewal and differentiation in the adult Drosphila intestine. Allison Bardin, Carolina Perdigoto, François Schweisguth. Dept Biol, Ecole Normale Superieure, Paris, France. A major challenge in stem cell research is to understand how different strategies are utilized in different biological contexts to allow both self-renewal and proper differentiation of stem cell progeny. Drosophila melanogaster provides several models of stem cell systems and has extensively developed genetic tools with which to address mechanisms of self-renewal and differentiation. The adult Drosophila melanogaster instestine harbors stem cells (ISCs) that provide newly differentiated cells throughout the adult lifetime, likely replacing those which turnover (Micchelli and Perrimon, Nature, 2006; Ohlstein and Spradling, Nature, 2006). The ISCs have been shown to employ the Notch signaling pathway to control the differentiation of their progeny. The Notch pathway is known to play important roles in many stem and progenitor cell systems (reviewed in Wilson and Radkte, Febs Lett., 2006). We are using the ISC to address how the Notch pathway is regulated during homeostasis in the adult intestine. In particular we are trying to understand how asymmetric fate of the daughter cells is controlled and what regulators of the Notch pathway contribute to this. We will present our ongoing work to understand the maintenance of stem cell identity, self-renewal and proliferation in the adult Drosophila intestine.

18 FoxK mediates TGF-beta signalling during midgut differentiation. Sergio Casas-Tinto1,2, Melisa Gomez-Velazquez1, Begoña Granadino-Goenechea2, Pedro Fernandez-Funez1. 1) Dept Neurology, UTMB, Galveston, TX; 2) Centro de Investigaciones Biologicas, CSIC, 28040 Madrid (Spain). The genetics and molecular mechanisms of endoderm differentiation are the least known of the three germ layers. It is clear, though, that inductive signals across germ layers play a key role in the development of the endoderm. In flies, the visceral mesoderm secretes TGF-beta/Dpp along with other signaling molecules that diffuse into the underlying midgut endoderm to control tissue- specific differentiation. TGF-beta signaling induces Mad phosphorylation, while the nuclear translocation of pMad regulates the expression of the Hox protein Labial. We report here the molecular and functional characterization of Drosophila FoxK, a transcription factor of the fork head box (Fox) family that responds to TGF-beta signalling and mediates labial regulation in the embryonic midgut endoderm. Specific antibodies raised against FoxK indicate a remarkable accumulation in the developing endoderm. The analysis of newly generated FoxK mutant alleles revealed that the embryos failed to generate midgut constrictions and lacked expression of Labial, a Hox protein critical for midgut differentiation. Our studies suggest that TGF-beta signalling directly regulates FoxK in midgut endoderm through functional Smad/Mad binding-sites on its 5’ region. Interestingly, FoxK can induce ectopic Labial expression only when co-expressed with the transcription factor Dfos/AP-1. Thus, FoxK and Dfos/AP-1 cooperatively regulate labial expression in midgut endoderm. Moreover, FoxK and Dfos/AP-1 can induce Labial expression even in the absence of pMad, indicating that the main role of Mad is to activate FoxK and Dfos/AP-1 in the endoderm. Then, FoxK and Dfos/AP-1 directly regulate labial to promote differentiation of parasegment 7 of the endoderm. Thus, we describe here a novel regulator of endoderm differentiation. Also, we propose a new mechanism for TGF-beta signalling that involves the sequential activation of tissue-specific transcriptional regulators during midgut endoderm differentiation.

19 The combinatorial control of muscle identity. Jonathan Enriquez, Laurence Dubois, Virginie Daburon, Michèle Crozatier, Alain Vincent. centre de biologie du developpement, Toulouse, France. Specification of muscle identity is a multistep process : Early positional information defines competence groups, from which muscle progenitors are selected, followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle whose morphological and functional properties have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. We addressed this question, using Collier (Col) expression as both a determinant and read-out of DA3 muscle identity. Characterisation of the col upstream region driving DA3 muscle-specific expression revealed the existence of two separate phases of cis regulation, correlating with conserved binding sites for different mesodermal transcription factors. Loss-of-function and gain- of-function experiments further showed that both Nautilus and Col were required for col activation in the myoblast nuclei that fuse to form the DA3 myofiber, thereby ensuring that all express the same identity program. Overexpression of Col and Nau, but neither Col nor Nau alone, can induce the formation of two DA3 muscles. Together, our results indicate that separate sets of cis-regulatory elements are required for expression of muscle identity factors in muscle progenitors and myofiber nuclei and support the concept of combinatorial control of muscle identity (Dubois et al., Development 2007, in press). Further analysis of col and nau mutant phenotypes showed that Col is required for the fusion process while Nau was required for the correct shape, orientation and epidermal attachment of the DA3 muscle, indicating that Nau and Col regulate, at least partly, different sets of genes. Finally, the role of homeotic genes in the final size of the DA3 muscle will be discussed. 90 PLATFORMS: Organogenesis

20 A key role of Pox meso in somatic myogenesis of Drosophila. Cheng Zhang1,5, Hong Duan1,3,5, Jianming Chen1,4, Helen Sink2, Erich Frei1, Markus Noll1. 1) University of Zurich, Institute of Molecular Biology, Zurich, Zurich, Switzerland; 2) Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, U.S.A; 3) Present address: Sloan-Kettering Institute, Department of Developmental Biology, 1275 York Avenue, New York, NY 10021, U.S.A; 4) Present address: Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, U.S.A; 5) These authors contributed equally to this work. Making a muscle in a vertebrate is both similar and different from that in Drosophila. Similar elements are deployed differently in flies and vertebrates. Are these differences real divergence of different developmental programs or rather variations of a common program is an interesting, yet presently unanswerable question. In vertebrates, Pax3 and Pax7 are important for the proper development of myogenic progenitor cells as well as for skeletal muscle development and regeneration. In Drosophila, the Pax gene Pox meso (Poxm), which belongs to the Pax1/9 family, was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. By studying the temporal and spatial expression patterns of Poxm and its loss- and gain-of-function phenotypes, we can demonstrate that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute, demarcates the ‘Poxm competence domain’, a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2, and VA3. Our results led us to reinterpret the roles of l(1)sc and twi in myogenesis and to propose a solution of the ‘l(1)sc conundrum’.

21 Two distinct progenitor populations remodel the Drosophila tracheal system during metamorphosis. Molly Weaver1,2, Mark Krasnow1,2. 1) Department of Biochemistry, Stanford University School of Medicine, Stanford, CA; 2) Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA. Drosophila larvae are composed of differentiated larval cells and undifferentiated progenitor cells called imaginal cells. At metamorphosis, most larval cells die whereas imaginal cells proliferate and differentiate into pupal and adult tissues. We find that there are two distinct populations of tracheal (respiratory system) progenitors : a classical population of imaginal cells that are set aside early in development in spiracular branch nests (SB tracheoblasts), and a novel population of differentiated larval dorsal branch (DB) stalk cells that transform into multipotent progenitors (DB tracheoblasts) late in larval life, and subsequently differentiate into three cell types under control of Breathless FGFR signaling. DB stalk cells are the first differentiated Drosophila cells found to naturally reenter the cell cycle, and provide a model for facultative stem cells in mammals, differentiated cells in the lung and other organs that are thought to replenish tissues after damage or disease.

22 Dynamic Regulation of the Eye Specification Network. Claire Salzer, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. The eye specification network, as it is currently modeled, consists of ten nuclear proteins that are woven into a complicated regulatory labyrinth. The regulatory relationships that are described for this cascade are based a number of criteria including in vivo molecular genetic analysis, in vitro biochemical and molecular assays as well as bioinformatic analysis of promoter sequences. Much of the in vivo data is focused on the effect that the removal of a network member has on the expression of the remaining genes. This analysis has led to the assignment of positive and inhibitory arrows throughout the cascade. We have generated retinal mosaic clones of several components of the network and have reexamined the regulatory relationships that exist amongst the pathway members. In particular we have focused on the relationship between sine oculis (so), eyes absent (eya) and dachshund (dac). The current model implies that the So-Eya protein complex promotes the expression of dac. We find that this is true only ahead of the morphogenetic furrow and in the first few columns of developing ommatidia. In more posterior regions of the retina both so and eya cooperate to repress dac. The implication is that the So-Eya complex can switch between the activation and repression states. Our results also demonstrate that several other regulatory relationships in the eye are dependent upon the geographical position within the developing eye field. We suggest that the eye specification network is much more dynamic than is currently envisaged by extant models. We will present and discuss our findings as well as present a more dynamic model that incorporates geographical positions. PLATFORMS: Organogenesis 91

23 Regulation of sexually dimorphic gonad development in Drosphila melanogaster. Nicole Camara, Mark Van Doren. Department of Biology, Johns Hopkins University, Baltimore, MD. The creation of sexual dimorphism during development is required for successful reproduction of the species, and is dependent on proper sex determination. The Drosophila gene doublesex (dsx) encodes a transcription factor that is required for all aspects of male and female somatic development outside the nervous system. DSX is a member of a family of DM containing proteins which are involved in sex-determination from vertebrates to invertebrates. Although dsx is understood to be a key sex-determination gene in Drosophila, little is known about how it functions to control sexually dimoprhic development. Here we investigae the role of dsx in gonad development in order to address remaining questions about dsx function. Is it required early or late for sex specific gonad development? Is it required autonomously or non-autonomously? What are the targets of DSX? We find that embryonic gonad development begins along a male pathway as evidenced by the presence of 3 male specific cell types in dsx mutant embryos. We propose that DSX may not be required for the initial specification of these cells, but may be required for their proper maitenance or function. Analysis of dsx mutant adult phenotypes indicates that both XX and XY dsx mutants can form either a male-like or female- like germline stem cell niche. Female germ cells appear more receptive to a male-like soma, than male germ cells to a female-like soma. We also present preliminary analysis of a new gene, CG5149, that may act downstream of dsx to control sexual dimorphism. CG5149 is expressed in the female soma and is absent from the male soma after gonad formation In later stages, CG5149 is also expressed in cells which become the male germline stem cell niche. This suggests that CG5149 may have distinct roles in male and female gonad development. CG5149 adults have a severe gonad defect with reduced germline in both males and females. CG5149 encodes a highly conserved protein of unknown function, and we are investigating its role in gonad development.

24 The Regulation and Function of Cad74A in Drosophila Oogenesis. Jeremiah J Zartman, Nir Yakoby, Chris A Bristow, Stanislav Y Shvartsman. Lewis Sigler Institute and Department of Chemical Engineering Princeton University, Princeton, NJ. During Drosophila oogenesis, a two-dimensional follicular epithelium encapsulating the developing egg gives rise to an elaborate three-dimensional eggshell, which includes tubular structures called dorsal appendages that project out from the main body. Egg morphogenesis and dorsal appendage formation has been studied extensively, but few connections have been made between signaling pathways, pattern formation, and the physical implementation of the morphogenetic program in this tissue. In an effort to address the gap between pattern formation and organogenesis, we have characterized the regulation and function of the non- classical cadherin, Cad74A, which is expressed starting in mid-oogenesis in all the columnar follicle cells contacting the oocyte except for two dorsolateral patches. The dorsolateral patches correspond to the cells that form the roof of the dorsal appendages. Using an antibody to Cad74A, we show that Cad74A repression in the dorsolateral patches is mediated by high levels of the transcription factor Br, which in turn is regulated by the integration of EGFR and Dpp signaling, the two pathways that are essential for patterning of the follicle cells. We also report the functional analysis of Cad74A which shows subtle phenotypes when expression levels are perturbed. On the basis of these results, we propose a model for Cad74A regulation and function during egg development. 92 PLATFORMS: Regulation of Gene Expression

25 The zinc finger protein Zelda is a key activator of the zygotic genome in Drosophila. Hsiao-Lan Liang1, Chung-Yi Nien1, Hsiao- Yun Liu1, Mark Metzstein2, Nikolai Kirov1, Christine Rushlow1. 1) Biology, New York Univ, New York, NY; 2) Human Genetics, Univ. of Utah, Salt lake city, UT. The control of embryogenesis is initiated by maternal gene products and later transferred to the zygotic genome in a process called the maternal to zygotic transition (MZT). Such processes involve the degradation of maternal transcripts and the activation of transcripts (Newport & Kirschner, 1982). Although general activators of the early zygotic genes have been postulated for years, none have actually been identified, in any organism. A recent finding that many early zygotic genes in Drosophila share a cis- regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAG-team sites (ten Bosch et al., 2006; De Renzis et al., 2007) suggests that a dedicated transcription factor may interacts with these sites to activate the zygotic genome. Here we report the discovery of a zinc-finger protein, Zelda (Zld), that binds specifically to TAG-team sites in the zen regulatory region, and is capable of activating transcription in transient transfection assays. Mutants lacking zld transcripts are defective in specific aspects of the cellularization process and fail to activate transcription of a large fraction of early zygotic genes, including zld and dpp, a sample of key cellularization genes, and the sex determination genes. These results suggest that Zelda may be a key activator of the genome activation in Drosophila.

26 Application of a cis-regulatory grammar for functional characterization of transcriptional regulatory elements in the Drosophila embryo. David N. Arnosti1, Ahmet Ay2, Chichia Chiu2, Evan Dayringer2, Rupinder Sayal1, Walid Fakhouri1. 1) Dept Biochemistry & Molec Biol, Michigan State Univ, East Lansing, MI; 2) Dept of Mathematics, Michigan State Univ, East Lansing, MI. To understand the specific and general features of cis regulatory elements controlled by key patterning regulators of the early embryo, we have quantitatively analyzed and modeled the activity of synthetic and semi-synthetic transcriptional modules regulated by short-range repressors Knirps, Giant, and Kruppel in the blastoderm embryo. Confocal laser imaging allows us to quantitatively “map” the gene regulatory surfaces associated with particular arrays of regulatory sequences. We assess a database of over 600 quantitatively analyzed embryos representing two dozen permutations of enhancers, and use this information to pursue two “bottom up” mathematical models. We have developed a three-tier quantitative mathematical model that associates potential functions with subelements of the enhancer, and combine them to provide predictions of the functional output of novel transcriptional elements. We have also adapted a fractional occupancy model that considers specific features of spacing and cooperativity in predicting gene activity. Using these approaches, we have accurately predicted the effects of spacing and stoichiometry between activators and repressors, a key feature of short-range repression, and are applying them to analyze the output of endogenous regulatory sequences. Our work complements “top down” approaches, and is aimed at informing and extending the power of current models of endogenous cis-regulatory elements to develop powerful bioinformatic tools applicable to population and evolutionary studies.

27 Enhancer identification by comparative genomics relies on abundant non-conserved DNA. Brant Peterson1, Emily Hare1, Venky Iyer1, Rick Kurashima3, Eric Jang3, Michael Eisen1,2. 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA; 2) Lawrence Berkeley National Lab, Berkeley, CA; 3) USDA Agricultural Research Service - PBARC, Hilo, HI. The identification of regulatory sequences in animal genomes is a significant challenge for computational and experimental genomics. The comparative genomic methods that have worked so well in vertebrates - in which unusually conserved non-coding sequences are often active in functional assays - have not worked well for members of the family Drosophilidae and other invertebrates. Here we apply these methods to three species of true fruit flies (Tephritidae) with genomes four to five times larger than D. melanogaster. We show that there are many discrete non-coding sequences conserved between these Tephritid species, and that nearly all (12/ 13) of the sequences we tested have regulatory activity in transgenic D. melanogaster embryos. We propose that the success of these methods in Tephritids, after their failure in Drosophila, is due to the presence of additional rapidly evolving (presumably largely non-functional) sequence between functional elements in the larger Tephritid genomes. Intriguingly, the expression pattern of one tested gene (giant) displays substantial divergence in embryos of the Tephritid B. dorsalis as compared to D. melanogaster, and furthermore the Tephritid enhancer is sufficient to reproduce the Tephritid pattern in the D. melanogaster embryo, indicating that the primary mechanism of expression evolution in this case is change in cis. We also demonstrate that mapping these conserved elements to the D. melanogaster genome clearly and accurately predicts functional regulatory elements from Drosophila using only primary sequence data, thus marking the first unambiguous success of a purely comparative approach to enhancer finding in D. melanogaster. We hypothesize that the general failure of comparative genomic methods in invertebrates to date is largely due to a bias for sequencing small genomes, and demonstrate that the sequencing of large genomes will greatly advance the identification of regulatory sequences. PLATFORMS: Regulation of Gene Expression 93

28 Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. Emily Hare1, Brant Peterson1, Venky Iyer1, Rudolf Meier2, Michael Eisen1,3. 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA; 2) Department of Biological Sciences, National University of Singapore, Singapore; 3) Genomics Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, CA. Although changes in developmental gene expression play an important role in the evolution of organismal form and function, the molecular basis for such changes is poorly understood. To characterize the evolutionary dynamics of regulatory sequences and constraints on their function, we have sequenced and characterized the regulatory activity of the even-skipped locus from five fly species in the acalyptrate family Sepsidae. We visualized the expression patterns of key regulators of anterior-posterior patterning in the sepsid Themira minor and found them to be qualitatively similar to their Drosophila counterparts, suggesting that this regulatory network is conserved between the families. As in Drosophila, there is considerable non-coding sequence similarity between even the most distantly-related sepsid species. There is, however, very little non-coding sequence similarity between sepsids and drosophilids. We used within- and between-family comparisons, as well as binding site content, to identify cis-regulatory regions in the sepsid eve loci and characterized their activities in transgenic D. melanogaster embryos. The RNA expression patterns driven by these enhancers largely overlap the endogenous D. melanogaster eve pattern with several interesting differences. That this functional conservation occurs despite the nearly complete absence of primary sequence or transcription factor binding site conservation demonstrates that the constraints on enhancer organization are fairly loose. Strikingly, each eve enhancer contains a small number of highly-conserved elements that overlap known functional sites in D. melanogaster. These highly-conserved elements are highly enriched for pairs of binding sites that overlap or neighbor each other, suggesting that functional constraint acts on a larger unit than the single binding site.

29 Cell fate specification in the blastoderm embryo involves developmental regulation of transcription elongation. Peter Gergen1, Lisa Prazak1, Xiaoling Wang1, Kevin Celestrin1, Hyowon Choi2, Giorgio Medranda1. 1) Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, NY; 2) Mount Holyoke University, South Hadley, MA. The simple combinatorial rules responsible for the metameric expression of sloppy-paired (slp1) provide several advantages for investigating the mechanisms of transcriptional regulation during cell fate specification in the Drosophla embryo. Recent studies show that the initial repression of slp1 in response to the pair-rule transcription factors occurs at a step downstream of PolII recruitment and transcripton inititation and involves the developmental regulation of transcription elongation. Biochemical experiments indicate that the negative elongation factor NELF participates in this process. Results of genetic experiments that investigate the in vivo function of NELF will be presented. We have also undertaken studies to identify the cis-regulatory determinants that contribute to the establishment and regulated elongation of a paused PolII complex on the slp1 promoter. These studies use PhiC31 integrase mediated site-specific integration of different reporter genes into the same chromosomal location in order to maximize the ability to reliably detect subtle and quantitative differences in gene expression. The results of experiments that investigate the functional interactions between two pair-rule responsive upstream enhancer elements and the slp1 promoter will be reported.

30 A comprehensive catalog of homeodomain DNA-binding specificities from D. melanogaster. Michael Brodsky1, Marcus Noyes1, Ryan Christensen2, Atsuya Wakabayashi1, Gary Stormo2, Scot Wolfe1. 1) Program in Gene Function & Expression, Univ Massachusetts Medical School, Worcester, MA; 2) Department of Genetics, Washington University, School of Medicine, St. Louis, MO. Homeodomain-containing proteins represent the second-largest family of transcription factors in animal genomes. A full description of homeodomains binding specificities is critical to understand how they regulate the expression of distinct target gene sets. We have characterized the DNA binding specificities of all 84 independent homeodomains from Drosophila, the first comprehensive characterization of homeodomain specificities from a metazoan genome. To visualize the range of DNA-binding specificities within this set, we performed a hierarchical clustering of transcription factors based on the similarity of their DNA recognition motifs. The majority of Drosophila homeodomains can be organized into 11 specificity groups. Some groups contain a large number of members (25 homeodomains are part of the “En group”), whereas others contain only 2 to 5 members. An additional 5 homeodomains have unique specificities that are not associated with any group. Thus, while many homeodomains recognize related DNA sequences, the homeodomain architecture can support a wide range of different DNA binding specificities. Correlation of DNA-binding specificities with residues at the protein-DNA interface, along with previous structural and biochemical analysis of homeodomains, has allowed us to construct a recognition code describing mechanisms of sequence discrimination by this family. We have confirmed the predictive power of this code with mutagenesis experiments in which one or a few amino acid changes predictably alter the DNA binding specificity of a homeodomain, including one example in which the preferred binding site is altered at four of six positions. By combining this recognition code and analysis of overall sequence similarities, we can use the Drosophila data to predict the specificities of the majority of human homeodomain proteins. These studies provide a framework to broadly predict the specificity of homeodomains from all metazoans. 94 PLATFORMS: Regulation of Gene Expression

31 Regulation of muscle identity by homeodomain transcription factors. Brian Busser1, Aditi Singhania1, Savina Jaeger2, Anton Aboukhalil2, Michael Berger2, Caitlin Gamble1, Stephen Gisselbrecht2, Martha Bulyk2, Alan Michelson1. 1) LDSB, NHLBI/NIH, Bethesda, MD; 2) Division of Genetics, Brigham & Women’s Hospital, Boston, MA. Homeodomain (HD) transcription factors (TFs) have been proposed to control the unique gene expression programs of individual muscle founder cells (FCs). We have investigated this hypothesis with an integrated genomics approach that combines genome- wide expression profiling, TF binding site determination, in silico evaluation of combinatorial TF codes, empirical testing of candidate enhancers, and both cis and trans tests of target gene regulation. We first showed by in situ hybridization that a small set of previously characterized FC genes is differentially responsive to over-expression of the muscle HD TFs Slouch, Muscle segment homeobox and Apterous. FC gene expression was activated and/or repressed by these TFs and responsiveness correlated with TF co-expression in wild-type embryos. Next, we extended the identification of HD-responsive genes on a genome-wide scale by expression profiling purified mesodermal cells from embryos in which an individual HD TF is over-expressed. These experiments revealed that different FC genes can be activated, repressed or remain unaffected by ectopic HD TFs. These effects are directly mediated at FC enhancers, as mutagenesis of HD binding sites inactivates known FC cis-regulatory modules (CRMs). Computational searches for combinations of TF motifs that are overrepresented in the noncoding regions of HD-responsive FC genes defined a HD-containing set of TFs. Predicted CRMs conforming to this TF code were functional FC enhancers. Unexpectedly, HD TFs also activated genes expressed in fusion competent myoblasts, which do not normally express these TFs. These results suggest that HD TFs regulate two distinct temporal waves of myogenic gene expression, one in the developing muscle FC, and a second in the mature multinucleated myotube. These studies provide new insights into the role of individual HD TFs in specifying cellular identity and into the transcriptional codes that regulate muscle gene expression.

32 Sequential developmentally programmed steps at target promoters reverse repression by Polycomb for terminal differentiation in a stem cell lineage. Xin Chen, Jose Morillo, Chenggang Lu, Margaret Fuller. Dev Biol, Stanford Univ, Stanford, CA. Emerging evidence indicates that repression by the Polycomb group (PcG) machinery may maintain terminal differentiation genes in a silenced state in precursor cells in embryonic and adult stem cell lineages. A major question remains how this epigenetically silenced state, capable of being maintained through many cell generations, is reversed to allow expression of differentiation genes appropriate to distinct cell types and developmental stages. Here we show that a series of events, orchestrated by the developmental program, reverses PcG repression to allow expression of terminal differentiation genes in the Drosophila male germ line adult stem cell lineage. In precursor cells, PRC2 components are expressed and terminal differentiation genes are transcriptionally silent, lack RNA PolII at the promoters and carry the H3K27me3 histone modification made by the E(z) enzyme. After the transition from proliferating spermatogonia to differentiating spermatocyte, the PRC2 components E(z) and Su(z)12 are downregulated and the testis TAF homologs (tTAFs) are expressed. However, action of tMAC, a testis specific version of the MIP/DREAM complex, is required for the recruitment of tTAFs and displacement of Polycomb (Pc) from the promoters of target differentiation genes. The tMAC is also required for relocalization of PRC1 components and tTAFs to the nucleolus. In addition, the PolII appears to arrive at the promoters of differentiation genes upon the switch from spermatogonia to spermatocyte, and is present even in tMAC or tTAF mutants, in which the targets are not expressed, suggesting that the PolII may be stalled at the promoter by Pc. These observations suggest terminal differentiation genes are turned on through a series of cell type and stage specific developmentally regulated events, in which repression by PcG set up in precursor cells initially blocks progression of RNA polymerase. After the cell fate switch to spermatocyte state, subsequent actions of cell-type specific transcriptional machinery then transform the chromatin landscape to allow terminal differentiation.

33 dHCF is required for Myc-dependent transcription regulation in Drosophila. Michael Furrer, Mirjam Balbi, Peter Gallant. Zoological Institute, University of Zürich, Zürich, Switzerland. dMyc is the Drosophila homolog of the vertebrate Myc onco-proteins, and it controls growth, proliferation and apoptosis during normal development. In order to exert its biological function, dMyc (in a complex with its partner protein dMax) binds to specific DNA sequences and activates or represses the expression of nearby genes. While a large number of dMyc targets have been identified, the mechanism by which dMyc controls their expression is not fully understood, although it seems to rely on the recruitment of chromatin modifiers. In order to identify transcriptional co-activators for dMyc, we have carried out an RNAi screen in S2 cells, and identified the Drosophila homolog of human host cell factor-1 (HCF-1) as an essential co-factor for dMyc. dHCF physically associates with dMyc and is required for the correct expression of dMyc targets in S2 cells. Downregulation of dHCF in vivo interferes with dMyc dependent growth and development, whereas dHCF overexpression synergizes with dMyc in the control of growth. These data suggest that dMyc controls the expression of its targets in part via recruitment of dHCF. This is in accordance with the role of dHCF as a component of several chromatin modifying complexes. dHCF physically interacts with the ATAC histone acetyltransferase complex, as well as with the trithorax-related Set1/Ash2 methyltransferase and the Sin3A containing histone deacetylase complexes. Taken together dHCF might act both as a positive and negative transcription regulator, by selectively modulating chromatin structure and, in addition, connect these chromatin-modifying activities to the transcription factor dMyc. PLATFORMS: Regulation of Gene Expression 95

34 Integration of inputs from multiple modular elements generates a gradient of transcriptional repression in response to the Dpp morphogen. Rahul Warrior1, Yao Li-Chin1, Phin Sopheap1, Rushlow Christine2, Arora Kavita1. 1) Dept Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Department of Biology, New York University, New York, NY 10003. Morphogen gradients play fundamental roles in patterning and cell specification during development by eliciting differential transcriptional responses in target cells. In Drosophila, Decapentaplegic (Dpp), the BMP2/4 homolog, downregulates transcription of brinker (brk) in a concentration-dependent manner to generate an inverse graded distribution of the nuclear repressor. Both Dpp and Brk are critical for directing Dpp target gene expression in defined domains and the consequent execution of distinct developmental programs. Thus determining the mechanism by which the brk promoter interprets the Dpp activity gradient is essential for understanding both Dpp-dependent patterning, and how graded signaling activity can generate different responses through transcriptional repression. We have uncovered several key features of the brk promoter that suggest it utilizes a complex enhancer logic not represented in current models. First, we find that the regulatory region consists of multiple, dispersed, compact modules that can independently drive brk-like expression patterns. Second, we show that each module contains binding sites for the Schnurri/Mad/Medea (SMM) complex, which mediates Dpp-dependent repression, linked to regions that direct activation. Third, we find that the SMM repression complex acts through a distance-dependant mechanism that uses the canonical co-repressor C-terminal Binding Protein (CtBP). Finally, we provide evidence that the brk promoter employs a modular organization in which multiple enhancer inputs acting in parallel are integrated to generate the final pattern. We suggest that this specialized architecture underlies the ability of brk to respond to the Dpp gradient in a precise and robust fashion.

35 The Mechanism of Dscam Mutually Exclusive Splicing. Brenton Graveley1, Sara Olson1, Marco Blanchette2,5, Jung Park1, Yiannis Savva1, Gene Yeo3, Joanne Yeakley4, Donald Rio2. 1) Dept Genetics & Dev Biol, Univ Connecticut Health Ctr, Farmington, CT; 2) Department of Molecular and Cell Biology, Center for Integrative Genomics, University of California, Berkeley, CA 94720-3204 USA; 3) 4Crick-Jacobs Center for Theoretical and Computational Biology, Salk Institute, LaJolla, CA 92037 USA; 4) Illumina Inc., 9885 Towne Centre Drive, San Diego, CA 92121-1975 USA; 5) Stowers Institute for Medical Research, Kansas City, MO 64110 USA. The Down Syndrome Cell Adhesion Molecule (Dscam) gene plays essential roles in neural wiring and pathogen recognition in Drosophila. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. We have focused on understanding the mechanism of mutually exclusive splicing in the exon 6 cluster which contains 48 variable exons. Using comparative genomics we have discovered a complex system of competing RNA structures that act to ensure that only one variable exon is spliced to the upstream constitutive exon. Here we show that the hnRNP protein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents SR proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.

36 The role of site accessibility in microRNA target recognition. Nicola Iovino1, Michael Kertesz2, Ulrich Unnerstall1, Eran Segal2, Ulrike Gaul1, (NI,MK,ES,UG)=equal contributions. 1) Laboratory of Developmental Neurogenetics, Rockefeller University, New York, NY; 2) Weizmann Institute, Rehovot, Israel. MicroRNAs, genomically encoded small RNAs of 21-24 bases, have recently emerged as an important class of post-transcriptional regulators, but the precise mechanisms underlying their interaction with their mRNA targets are still poorly understood. Most studies so far have focused on the quality of the sequence match between microRNA and target and ignored the secondary structure of the target mRNA. We decided to systematically explore the importance of target site accessibility in microRNA-mRNA interactions. Using microRNAs expressed in S2 cells, we developed a sensitive linear reporter assay, in which neither microRNA nor target mRNA are overexpressed and the measured reductions in reporter activity are solely attributable to translational repression. We used this assay to measure over 60 microRNA-target interactions, spanning a wide range of target types and mutation designs, and found that mutations diminishing target accessibility significantly reduce microRNA-mediated translational repression, with effects comparable to those of mutations that disrupt sequence complementarity. Based on these observations, we devised a parameter- free thermodynamic model for microRNA-target interaction that computes the difference between the free energy gained by formation of the microRNA-target duplex, and the energetic cost of unpairing the target to make it accessible to the microRNA. We integrated site accessibility into a genome-wide target prediction program (PITA) and applied it to all 3’UTRs of fly, worm, mouse and human. We find that, in all four organisms, microRNAs sites show a marked preference for highly accessible regions, suggesting that genomes accommodate site accessibility by preferentially positioning targets in highly accessible regions. Thus, target accessibility proves to be a key factor in microRNA function. 96 PLATFORMS: Regulation of Gene Expression

37 Mextli, a novel eIF4E-binding protein from Drosophila. Greco Hernandez1,2, Gritta Tetweiler1,2, Mathiu Miron1,2, Paul Lasko1, Nahum Sonenberg2. 1) Dept. of Biology, McGill University; 2) Dept. of Biochemistry. McGill University. Eukaryotic translation initiation factor eIF4E is a key regulator of translation. Regulation of eIF4E is central to overall control of protein synthesis. eIF4E-binding proteins (4E-BPs) play a critical role in the regulation of eIF4E. They bind the dorsal convex surface of eIF4E to form a translationally inactive eIF4E/4E-BP complex. Upon stimulation of cells with hormones or growth factors, 4E-BPs become phosphorylated and dissociate from eIF4E, rendering eIF4E available to interact with eIF4G to form the translationally active eIF4G/eIF4E complex. Recently, the interaction of eIF4E with other proteins, including 4E-transporter (4E-T) and maskin, has been shown to regulate specific cellular or developmental processes. For these reasons, we are searching for additional eIF4E binding proteins in Drosophila. From a far-western genomic screen on a 20-22 h-old embryonic cDNA library, using 32P-labeled FLAG-HMK-eIF4E1 as a probe, we identified a novel eIF4E-binding protein with no similarity to other eIF4E-binding proteins such as eIF4G, 4E-T and the 4E-BPs. It possesses a canonical eIF4E binding site YXXXXLL at its carboxy-terminal end. When this motif is mutated to AXXXXAA, the interaction with eIF4E1 is abrogated. The novel eIF4E binding protein has a predicted molecular weight of 70.1 kDa and is encoded by three alternatively-spliced mRNAs encoded by a single gene. Interaction data between this protein and other eIF4E-related cap-binding proteins present in Drosophila, as well as other functional features of this protein will be presented.

38 Dorsal interacting protein 3 potentiates activation by Drosophila Rel-homology domain proteins. Girish Ratnaparkhi, Albert Courey. Chemistry & Biochemistry, UCLA, Los Angeles, CA. Dorsal interacting protein 3 (Dip3), identified as a Dorsal (DL) interactor in a yeast two-hybrid screen, contains both MADF and BESS domains (Bhaskar & Courey, Gene, 2002, 16:173). The BESS domain of Dip3 binds to the DL Rel homology domain, and we show that Dip3 synergizes with DL, Dorsal-like immunity factor (Dif) and Relish (Rel), three Rel family transcription factors encoded in the Drosophila genome. Less than 1% of eggs laid by homozygous viable dip31 flies show defects in dorso-ventral (D/V) patterning. However, Dip3 mutations enhance the patterning defects that result from dl haploinsufficiency, indicating a dispensable role for Dip3 in D/V patterning. Since Rel family factors play prominent roles in innate immunity, we examined the role of Dip3 in the immune response. dip31 larvae and adult flies are sensitive to immune challenge. Adult flies challenged with a mixture of gram positive (Micrococcus luteus) and gram negative (Escherichia coli) bacteria but not fungus (Beauveria brassiana) have shortened lifetimes- about half of the flies die within a month of immune challenge, while wild-type flies show little mortality during the same period. Quantitative RT-PCR was used to demonstrate that dip31 larvae show significant reduction of expression of the antimicrobial defense genes drosomycin, diptericin, defensin and cecropin-A. Chromatin immunoprecipitation experiments in S2 cells containing activated Toll and Immune deficient signaling pathways indicates the presence of Dip3 at the promoters of these genes. Binding requires the presence of Rel proteins at these promoters. On polytene chromosomes, Dip3 co-localizes in DAPI deficient regions to a subset of the bands occupied by RNA Polymerase II confirming its role in transcriptional activation. Immunolocalization of Dip3 at the chromocenter of polytene chromosomes suggests that in addition to its role in activation, Dip3 may also have a role at the centromeric region of cleavage stage chromosomes. PLATFORMS: Evolution and Quantitative Genetics 97

39 Concerted cis-regulatory evolution at multiple neuroectodermal loci. Justin Crocker, Albert Erives. Biology, Dartmouth College, Lebanon, NH. An outstanding question in evolutionary developmental biology is how the genetic mechanisms underlying developmental rates co-evolve with changes in life-history. This question is particularly relevant for Drosophilid embyrogenesis in which precise, spatiotemporal morphogen gradients pattern embryos that differ both by size (1-2x) and timing (1-2x) in different species. Evolutionary compensation of the production and decay rates of both maternal morphogens and early zygotic genes must somehow be achieved in order to progress through the equivalent embryonic stages in each species. Here, we compare the activities of multiple neurogenic ectodermal enhancers (NEEs) in Drosophila melanogaster and Drosophila virilis. Early syncytial Drosophila virilis embryos take 4 hours to complete early cell divisions, culminating in cellularization, and corresponding to a developmental rate that is twice as slow as Drosophila melanogaster. Nonetheless, the NEE-driven Drosophila virilis loci are expressed in a similar position and span along the dorsal-ventral axis of the stage 5 embryo. However, we show that the activities of virilis NEEs in transgenic melanogaster are more robust as measured by span of expression along the D/V axis compared to melanogaster NEE-driven transgenes. We trace this enhanced expression to configurations of Dorsal and Twist binding sites present in the set of NEEs and demonstrate that by mimicking the spatial configurations of virilis NEEs in melanogaster NEES, the melanogaster NEEs faithfully recapitulate the dorsal- ventral domains of expression of virilis NEEs in melanogaster embryos. We conclude that concerted cis-regulatory evolution across the NEEs has occurred in both D. virilis and D. melanogaster lineages in order to compensate through stabilizing selection evolutionary changes occurring in trans.

40 Estimating the fraction of sites under positive and negative selection via explicit population genetic hidden Markov models. Andrew Kern, David Haussler. Center For Biomolecular Science and Engineering, UC Santa Cruz, Santa Cruz, CA. Patterns of genomic variation within and between species are jointly determined by stochastic forces, such as drift and mutation, along with deterministic forces, such as selection. A long-standing question in the field of population genetics is to what extent does selection shape population genetic variation within the genome. Furthermore, the genomic targets of natural selection are scarcely known from natural populations. Fusing Hidden Markov Models (HMMs) with the classical population genetic theory of genic selection, we have developed and implemented a fully probabilistic population genetic HMM (popGenHMM), which allows for inquiry into the genomic targets of selection. In particular we use 2-state (selected and neutral states) and 3-state (neutral, positively selected, and negatively selected states) models whereby allele frequencies are emitted from each state with probabilities determined by sampling from the stationary distribution of allele frequencies as determined via diffusion approximations. Transition probabilities between states are assumed to be constant per base pair, thus allowing for a biologically reasonable reduction in the correlation between states over physical distance. Training of the models proceeds via an expectation maximization (EM) algorithm, followed by model selection using the Bayesian information criterion (BIC), Viterbi decoding, and subsequent scoring of predicted selected elements. We present results of our popGenHMM when applied to early release data from the Drosophila Population Genomics Project (http:/ /dpgp.org), consisting of roughly 7Mb of data resequenced from chromosomes 2L and X in 50 lines of Drosophila melanogaster. 3- state models are shown to fit the data significantly better than 2-state models, and large differences in estimates of selection parameters are observed between the X chromosome and 2L. Correlations to genomic annotations and global parameter estimates of the models are discussed.

41 cis-regulatory variation is typically poly-allelic in Drosophila. Jonathan Gruber, Anthony Long. Dept Ecology & Evol Biol, Univ California, Irvine, Irvine, CA. The expression of a particular suite of genes is necessary for most organismal traits, though both traits and expression levels vary heritably in populations. As a preliminary inquiry toward determining whether heritable variation in gene expression may be the general source of heritable phenotypic variation, we describe standing variation of cis-regulation among 16 strains of Drosophila melanogaster. With robust biological replication and multiple assays, we obtained precise Allelic Expression (AE) quantification from the Oligo Ligation Assay high-throughput SNP genotyping platform. We observed significant Differential Allelic Expression through at least one assay in 17/18 genes. Moreover, a novel crossing design allows us to combine AE estimates of the entire panel across assays. Of the differentially expressed genes queried with multiple and concordant assays, a majority show statistical support for three or more alleles. Our results suggest that variants affecting cis-regulation are a common feature of Drosophila genes, and that this variation is often the result of more than one segregating, cis-acting polymorphism. 98 PLATFORMS: Evolution and Quantitative Genetics

42 Evolutionary constraint and adaptation in the metabolic network of 12 Drosophila species. Anthony Greenberg, Sarah Stockwell, Andrew Clark. Dept Molec Biol & Genetics, Cornell Univ, Ithaca, NY. Taking advantage of the newly-available whole-genome sequences of 12 Drosophila species, we examined how protein function and metabolic network architecture influence rates of enzyme evolution. We found that despite high overall constraint, there were significant differences in rates of amino acid substitution among functional classes of enzymes. This heterogeneity arises because proteins involved in the metabolism of foreign compounds evolve relatively rapidly, while enzymes that act in “core” metabolism exhibit much slower rates of amino acid replacement, suggesting strong selective constraint. Network architecture also influences enzymes’ rates of amino acid replacement. In particular, enzymes that share metabolites with many other enzymes are relatively constrained, although apparently not because they are more likely to be essential. Our analyses suggest that this pattern is driven by strong constraint of enzymes acting at branch points in metabolic pathways. We conclude that metabolic network architecture and enzyme function separately affect enzyme evolution rates. We are currently extending these findings by experimentally estimating fluxes through energetic pathways in 92 wild-caught D. melanogaster lines.

43 Population transcriptomics of host shifts in the cactophilic Drosophila mojavensis. Luciano Matzkin, Therese Markow. Dept Ecology & Evolutionary Biol, Univ Arizona, Tucson, AZ. In the presence of environmental change, natural selection can shape the transcriptome. Those genotypes that are better able to modulate gene expression to maximize fitness will be favored. It is imperative to examine gene expression at the population level as it responds to an environmental shift in order to distinguish random or neutral gene expression variation from the pattern produced by natural selection. Drosophila mojavensis is a cactophilic fly endemic to the deserts of North America. This species contains four genetically isolated cactus host races each individually specializing in the necrotic tissues of different cactus species. The necrosis of each cactus species provides each of the resident D. mojavensis populations with a distinct chemical environment. Using a partial genome cDNA array we previously investigated the role of transcriptional variation in the adaptation of D. mojavensis to its hosts using one isofemale line. That dataset produced a set of candidate loci that were differentially expressed in response to host shifts, some of which have a non-neutral pattern of evolution. Using the recently sequenced and annotated genome of D. mojavensis, we recently developed a new gene expression array for all the annotated genes. This new microarray consists of 69,997 60-oligonucleotide probes representing 17,504 predicted genes. We now have employed this new array to reveal the effects of genotype, environment and their interaction on gene expression by exposing eight recently collected isofemale lines of D. mojavensis from Sonora to organpipe (native host) and agria (alternative host). In addition we quantify the amount of covariation of gene expression between genes.

44 The evolution of non-coding RNAs from insect Hox complexes. Matthew Ronshaugen. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. Large non-coding RNAs (ncRNAs) were identified in the Drosophila melanogaster Hox complex more than 20 years ago. Further study has ultimately led to the identification of more than 10 distinct non-coding transcription units. The majority of these ncRNAs are associated with previously described enhancers, Polycomb/Trithorax response elements that regulate Hox expression or miRNAs that attenuate Hox function. Recently, the human Hox complexes were shown to contain numerous ncRNAs, some of which similarly modulate Hox expression or function. With the exception of the Hox miRNA genes little is known about the evolution and conservation of these ncRNAs. It is not even clear if they are a general feature of Hox complexes outside of flies and humans or if any aspect of their expression or function is conserved. To begin to address this question I have used a comparative tiling microarray approach focusing on three developmentally diverse insects, the fruit fly Drosophila melanogaster, the honeybee Apis mellifera, and the flour beetle Tribolium castaneum. Tiling microarrays show that during embryonic development large ncRNAs are common to all three of the Hox complexes. I find that global patterns of intronic and intergenic transcription differ between the short germ beetle and long germ fruit fly. In situ analyses of these ncRNAs led to the surprising observation that a number of these ncRNAs overlap and are transcribed antisense to another gene. This investigation establishes that ncRNAs are common to many metazoan Hox complexes and may play a role in coordinating global Hox gene expression. It also begins to address whether variation in these ncRNAs has contributed to morphological evolution of the metazoa. PLATFORMS: Evolution and Quantitative Genetics 99

45 Structure and evolution of gene network involved in specification and function of extraembryonic membranes in insects. Yury Goltsev1, Gustavo Rezende1,2, Denise Valle2, Gregory Lanzaro3, Mike Levine1. 1) UC Berkeley; 2) Instituto Oswaldo Cruz, Brazil; 3) UC Davis. In many insects the activity of early D/V patterning gene network serves two important goals. 1-It provides keys for specification of various tissues in embryo proper. 2-It outlines and subdivides the territory dedicated for extraembryonic membranes (EM), which assist morphogenesis and play protective role during development. Using An. gambiae (two EMs - amnion and serosa) and D. melanogaster (one EM - amnioserosa) we study the evolution of D/V network as well as downstream effector genes partaking in EM function. In course of our studies in mosquito we have found significant rearrangement of DV network as it is known from fly embryo. Thus for example the patterns of dorsal genes such as of tup and doc clearly indicate specification of two dorsal lineages in mosquito as opposed to one in fly. Moreover reduced affinity of Dorsal sites in enhancer of mosquito sog gene leads to ventralization of sog expression and expansion of dorsal dpp signaling domain which largely correspond to overall zone occupied by the progenitors of two EMs. We went further and by microarray screen identified and examined the genes specifically enriched in mosquito serosa. We found genes belonging to wax production, chitin synthesis and crosslinking pathways which are apparently involved in production of additional (synthesized after oviposition) eggshell layer - serosal cuticle (SC). SC is described in other insects and is shown to have composite two-layered structure. The most outbound is a wax layer and more proximal is chitin-containing layer. Our desiccation resistance assays revealed that SC plays unique and primary role in protection of mosquito egg against arid conditions. Specifically we see that the mosquito egg gains desiccation resistance only after the SC is synthesized. Altogether we describe genetic basis of evolution and variability in specification of dorsal tissues in Dipterans and shed light on the molecular mechanisms of desiccation resistance in An. gambiae eggs.

46 Engineering the genomes of wild insect populations. Bruce Hay1, Chun-Hong Chen1, Haixia Huang1, Catherine Ward1, Jessica Su1, Ming Guo2. 1) Deptment of Biology, MC156-29, California Institute of Technology, Pasadena, CA; 2) Departments of Neuology and Pharmacology, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095. There are many contexts in which it would be useful to be able to manipulate the genomes of wild populations. For example, mosquito-borne diseases such as malaria and dengue fever infect hundreds of millions of people each year, and current approaches for prevention, which often focus on vector suppression, are not adequate. An alternative strategy for controlling the transmission of these diseases involves replacement of the wild insect population with transgenic animals unable to transmit disease. An essential component of such a strategy is the presence of a drive mechanism that will ensure the rapid spread of genes conferring disease refractoriness, which may carry a fitness cost, throughout the wild population. Maternal-effect selfish genetic elements (Medea elements) are attractive candidates to mediate drive. Medea elements select for their own survival by inducing maternal-effect lethality of all offspring not inheriting the element-bearing chromosome from the maternal and/or paternal genome. This behavior is predicted to lead to rapid spread of the element within the population even if it carries an associated fitness cost. Medea elements have been shown to exist in the flour beetle Tribolium castenaeum, but their molecular nature is unknown (Beeman et al., Science 256: 89-92). We have developed synthetic Medea elements that can drive population replacement in Drosophila and that are resistant to recombination-mediated dissociation of drive and effector functions (Chen et al., Science 316: 597-600). The genetic and cell biological principles utilized should be applicable to a number of other insect species, and have the potential to allow for iterative cycles of population replacement. We will discuss the development of Medea elements, as well as our progress in engineering reproductive isolation.

47 Genetic and genomic analyses of the wing polyphenism and polymorphism in the pea aphid (Acyrthosiphon pisum). Jennifer Brisson1, Greg Davis2, David Stern2, Sergey Nuzhdin3. 1) Section of Ecology & Evolution, University of California, Davis, Davis, CA; 2) Princeton University; 3) University of Southern California. The pea aphid, an emerging genomic model system, exhibits dramatically different adult phenotypes of winged or unwinged morphs that are induced by environmental conditions in asexual females (a polyphenism) and by a single unidentified genetic locus in males (a polymorphism). Specifically, during the spring and summer months, females reproduce parthenogenetically, producing clonal daughters for ten to twenty generations. These females typically lack wings. Under stressful conditions, however, such as when the host-plant becomes overcrowded, mothers produce daughters that develop wings and are capable of dispersing to other host plants. Males also exhibit winged and unwinged morphs, although in contrast to females, the male wing polymorphism is determined by a single unidentified locus on the X chromosome called aphicarus (api). Winged and unwinged male siblings are genetically identical except for their X-chromosomes, which carry either the api-unwinged or api-winged allele (named for their effect on males). Interestingly, genetic variation for the female wing polyphenism segregates with the male wing polymorphism suggesting that the developmental networks underlying the polyphenism and polymorphism are not independent. Further, this linkage is in reverse phase such that clones that produce winged males in the sexual phase of the life cycle are less likely to produce winged females during the asexual phase of the life cycle. Here we present our ongoing efforts to characterize the female wing polyphenism and male wing polymorphism using genetic and genomic approaches. 100 PLATFORMS: Evolution and Quantitative Genetics

48 Do insecticides alter natural host-parasitoid interactions in Drosophila? Neil Milan, Todd Schlenke. Dept. of Biology, Emory University, Atlanta, GA. Our previous work has shown that natural host plant toxins can have an effect on the interaction between fruitflies and their parasitoid wasps. Resistance to host plant toxins can help flies escape wasp parasitism in one of two general ways: volatile toxins can repel wasps from the toxic food (thus reducing the attack rate), and some toxins appear to limit growth of wasps inside the flies (thus increasing fly immune success). We now extend this work to two man-made toxins that many natural Drosophila melanogaster populations have evolved resistance against, the insecticides DDT and malathion. We are interested in whether Drosophila’s natural parasites have evolved similar insecticide resistance capabilities as their hosts, or whether evolution of resistance against DDT and malathion by D. melanogaster has allowed resistant fly populations a temporary reprieve from normal parasitoid-induced mortality. This work should shed light on a potential drawback of insecticide use to control pests: that natural parasites may suffer worse than the hosts the toxins are meant to control.

49 Multiple infections of Spiroplasma bacteria in Drosophila. Tamara S. Haselkorn, Therese A. Markow, Nancy A. Moran. Dept Ecology & Evolutionary Biol, University of Arizona, Tucson, AZ. Bacterial endosymbionts are common in insects and interact with their hosts in myriad ways, ranging from mutualistic to parasitic, and can have dramatic effects on their host’s evolution. Drosophila harbor far fewer heritable bacterial symbionts than other insects, namely only Wolbachia and Spiroplasma. While the incidence and effects of Wolbachia have been studied extensively, the prevalence and significance of Spiroplasma infections in Drosophila are far less clear. These small, gram-positive, helical bacteria infect a diverse array of arthropod hosts, conferring a variety of fitness effects, and are also well-known plant pathogens. Spiroplasma is a male-killer in certain Drosophila species, such D. melanogaster and species of the willistoni group. In other species of Drosophila, however, it is not a reproductive manipulator, and its effect is unclear. Previous studies have identified different Spiroplasma haplotypes circulating in Drosophila populations. We used a multi-locus sequence analysis to reconstruct a robust Spiroplasma endosymbiont phylogeny, assess genetic diversity, and look for evidence of recombination. Seven loci, many of which are bacterial housekeeping genes located in different areas of the Spiroplasma genome, were sequenced from over 70 Spiroplasma-infected individuals from seven different Drosophila species. Results from this intensive sampling and sequencing effort reveal at least five separate introductions of Spiroplasma into Drosophila. At least three phylogenetically distinct Spiroplasma haplotypes have infected Drosophila.

50 The phenotypic effects of interspecific cytonuclear interactions. Colin Meiklejohn, Kristi Montooth, Dawn Abt, David Rand. Dept EEB, Brown Univ, Providence, RI. Despite the extensive amount of comparative sequence data that has been generated for mitochondrially encoded genes, functional consequences of mitochondrial sequence evolution are known from only a handful of systems. In order to better understand the significance of mtDNA divergence for organismal phenotypes, we created a panel of D. melanogaster lines that contain mtDNA from multiple strains of D. melanogaster (both cosmopolitan and Zimbabwe races), D. simulans, and one haplotype of D. mauritiana. The performance of four X chromosomes relative to a standard in two autosomal backgrounds was measured in combination with these nine mtDNAs in order to identify X chromosome-mtDNA interactions affecting fitness. Despite the fact that hundreds of mutations have fixed between the D. melanogaster and D. simulans mitochondrial lineages, the foreign mitochondria show very slight effects on X chromosome segregation or any other assayed phenotypes, with one exception. A single D. simulans mtDNA haplotype causes a two and a half day delay in egg to adult development time, reduces female fecundity by 50%, lowers the activity of the cytochrome c oxidase complex, and produces short, thin macrochaete, but only in one D. melanogaster autosomal background. The sequence of this mitochondrial haplotype is virtually identical to another D. simulans mtDNA that shows no phenotypic effects, implicating a single mitochondrial mutation in this severely disruptive mito-nuclear epistatic interaction. PLATFORMS: Evolution and Quantitative Genetics 101

51 Satellite sequence de-condensation as a cause of hybrid lethality. Patrick M. Ferree, Daniel A. Barbash. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. Haldane’s Rule states that deleterious phenotypes observed in interspecific hybrids are more likely to occur in the heterogametic sex (XY and ZW). A well-known exception to this rule is the embryonic lethality of (XX) hybrid females from D. simulans mothers and D. melanogaster fathers. A mutation suppressing this lethality, zygotic hybrid rescue (zhr), was previously mapped to the pericentric heterochromatin of the D. melanogaster X chromosome. This mutant line contains few copies of the pericentric 359 base pair (bp) repeat, a member of the 1.688g/cm3 satellites, suggesting that this non-coding satellite DNA may be a major component of the zhr locus that causes hybrid embryonic lethality. The 359 bp satellite is a particularly interesting candidate because D. simulans does not contain this repetitive sequence. We conducted a detailed cellular analysis to determine the underlying mechanism(s) causing embryonic lethality. Our studies show that during nuclear cycles 10-13 hybrid embryos exhibit major defects in nuclear spacing and mitotic synchrony. We also observe lagging chromatin at the metaphase plate. This defect does not reflect a general problem of chromatin condensation in hybrids. Rather, we have used fluorescence in situ hybridization (FISH) analysis to show that this lagging chromatin derives exclusively from the D. melanogaster X. Furthermore, we find that this lagging chromatin includes the 359 bp satellites, which appear severely under-condensed. To demonstrate the specificity of this defect we show that a translocation containing the 359 bp satellites to the Y chromosome results in Y-specific condensation defects and reverses the lethality from females to males. Our results suggest a direct role of the D. melanogaster X pericentric region, and specifically the 359 bp satellites, in the developmental failure of hybrid female embryos. Previous studies have identified several protein-coding genes as causing hybrid lethality. Our data suggest that rapidly evolving satellite sequences also play a major role in driving hybrid incompatibility between diverging species.

52 Adaptive evolution of the aging gene Insulin-like Receptor. Annalise Paaby1, Mark Blacket2, Ary Hoffmann2, Paul Schmidt1. 1) Department of Biology, University of Pennsylvania, Philadelphia, PA; 2) Centre for Environmental Stress and Adaptation Research, University of Melbourne, Melbourne, Australia. Several candidate genes for aging have been identified by extended longevity mutant phenotypes, including two members of the insulin signaling pathway: the Insulin-like Receptor (InR) and its substrate, chico. Research in the biology of aging has made important advances in our understanding of how such aging genes, and the insulin signaling pathway in particular, determine aging and longevity. However, it is unknown whether these aging loci actually contribute to genetic variance for lifespan in natural populations. For example, a gene under strong constraints may affect lifespan in its function but provide no genetic variation of functional significance. Here we present polymorphism data for InR and chico from natural populations of D. melanogaster across a wide latitudinal geography. InR shows evidence of adaptive evolution on both long and short timescales, exhibiting a history of divergent protein evolution since D. melanogaster shared a common ancestor with D. simulans two million years ago and a pattern of amino acid polymorphism by geography that suggests a response to contemporaneous selection pressures. These patterns are evident across reciprocal latitudinal clines on two continents, making isolation by distance an unlikely explanation. Our results suggest that two members of the insulin signaling pathway, both identified as candidate genes for aging by extended longevity mutant phenotypes, experience different selection pressures and contribute unequally to natural variation in lifespan phenotypes. 102 PLATFORMS: Cytoskeleton and Cell Biology

53 The role of cell surface internalization in epithelial polarity and proliferation control. Sarah L. Windler, David Bilder. Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA. In flies and mammals, loss of cell polarity is correlated with the transformation from normal to neoplastic tissue, but the link between polarity and proliferation is not understood. In order to learn more about the mechanisms governing epithelial cell polarity and proliferation control, we have developed an efficient genetic screen to identify fly neoplastic tumor suppressor genes (TSGs). From this screen, we isolated mutations that disrupt the endocytic adaptor complex, AP-2. In addition, we found that mutations in other genes that control the internalization step of endocytosis - including clathrin heavy chain (chc), and shibire (shi) - also function as neoplastic TSGs. Using a live trafficking assay, we demonstrate that cells mutant for AP-2α, chc, or shi have defects in Notch endocytosis. Even though these genes all function at the internalization step of endocytosis, different mutants show surprisingly different Notch localization, indicating that Notch may be internalized in an AP-2- and Clathrin-independent as well as -dependent manner. We further investigated the mechanism(s) by which these endocytic internalization mutants cause neoplastic transformation. Surprisingly, though accumulation of the apical polarity determinant Crumbs appears sufficient to drive neoplastic transformation, it does not appear to be necessary for the neoplastic phenotype. Alternative mechanisms responsible for polarity and proliferation defects in the endocytic mutants will be discussed.

54 Centrosomin is regulated by multiple kinases at mitotic centrosomes during development in D. melanogaster. Robert Eisman, Lei Gong, Melissa Phelps, Thomas Kaufman. Dept Biol, Jordan Hall A505, Indiana Univ, Bloomington, IN. Progression of the cell cycle, and the changes in protein composition of the centrosome during this process, are regulated by the phosphorylation/dephosphorylation of proteins. Several of the key regulatory kinases and phosphatases required for cell cycle progression are partially characterized, but the identity and the effects of phosphorylation on the function of specific target proteins remain largely unknown. We have shown that Centrosomin (Cnn), an essential core component of the D. melanogaster mitotic centrosome, is phosphorylated during embryogenesis. To understand the mitotic regulation of Cnn during development, we have begun an in-depth analysis of the phosphorylation state of Cnn at different stages of development and in different tissues and Drosophila cell lines. Two-dimensional western analysis reveals that Cnn is phosphorylated at multiple residues, and the pattern of phosphorylation varies depending on Cnn isoforms present, stage of development, and tissue or cell type. We have used both genetic and molecular techniques to identify phosphorylated residues within Cnn isoforms and to identify the kinases that regulate Cnn function during syncytial development. Additionally, we have systematically mutated specific phosphorylated sites in Cnn and have characterized the phenotypic consequences of constitutive phosphorylation and dephosphorylation at these residues during mitosis in D. melanogaster. This work has begun to elucidate the complex posttranslational regulation of Cnn at the mitotic centrosome and provides new insight into the molecular function of Cnn during centrosome replication and maturation throughout development.

55 In vivo quantitative imaging of coordinate cell movements within developing Drosophila embryos. Amy McMahon1, Willy Supatto2, Scott Fraser2, Angela Stathopoulos1. 1) Biology, California Institute of Technology, Pasadena, CA; 2) Beckman Imaging Center, California Institute of Technology, Pasadena, CA. Gastrulation is a conserved yet highly complicated embryonic process combining cell migration and morphological changes, which results in the establishment of different germ layers. In the Drosophila embryo, gastrulation involves dynamic cell movements of presumptive mesoderm cells: (I) invagination of these cells into the embryo, (II) an epithelial-to-mesenchymal transition, and (III) spreading of mesoderm cells within the embryo to form a monolayer. To date, the most authoritative studies of mesoderm migration have relied on observations from fixed embryos, leaving many questions regarding this process unanswered. To resolve this, we are performing in vivo analysis of embryos with 2-Photon Laser Scanning Microscopy. We have optimized this technique to image up to 90 microns within an embryo, allowing us to capture the entire process of gastrulation with sufficient spacial and temporal resolution. To our knowledge, this is the first time that mesoderm migration in its entirety has ever been observed in Drosophila. Furthermore, by using 3D cell tracking software, we can collect quantitative data regarding the behavior of individual or groups of cells as they move. We have also devised new methods for analyzing large data sets and decoupling different types of movement within embryos. By disassembling the migration into its key components, we have uncovered new insights: 1) that the dorsal spreading of the mesoderm is directed, while anterior-posterior movement is passive, and 2) that cells at the leading edge originate from a particular position within mesoderm cell collective and remain at the edge until the entire migration is complete. This approach of combining live cell imaging with quantitative analysis will be extended to study mutant backgrounds, providing insights into the mechanisms controlling mesoderm migration. We contend that live imaging of embryos will provide novel insights into the coordination of collective cell migration. PLATFORMS: Cytoskeleton and Cell Biology 103

56 Drosophila APC2 APC1 null epithelial clones exhibit wingless pathway dependent cell shape changes and epithelial misfolding. Sandra G. Zimmerman, Lauren M. Thorpe, Carolyn A. Mallozzi, Vilma R. Medrano, Brooke M. McCartney. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA. Mutations in the colon cancer tumor suppressor, Adenomatous polyposis coli (APC), contribute to cancer initiation through APC’s role as a negative regulator of Wnt/Wingless (Wg) signaling and through APC’s role in cytoskeletal functions. The precise mechanisms by which APC proteins affect signaling and cytoskeletal function are not well understood. To determine the cellular consequences of complete loss of APC function, we have generated APC2 APC1 double null clones (APC2g10 APC1Q8) in the Drosophila wing imaginal epithelium. APC2g10 APC1Q8 tissue begins to exhibit morphological abnormalities, including smooth clone borders, apical constrictions, and slight invagination approximately 45 hours after clone induction. Smooth clone borders suggest changes in adhesion, but we did not observe significant changes in levels or localization of DE-Cadherin, Armadillo, or α-catenin at the adherens junctions. Throughout larval and pupal development, APC2g10 APC1Q8 clones develop progressively more dramatic invaginations and misfolding, resulting in outpocketing of the mutant tissue from the surrounding epithelium. In addition to these morphological changes, outpocketing clones overproliferate and are largely restricted to areas of the disc that do not normally activate Wg signaling. Consistent with the hypothesis that the outpocketing is due to ectopic Wg pathway activation, we have shown that blocking the Wg pathway in APC2g10 APC1Q8 clones suppresses the outpocketing. Further, activation of the Wg pathway independent of APC function is sufficient to induce the outpocketing. Our current model is that activation of the Wg pathway in the APC2g10 APC1Q8 clones induces apical constriction, resulting in inappropriate invagination of the epithelium. We are currently investigating the relationship between Wg pathway activation and the regulation of actin dynamics and apical constriction.

57 Hook-like is a negative regulator of endocytic trafficking. Adam Haberman, Sanchali Ray, Helmut Krämer. Cntr Basic Neurosci, UT Southwestern Med Cntr, Dallas, TX. In a screen to identify genes required for transport of proteins to lysosomes and lysosome-related pigment granules, we identified a mutation in hook-like (hkl). Hkl mutants have reduced eye pigmentation, but enhanced degradation of the endocytosed proteins Boss and Delta. Regions of Hkl have strong homology to the apoptotic protein Acinus, including 81% similarity to the region of Acinus that has been proposed to fragment chromatin during apoptosis. However, no apoptotic phenotypes have been identified in hkl mutants. Hkl mutants disrupt localization of the early endocytic protein Avalanche and Hook (Hk). Hk mutants also cause enhanced lysosomal degradation of endocytosed proteins, however hk and hkl phenotypes differ in two significant ways. Hkl does not cause the hooked bristles that are characteristic of hk mutants, and hkl modifies developmental signaling while hk does not. Loss of a single copy of hkl enhances the wing notch phenotype of a partial loss-of-function N allele and rescues the rough eye phenotype of the gain-of-function Ellipse allele of EGFR. Hkl trafficking and signaling phenotypes are opposite of those seen in mutations of genes required for uptake of activated receptors into MVBs, including hrs, erupted, and VPS25. Mutations in these genes cause accumulation of endocytosed proteins and an enhancement of N and EGFR signaling. Hkl mutants cause a reduction of endocytosed proteins and impair N and EGFR signaling. Thus, Hkl may function to slow or restrict endocytic trafficking at or upstream of MVBs. Interestingly, both hk and hkl are required for starvation-induced autophagy, as has also been seen for many positive regulators of MVB internalization, underscoring the importance of endosomes in autophagy.

58 Diaphanous, a regulator of cell contractility and motility. Catarina Homem, Mark Peifer. Dept Biol, Univ North Carolina, Chapel Hill, NC. Embryonic development requires coordinated tissue movements and cell shape changes. In order for all these processes to occur normally, cells must maintain adhesiveness, yet their underlying cytoskeleton has to be very dynamic. Since adhesion, cytoskeletal dynamics and cell contractility are interrelated, it is increasingly important to study these processes in whole animals during morphogenesis. It is unclear how changes in the actin cytoskeleton are coordinated with contractility and altered cell adhesion. We identified a novel mechanism for coordinate regulation of adhesion with the actomyosin cytoskeleton during embryogenesis. Diaphanous-related formins like Drosophila Diaphanous (Dia) are important regulators of actin polymerization. We examined Dia’s role during morphogenesis using both gain- and loss-of-function approaches. We used constitutively-active Diaphanous to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. These data are consistent with regulated Dia activity playing a normal role in amniooserosal cell apical constriction and epidermal cell shape changes. We tested this hypothesis by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated affects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis. We are currently extending this work by examining the role Dia plays in migrating cells in both oogenesis and embryogenesis. 104 PLATFORMS: Cytoskeleton and Cell Biology

59 DRhoGEF2 regulates contractile force during segmental groove morphogenesis. Shai Mulinari, Mojgan Padash Barmchi, Udo Häcker. Experimental Medical Science, Lund University, Lund, Sweden. Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the Actin-based cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors (RhoGEFs). One of these exchange factors, DRhoGEF2, has been shown to play an important role in the constriction of Actin filaments during pole cell formation, blastoderm cellularization and invagination of the germlayers at gastrulation. Here we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become morphologically distinguishable as tissue infoldings during mid-embryogenesis. Formation of segmental grooves is associated with apico-lateral accumulation of F- Actin and the Rho1-effector Diaphanous (Dia) in groove founder cells. At groove regression, DRhoGEF2, Myosin II and F-Actin but not Dia are enriched in cells posterior to the groove that undergo apical constriction. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes that occur during segmental groove formation and subsequent regression. DRhoGEF2-dependent groove formation requires the segment polarity genes engrailed and hedgehog. Overexpression of DRhoGEF2 in epidermal cells is sufficient to induce cortical Myosin II accumulation and cell contraction resulting in a deepening of segmental grooves that can be suppressed by Rho1 inactivation. Expression of activated DiaCA induces Myosin II accumulation and deepening of segmental grooves similar to DRhoGEF2-overexpression. However, unlike DRhoGEF2, DiaCA induces F-Actin polymerization, filopodia formation and strengthens cell-cell contacts. Our morphological analysis furthermore suggests, that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-Actin nucleation during segmental groove morphogenesis. Our data suggest that DRhoGEF2 may contribute to establish a balance of activity between different Rho1-effector pathways.

60 The golgi SNARE, Gos28, is essential for rhodopsin transport and photoreceptor survival. Erica E Rosenbaum, Natalia S Rozas, Nansi Jo Colley. Dept. of Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin, Madison, WI. In sensory neurons, successful transport of signaling molecules through the secretory pathway is essential for cell function and survival. SNARE proteins play a critical role in the final docking and fusion events of vesicular transport by forming distinct SNARE core complexes typically comprised of one v-SNARE motif (associated with the vesicle) and three t-SNARE motifs (associated with the target compartment). Based on in vitro studies, the Golgi SNARE protein, Gos28, has been implicated as a t-SNARE in anterograde transport from the ER to Golgi, transport within the compartments of the Golgi and retrograde transport of vesicles from the recycling endosome back to the Golgi. Here, we demonstrate a role for Gos28 in the vesicular trafficking of the major rhodopsin (Rh1) during its biosynthesis in Drosophila photoreceptor cells. Two mutant alleles of gos28 were identified, an EMS-generated allele and an allele containing a transposable P-element insertion. Antibodies directed to the Gos28 protein recognize a 24kD band in wild-type flies that is absent in the gos28 alleles. Mutations in Drosophila gos28 lead to severe defects in Rh1 levels and cause Rh1 to accumulate throughout the secretory pathway. Very little Rh1 reaches its final destination in the light-sensitive rhabdomeres, which normally contain rhodopsin and the other components of the phototransduction cascade. In the gos28 mutants, all other photoreceptor cell proteins tested are expressed normally, suggesting that Gos28 is specifically required for the transport of Rh1-containing vesicles. Mutations in gos28 also lead to a severe late-onset retinal degeneration. Our results illustrate a critical role for Gos28 in Rh1 trafficking and provide genetic evidence that defects in gos28 lead to inappropriate accumulation of Rh1 throughout the secretory pathway and subsequent retinal degeneration. Our gos28 mutants offer a powerful model for studying the in vivo role of Gos28 in vesicular transport. Drosophila Gos28 displays 48% homology with human Gos28, making it a candidate gene for retinal degeneration in humans.

61 Eating yourcellf into shape: Atg1 and autophagy in cell shape changes. Pavan Kadandale, Amy Kiger. Dept. of Cell & Developmental Biology, UCSD, La Jolla, CA. Atg1 is a conserved Ser/Thr kinase that is a key regulator of autophagy, the critical process by which cells target long-lived proteins and organelles to the lysosomes for degradation. Intriguingly, independent studies have established a role for Atg1 in cellular morphogenesis. Previous work shows that Atg1 is required for the proper elongation and branching of neurites. We show that Atg1 function is required for the ecdysone-induced elongation of Kc cells, as well as the spreading response of primary, dissected larval hemocytes. This shared requirement for Atg1 in both cellular morphogenesis and autophagy suggests a link between these two critical processes and a novel role for autophagy in the regulation of cell shape. We are, therefore, delineating the molecular mechanisms of Atg1-mediated functions and testing whether cell shape changes require autophagy. We show that Atg1 is required for both cytoskeletal remodeling and membrane trafficking in cells undergoing shape changes. Atg1 function also impacts phosphoinositide phosphate (PIP) regulation, as shown by a genetic interaction between Atg1 and mtm (myotubularin, a phosphoinositide phosphate phosphatase), and changes in the phosophoinositide 3-phosphate (PI3P) pools in Atg1 mutant cells. Blocking autophagy, either using a known inhibitor (3-Methyladenine) or by mutating another autophagy-related gene (atg3), also prevents the spreading response of primary larval hemocytes. In conjunction with others’ work, our results establish a link between Atg1 and PI3P, both of which are regulators of autophagy and cytoskeletal and membrane reorganization. Our data also indicates a requirement for autophagy in cell shape changes. Taken together, this suggests that interactions between Atg1 and PI3P pools are required for cytoskeletal and membrane remodelling via autophagy, ultimately resulting in the change of cell shape. Further work will establish the mechanisms by which the complex interplay between Atg1 functions, PI3P regulation and autophagy control cell shape changes. PLATFORMS: Cytoskeleton and Cell Biology 105

62 Dynamics of sarcomere assembly in the flight muscles. John Sparrow, Zacharias Orfanos. Dept Biol, Univ York, York, United Kingdom. Differentiation of striated muscle involves the organized assembly of many different muscle proteins into sarcomeres, the large regular macromolecular contractile complexes. Sarcomeres of the Drosophila indirect flight muscles (IFM) have an extremely regular structure compared to vertebrate striated skeletal muscle, providing a unique system for genetic studies of striated muscle differentiation and disease. Using a GFP protein trap of the Z-disc protein Salimus we have confirmed earlier EM studies that once the initial Z- body arrays (proto-sarcomeres) appear in the muscles at 30-35h after puparium formation (APF) they develop in synchrony to mature sarcomeres in all indirect flight muscles (DLM and DVM). Importantly no new sarcomeres are added after 40h APF. Proto- sarcomeres initially elongate, but then at 65-70h APF the Z-discs and sarcomeres begin to widen laterally, while sarcomere elongation continues to reach mature sarcomere length at 90-100h APF. In the absence of IFM-specific ACT88F actin (Act88F6 null mutation) Z-body/actin filament arrays also appear at 35h APF, suggesting that another actin isoform is responsible, but these structures are no longer apparent by 50h APF, confirming that ACT88F actin is required even for early sarcomere development. In the absence of sarcomere myosin heavy chain (Mhc7; IFM-specific Mhc null) some early, delayed, sarcomere elongation occurs, and, although mature sarcomere lengths are never achieved, the widening of Z-discs does occur. This argues that thick filament assembly is required for sarcomere elongation, but not for Z-disc widening. Using antibodies and sarcomeric protein GFP traps we have studied the dynamics of protein assembly into developing sarcomeres. Our results show that myosin assembles into the thick filaments at the M-line and that binding of flightin, an IFM-specific myosin binding protein, follows myosin incorporation, but with a detectable delay. Based on these and other data we will present a model of sarcomere development in IFM.

63 Genetic control of cell morphogenesis during formation of the Drosophila cardiac tube. Caroline MEDIONI1, Martine ASTIER1, Monika ZMOJDZIAN2, Krzysztof JAGLA2, Michel SEMERIVA1. 1) CNRS-UMR6216, IBDML, MARSEILLE, France; 2) INSERM U384, Faculté de Médecine, Clermont-Ferrand, France. Tubulogenesis is an essential component of organ development, yet the cellular mechanisms controlling tube formation are poorly understood. As a model of tubulogenesis, we analyzed the formation of the Drosophila cardiac lumen which arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our detailed 3D and time lapse imaging of cell behaviour and the distribution of cell polarity markers reveals that lumen formation occurs by repulsion of pre-patterned secondary basal membrane domains rather than by fusion of apical membrane vesicles to the luminal compartment. Cardioblasts forming the lumen are devoid of an apical domain. Lumen formation is therefore driven by cell shape remodeling in contrast to all previously described models of tubulogenesis. In support of these findings, blocking the Slit/Robo pathway prevents cardioblast cell-shape changes and inhibits lumen formation. Our data reveal a new mechanism of tube formation which may be conserved during vertebrate heart tube formation and in vasculogenesis.

64 steamer duck inhibits epidermal cell-cell fusion in Drosophila larvae. Yan Wang, Michael Galko. The Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX. During cell-cell fusion pairs or groups of cells merge their plasma membranes to form a single cytoplasm that accommodates all their intracellular components, including multiple nuclei. This event is critical for fertilization and the subsequent development of diverse tissues, such as muscle, placenta and bone. Cell-cell fusion is also important for physiological and pathological events such as macrophage engulfment of foreign bodies, tissue repair and regeneration, and cancer progression. Despite its importance, the proteins directly executing cell-cell fusion remain obscure and its molecular basis is not well understood. When third instar Drosophila larvae are wounded, epidermal cells near the wound fuse with each other. We are exploiting this inducible cell-cell fusion assay to identify genes required for cell-cell fusion. We constructed a reporter line whose epidermal cell membranes and nuclei are fluorescently labeled and can be visualized in live larvae. Using this reporter we conducted a targeted pilot screen of 30 candidate wound healing genes in which gene function is conditionally knocked down by UAS-RNAi within the larval epidermis. This screen identified a negative regulator of epidermal cell-cell fusion called steamer duck (stck). stck encodes the protein PINCH, a known component of integrin-dependent cell adhesion structures at embryonic muscle attachment sites and between the adult wing epithelial layers. Immunofluorescence with an anti-PINCH antibody showed that PINCH is localized to epidermal cell membranes and that UAS-stck RNAi efficiently depleted epidermal PINCH protein. The hyperfusion phenotype caused by stckRNAi is not wound-dependent because it also was observed in unwounded larvae. However, we observed that PINCH and its known binding partner, integrin-linked kinase (ILK) are relocalized from the plasma membrane upon wounding. We are currently testing a model whereby destablization of epidermal cell-cell adhesion, either through wounding or loss of epidermal PINCH protein, leads to activation of a latent cell-cell fusion machinery in these cells. 106 PLATFORMS: Cytoskeleton and Cell Biology

65 The Frizzled and Fat/Dachsous pathways control wing topography. Simon Collier, Kristy Doyle, Justin Hogan, Eric Aten. Dept Biological Sci, Marshall Univ, Huntington, WV. The adult wing membrane is characterized by ridges that have an anteroposterior (A-P) orientation in the anterior wing and a proximodistal (P-D) orientation in the posterior wing. We have found that the formation and orientation of both A-P and P-D ridges is controlled by the Frizzled Planar Cell Polarity (Fz PCP) signaling pathway. The Fz PCP pathway also specifies P-D hair polarity in both the anterior and posterior wing. This raises a question; if the Fz PCP pathway polarizes wing cells for both hair polarity and ridge orientation, why is there a different relationship between hairs and ridges in the anterior wing compared to the posterior wing? We will present evidence that the specification of A-P and P-D wing ridges occurs at different times during pupal development. In an early phase of Fz PCP signaling (prior to 18 hours a.p.f.), P-D ridges are specified in both the anterior and posterior wing. In a late phase of Fz PCP signaling (prior to 32 hours a.p.f.), anterior ridges are reoriented to A-P and P-D hairs are specified across the wing. Our data suggest that the two phases of Fz signaling are characterized by differential use of Prickle protein isoforms. The Sple isoform is primarily active in the early phase and the Pk isoform in the late phase. A significant feature of this two-phase model is that only the anterior ridges are reoriented to A-P in the late phase. This implies that there is some activity preventing the reorientation of ridges in the posterior wing. We propose that the Fat/Dachsous signaling pathway plays this role. Loss of Fat, Dachsous or Four- jointed activity during wing development allows the formation of A-P ridges in the posterior wing. We will present data from timed knockdown and over-expression experiments to support this conclusion.

66 Stripe non-autonomously controls the orientation of actin-based protrusions. Stacie Dilks, Stephen DiNardo. University of Pennsylvania, Philadelphia, PA. The ability of cells to organize actin filaments into stable structures is vital to many cellular functions such as nutrient absorption and sensory input. Although the actin-based protrusions that underlie these processes often have elaborate shapes, it is unknown how cells form shaped protrusions. In the embryo, actin-based protrusions (called denticles) are produced by seven rows of ventral epidermal cells, and differ in shape and hooking polarity from row to row. Specifically, cell rows 1 and 4 are the only two rows in the denticle field that hook to the anterior. This leads us to wonder what is unique about denticle rows 1 and 4 that causes them to reverse their polarity and hook to the anterior. To date, no cell fate determinant or transcription factor is known to be unique to these two cell rows that might provide clues to their polarity reversal. Stripe (sr), a transcription factor required for larval muscle attachment, is expressed in a row-specific pattern in the epidermis. Interestingly, sr is expressed in denticle rows 2 and 5, immediately posterior to the two anterior-facing denticle rows. We now show that sr expression is required, non-autonomously, for the anterior denticle polarity seen in cell rows 1 and 4. Stripe is a downstream target of both the Hh and EGFR signaling pathways, and is the relevant target for anterior denticle polarity. Global overexpression of sr also results in a loss of anterior polarity, indicating that anterior polarity is not conferred on a cell simply via its proximity to a stripe- expressing cell, but through a more complex mechanism. By ectopically expressing stripe in a row-specific manner, we show that cells assign hooking polarity by integrating information from both neighboring cell rows. Finally, preliminary data indicates that sr may mediate its effect on denticle polarity via the cytoskeletal linker protein shortshop, as mutants in this sr target gene exhibit polarity defects that mimic stripe. PLATFORMS: Gametogenesis 107

67 Transition of male primordial germ cells to functional germline stem cells. Matthew Wawersik1, Rebecca Sheng2, Trevor Posenau1, Juliann Gumulak-Smith1, Erika Matunis2, Mark Van Doren3. 1) Biology Dept., College of William & Mary, Williamsburg, VA; 2) Dept. of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD; 3) Dept. of Biology, Johns Hopkins University, Baltimore, MD. Stem cells maintain tissue homeostasis by producing differentiating daughters that replace mature cells lost to injury or turnover. Adult germline stem cells (GSCs) are descendants of primordial germ cells (PGCs) that are specified during embryogenesis. While studies have examined the molecular requirements for PGC specification, migration, and coalescence, very few have examined the timing and regulation of GSC establishment. We have characterized the transition from PGCs to functional GSCs in the developing male Drosophila gonad. We find that anterior PGCs adjacent to the embryonic hub express GSC markers and behave functionally like GSCs by the end of embryogenesis, while PGCs in the posterior of the gonad appear to differentiate. In contrast to female PGCs that remain quiescent until the late third instar larval stage, GSCs in the male gonad produce transit amplifying daughter cells as mid first instar larvae. We also find that GSC establishment is concurrent with hub formation, suggesting that niche formation is a key step that initiates the PGC to GSC transition. Finally, analogous to adult GSCs, our data indicate that newly formed GSCs require JAK/STAT signaling to be maintained, while ectopic activation of the JAK/STAT pathway prevents germ cell differentiation. Together, these data indicate that the JAK/STAT pathway plays an important role in regulating the establishment of function GSCs in late- stage male embryonic gonads.

68 Live imaging of asymmetric centrosome migration and spindle orientation in Drosophila male germline stem cells. Jun Cheng1, Nahid Hemati2, Yukiko M. Yamashita2,3, Alan J. Hunt1. 1) Department of Biomedical Engineering; 2) Center for Stem Cell Biology, Life Sciences Institute; 3) Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI. Proper orientation of the mitotic spindle is essential for establishing the axis of asymmetric stem cell division. However, the mechanism and regulation of spindle orientation are poorly understood. The Drosophila male germline stem cells (GSCs) is one of the best studied model systems in which centrosome positioning and spindle orientation, leading to asymmetric stem cell division, are well documented, but mostly inferred from observations on fixed-cell preparations. We have established conditions for time- lapse live-cell imaging, allowing direct observation of centrosome movement and spindle formation, allowing the behavior to be tracked in detail. Using fluorescent protein-tagged α-tubulin and centrosomal proteins, the mitotic spindle orientation and centrosome migration during interphase were examined by time-lapse live-imaging. The results clearly confirmed that GSCs underwent asymmetric mitosis with the mitotic spindles orthogonal to the adjacent hub cells (niche cells). The somatic cyst progenitor cell (CPC) divisions were also recorded, which indicated that their mitotic spindles were not oriented during mitosis, suggesting that distinct cellular/molecular mechanisms are involved in the control of asymmetric divisions of GSCs and CPCs. Moreover, in our preliminary study, we observed that GSCs with misoriented centrosomes did not undergo mitosis for a prolonged time period, and entered mitosis only after the misoriented centrosomes rotated back into normal orientation, which implies that the centrosome orientation may be monitored in GSCs to ensure the asymmetric stem cell division.

69 Live Imaging of Drosophila Spermatogonia Dedifferentiating into Germline Stem Cells. Xuting Rebecca Sheng, Crista Brawley, Erika Matunis. Dept Cell Biol, Johns Hopkins Univ, Baltimore, MD. In the Drosophila testis, germline stem cells (GSCs) and somatic stem cells (SSCs) adhere to a group of stromal hub cells that comprise the stem cell maintaining microenvironment, or niche. Both stem cell populations produce daughter cells that are displaced away from the niche. Cellular differentiation within a stem cell lineage is commonly considered irreversible. However, if stem cells are depleted, their daughters (transit-amplifying cells) may revert back into stem cells using a process called dedifferentiation. Dedifferentiation occurs in both the Drosophila testis and ovary, but the mechanism is poorly understood. To establish a novel system where GSC daughters (spermatogonia) dedifferentiated into GSCs, we first ectopically expressed the differentiation factor Bag-of-marbles (Bam) in the testis. Bam expression resulted in GSC loss through differentiation, while SSCs appeared unaffected. We then withdrew ectopic Bam expression from flies depleted of GSCs and saw that GSCs were regenerated by the remaining spermatogonia. To bypass limitations of resolving specific cellular changes in fixed tissue, we cultured and imaged GFP-labeled germline cells during GSC recovery. This revealed that both single and interconnected clusters of spermatogonia near the hub make actin-rich protrusions resulting in hub contact. Upon initial contact with the hub, spermatogonia were able to increase the amount of hub contact despite the presence of SSCs adjacent to the hub. Furthermore, we observed occasional spermatogonia with protrusions that appeared to move closer towards the hub indicating that normally non-motile germline cells may become motile in order to replace lost stem cells. This suggests that dedifferentiating cells can actively displace SSCs when re-establishing occupancy of the niche and that signals from the hub may cause dedifferentiating spermatogonia to “hone” into the niche. 108 PLATFORMS: Gametogenesis

70 Asymmetric activation of Rac in germ line stem cells of the ovary controls both the plane of division and the response to BMP signals. Wen Lu1, M. Olivia Casanueva2, Chip Ferguson1,2,3. 1) Committee on Genetics; 2) Committee on Developmental Biology; 3) Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL. How adult stem cells maintain the balance between self-renewal and differentiation is a key question in stem cell biology. The germ line stem cells (GSCs) of the ovary reside at the anterior tip of each ovariole adjacent to somatic cells that form a “niche” responsible for GSC maintenance. Bone Morphogenetic Proteins (BMPs) secreted by the surrounding somatic tissues act as niche signals necessary for GSC maintenance. Our data support the hypothesis that interactions between the niche cells and the GSCs polarize the GSCs leading to the asymmetric activation of the small GTPase Rac at the GSC - niche interface. Activated Rac both controls the plane of GSC division, most likely by directing the localization of the cytoskeletal-associated protein APC2, and promotes BMP signaling in the GSC by elevating the activity of the JNK pathway. Together, these two activities of Rac cooperate to ensure a robust pattern of asymmetric self-renewal divisions. The future goals of this research are to determine how Rac is asymmetrically activated at the GSC - niche interface, and to explore the mechanisms by which elevated JNK signaling promotes BMP signal transduction in the GSC.

71 Insulin signals regulate GSC maintenance via the control of niche size. Hwei-Jan Hsu, Daniela Drummond-Barbosa. Department of Cell and Developmental Biology, Vanderibilt Univerisity Medical Center, Nashville, TN. Ovarian germline stem cells (GSCs) reside within a specialized microenvironment (or niche) that provides signals required for GSC maintenance. GSC activity is also modulated by external factors, such as diet, but this process is less well understood. Our previous work documented that neural-derived insulin-like peptides (Dilps) directly control ovarian GSC division in response to diet; however, it was not know whether insulin signals are also involved in the regulation of GSC maintenance. As female flies age, they progressively lose GSCs, and this is paralleled by a reduction in their niche size. Interestingly, we found that flies kept under a protein-poor diet lose their GSCs faster with age than flies kept under a protein-rich diet, suggesting that insulin signaling may affect GSC number and niche size. To specifically address if insulin signaling regulates GSC maintenance, we examined the GSC number and niche size in insulin receptor mutants and found that these mutants have fewer GSCs and smaller niche size than control females. To test if insulin signaling directly controls GSC maintenance, we performed insulin receptor mutant mosaic analysis in GSCs. Our results show that insulin receptor mutant GSCs exhibit a half-life comparable to that of control GSCs, suggesting that insulin signals regulate the survival of niche cells to indirectly promote the maintenance of GSCs. Remarkably, overexpression of Dilp2 maintains a higher number of GSCs and niche cells in aging females, suggesting that reduced insulin signaling underlies the gradual reduction in niche size and GSC loss that occurs naturally with age.

72 Stem Cell Maintenance Through Competition in the Follicle Stem Cell Niche. Todd Nystul, Allan Spradling. Embryology Dept, Carnegie Inst, Baltimore, MD. Follicle stem cells (FSCs) reside in the germarium of the Drosophila ovary, and are the progenitors of the follicular epithelium that surrounds the developing germline. We have used the FSCs as a model for understanding the behavior and maintenance of an epithelial stem cell in its native, in vivo environment (1). Using a newly developed dual-marked clonal system, we confirmed that there are exactly two FSCs per germarium, and found that they reside in separate, non-adjacent niches. This differs from the male and female germline stem cell niches, where multiple stem cells are contained within a fixed, cellular niche. Surprisingly, though the FSC niche is also fixed in place, it lacks a fixed stromal-cell component. We found that FSCs can be reliably identified without clonal analysis by their distinctive shape and consistent location in the germarium, which allowed us to analyze the behavior of early FSC daughters. Approximately half of the FSC daughters move away from the niche toward the posterior, while the other half migrate laterally and contact the opposite FSC niche before integrating into the growing epithelium. FSCs are regularly lost and replaced during adult life, and we observed that the laterally migrating FSC daughters facilitate this replacement by competing with resident stem cells for niche occupancy. We are currently screening for mutations that affect FSC function, and have identified several candidate genes which may alter the competitiveness of FSCs and their daughters for niche occupancy. 1. Nystul, TG and Spradling, AC. (2007). An Epithelial Niche in the Drosophila Ovary Undergoes Long-Range Stem Cell Replacement. Cell Stem Cell 1, 277-285. PLATFORMS: Gametogenesis 109

73 A Bam complex mediates a cap-dependent translational switch to promote stem cell differentiation. Jean Maines, Yun Li, Dennis McKearin. Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX. The balance between germline stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos-Pumilio (Nos-Pum) translational repressor complex, which promotes GSC self-renewal, and the expression of Bam, a key differentiation factor. We explored the mechanistic basis of Bam’s function promoting germ cell differentiation. We found that bam behaves genetically as a negative regulator of nos. Furthermore, Bam and Nos proteins are expressed in reciprocal patterns in young germ cells and sequences in the nos 3’ UTR are critical for Nos elimination from Bam expressing cells. Ectopic Bam expression causes differentiation of germline stem cells, and strikingly, this activity also acts through the nos 3’-UTR, since expression of a nos transgene with a tubulin 3’-UTR prevents Bam-induced GSC loss. Bam forms a complex with Bgcn, a member of the RNA helicase family, suggesting that the Bam-Bgcn complex may associate directly with RNA. Finally, we found that Bam is a component of an mRNA cap-binding complex, suggesting that a Bam complex blocks translation of nos, and perhaps other target RNAs, via cap-mediated translational repression. We conclude that Bam promotes germ cell differentiation by translational repression of nos, and declining Nos levels, in turn, promote differentiation by derepressing translation of differentiation-promoting factors. These findings emphasize the importance of translational repression in regulating the balance between stem cell self- renewal and differentiation, particularly in the germ cell lineage.

74 Role of Drosophila Ime4 and Ime2 in the Initiation of Meiosis. Cintia Hongay, Gerald Fink, Terry Orr-Weaver. Fink and Orr- Weaver Labs, Whitehead Inst, Cambridge, MA. Meiotic cell division is restricted to specialized cells of sexually reproducing eukaryotes and is required to preserve the species- specific chromosomal number upon fertilization. The early events that commit cells from a mitotically dividing population of germ stem cells to the unique divisions of meiosis are mostly unknown. In yeast, a unicellular organism, we have found that IME4 (Inducer of Meiosis 4, putative RNA methyltransferase) is a mitotic:meiotic switch: it is required in diploids to enter meiosis and, if expressed in haploids, it is sufficient to license them to initiate meiosis (Hongay et al., 2006). Expression of IME4 in haploid cells is followed by expression of IME2 (encoding a protein kinase), an event recognized as a hallmark of commitment to meiosis and subsequent sporulation in budding yeast. There are highly conserved homologs of these two genes in D. melanogaster, M. musculus, H. sapiens, and other metazoans, but their functions are presently unknown. We have detected gonadal expression of dIME2 and dIME4 in Drosophila suggesting a role in meiosis and thus evolutionary conservation of their functions in metazoans. In situ hybridization shows that dIME4 RNA is localized to the apical region of the testes, coinciding with the area where the decision to undertake a meiotic cell fate occurs. In addition, we have obtained two ime4 hypomorphs using a P-element excision strategy that are male and female sterile, a phenotype that we corroborated with RNAi against dIME4. These results support a role for dIME4 and potentially dIME2 in Drosophila meiosis. Hongay, C. F., Grisafi, P. L., Galitski, T., and Fink, G. R. (2006). Antisense transcription controls cell fate in Saccharomyces cerevisiae. Cell 127, 735-745. 110 PLATFORMS: Signal Transduction

75 Specificity of Signaling by Drosophila Fibroblast Growth Factors. Angelike Stathopoulos, Phoebe Tzou, Snehalata Kadam. Div Biol, MC 114-96, Caltech, Pasadena, CA. FGF signaling is used reiteratively throughout development of animals to control a diverse set of cellular processes including migration and differentiation. Over 120 FGF-FGF receptor (FGFR) combinations are possible in vertebrates, and we will present evidence that only 3 such combinations function in Drosophila. The Pyramus (Pyr) and Thisbe (Ths) genes from Drosophila encode the first pair of invertebrate FGFs that bind to the same FGFR isoform. We aim to determine whether different FGF ligands are required to effect qualitatively different responses downstream of FGFR activation and have a unique advantage using the simpler Drosophila model system. To this end, we have isolated single mutants affecting either Pyr or Ths genes and compare them with a deficiency mutant that we have previously characterized [DfBSC25], removing both genes. We find that both FGF genes are required to control migration of the mesoderm during gastrulation. Furthermore, the unique phenotypes of these single mutants suggest that both ligands work cooperatively to coordinate this process by different mechanisms. In addition, FGF signaling is required for regulating cell differentiation, as in the specification of pericardial, dorsal somatic mesoderm, and longitudinal visceral mesoderm founder (LVMF) cells occurring during embryogenesis. In pyr single mutants, pericardial and dorsal somatic mesoderm cells are mostly absent, whereas in ths mutants these same cells are indeed specified; both pyr and ths mutants affect LVMF specification. Our results indicate that Ths and Pyr have differential roles in cell migration and specification and that, in some biological contexts, they cannot substitute for one another. We have furthered our study mechanistically by defining the functional domains of these two FGFs. Unexpectedly, our preliminary results suggest that specificity is not imparted by the FGF ligand homologous regions of the proteins. Implications of these results toward specificity of FGF signaling in Drosophila compared with vertebrates will be discussed.

76 Endosomal Entry Regulates Notch Receptor Activation in Drosophila. Thomas Vaccari1, Han Lu1, Ritu Kanwar2, Mark Fortini2, David Bilder1. 1) Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA; 2) Center for Cancer Research, National Cancer Institute, Frederick, MD. Signaling through the transmembrane receptor Notch is widely utilized throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we have systematically assessed Notch localization, processing and signaling in a comprehensive set of Drosophila mutants that block access of cargo to different endocytic compartments. We will present data that indicate that processing and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. This dramatic activation switch does not involve Notch ligands and indicates that endosomal access of the Notch receptor promotes signaling. In mutants that block endosomal entry we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.

77 Pentagone, a novel BMP/Dpp target gene, involved in patterning and growth in Drosophila. Robin Vuilleumier1, Markus Affolter2, George Pyrowolakis1. 1) Developmental Biology Unit, Biology I, University of Freiburg, Freiburg, Germany; 2) Dept. of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland. In Drosophila, patterning and growth of appendages is controlled by a small number of highly conserved signaling molecules. Among them, Decapentaplegic (Dpp), the ortholog of the BMP2/4 ligands in vertebrates, activates a conserved signal transduction pathway to transcriptionally regulate target genes. Recently, we uncovered a branch of the BMP/Dpp-signaling pathway which culminates in direct repression of a number of key developmental genes. This process depends on a highly conserved 16 bp long cis-regulatory element, the Silencer Element (SE). We use the consensus sequence of the SE as bait to perform genome-wide, in silico, screens to identify genes that are potentially repressed by the BMP/Dpp-signaling pathway. Here we report the identification and characterization of a novel BMP/Dpp target gene, pentagone (pent). In embryos and larval imaginal discs, pent expression is restricted to regions with low or absent BMP/Dpp signaling activity. We present in vitro and in vivo evidence that pent is directly repressed by the BMP/Dpp signaling pathway through the SEs present in the regulatory region of the gene. In addition, loss- and gain-of-function studies demonstrate that pent plays a crucial role for normal patterning and growth of lateral regions of the Drosophila wing. Interestingly, pent mutants display a comparable phenotype to mutants in components of the BMP/Dpp-signaling pathway. Because of these observations and the fact that pent encodes a secreted, multi-domain protein, we are currently testing the hypothesis that Pent regulates the activity of the Dpp-morphogen by modulating ligand distribution, availability and/or reception. PLATFORMS: Signal Transduction 111

78 A translational block to HSPG synthesis permits BMP signalling in the early Drosophila embryo. Douglas Bornemann1, Sangbin Park2, Sopheap Phin1, Rahul Warrior1. 1) Dept Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Department of Developmental Biology, Stanford University School of Medicine, Stanford, California. Heparan sulfate proteoglycans (HSPGs) are extracellular macromolecules found on virtually every cell type in eumetazoans. HSPGs are composed of a core protein covalently linked to glycosaminoglycan (GAG) sugar chains that bind and modulate the signaling efficiency of many ligands including Hedgehog (Hh), Wingless (Wg), and Bone Morphogenetic Proteins (BMPs). Here we show that in Drosophila, loss of HSPGs differentially affects embryonic Hh, Wg and BMP signaling. We find that a stage-specific block to GAG synthesis prevents HSPG expression during establishment of the BMP activity gradient that is critical for dorsal embryonic patterning. Subsequently proteoglycan synthesis is initiated coincident with the onset of Hh and Wg signaling which require HSPGs. This temporal regulation is achieved through translational control of HSPG synthetic enzymes through Internal Ribosome Entry Sites (IRES). IRES-like features are conserved in GAG enzyme transcripts from diverse organisms arguing that this represents a novel evolutionarily conserved mechanism for regulating GAG synthesis and modulating growth factor activity.

79 Interactions between the retinal determination protein Eyes absent and the Abelson tyrosine kinase suggest a novel function for Eya in cytoskeletal regulation during axonogenesis. Wenjun Xiong, Noura Dabbouseh, Ilaria Rebay. Ben May Dept. for Cancer Research, The Univ. of Chicago, Chicago, IL. A fundamental question in developmental biology is how multiple signaling pathways converge on downstream transcriptional effectors to initiate and ensure proper developmental programs. The discovery that Eyes absent (Eya), originally described as a transcriptional cofactor, also functions as a protein tyrosine phosphatase, adds further complexity to the interplay between different signaling inputs and the retinal determination gene network. Although both activities of Eya are required for Drosophila eye development, the details of their regulation have yet to be revealed. In particular, little is known about the functions and regulation of Eya phosphatase activity. In a genetic screen designed to identify tyrosine kinases upstream of Eya, the Abelson tyrosine kinase (Abl) was implicated as a positive regulator of Eya’s function in retinal determination. The genetic synergy between Eya and Abl contributes to multiple developmental programs, including axon pathfinding in the embryonic CNS and the axon targeting process in the larval visual system. In biochemical assays, Abl directly phosphorylates Eya at multiple tyrosine residues, and can increase Eya tyrosine phosphorylation in vivo. As a result of this tyrosine phosphorylation, Eya relocates from the nucleus to the cytoplasm where we hypothesize it participates in phosphotyrosine-mediated signaling networks that regulate cytoskeleton dynamics. Supporting this model, we have found that nuclear-restriction of Eya compromises its function in vivo, and that coexpression of membrane- tethered Eya can reconstitute full Eya activity. Together our data suggest a model in which Eya, in addition to operating as a nuclear transcription factor, also participates as a phosphatase in cytoplasmic phosphotyrosine signaling networks that regulate neuronal morphogenesis.

80 A cell autonomous requirement for the Dally-like core protein in Hedgehog signaling. Elizabeth H. Williams1, William N. Pappano2, Philip A. Beachy1. 1) HHMI, Dept of Developmental Biology, Stanford Univ Sch Med, Stanford, CA; 2) HHMI, Dept of Molecular Biology & Genetics, Johns Hopkins Univ Sch Med, Baltimore, MD. The Drosophila glypicans Dally and Dally-like (Dlp) have well characterized roles in cell non-autonomous movement of morphogens such as Hedgehog (Hh) and Wingless (Wg). A genomic RNAi screen in cultured cells revealed that Dlp, but not Dally, also is required for cell autonomous Hh response. This role and specificity similarly are observed during embryonic patterning. While the heparan sulfate proteoglycan (HSPG) moieties of mature Dlp are necessary for its role in morphogen movement, we have found that these sugar modifications are dispensable for its role in cell autonomous Hh signaling. In a cultured cell Hh signaling assay, a Dlp variant DlpΔGAG, which lacks detectable HS modification, fully rescues RNAi depletion of endogenous dlp transcript. The Dlp core protein also is sufficient for some of the essential in vivo roles of Dlp during embryonic and larval development since expression of a DlpΔGAG transgene rescues the zygotic larval lethality of the dlpA187 null allele comparably to expression of a wild-type Dlp transgene. In addition, segment polarity in the larval cuticle, which requires both intact Hh and Wg signaling, is partially rescued in dlpA187 germline clones by DlpΔGAG expression. This cell autonomous activity for Dlp in Hh signal transduction maps to the N- terminal globular domain and requires membrane association but not specifically the glycosyl phosphatidylinositol (GPI) linkage characteristic of glypicans. The most closely related mammalian glypicans fully rescue RNAi depletion of endogenous dlp transcript in a cultured cell Hh signaling assay, demonstrating conservation of this activity through evolution. Beyond the established roles of the HS moieties of HSPGs in signal response and movement through tissues, our studies define a conserved activity for the core protein of the Dlp HSPG in cell autonomous Hh signal transduction. 112 PLATFORMS: Signal Transduction

81 Sequential actions of feedforward and feedback loops pattern Drosophila egg: genetic experiments and computational modeling. Nir Yakoby1,2, Jessica Lembong1,2, Trudi Schüpbach3, Stanislav Y. Shvartsman1,2. 1) Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ; 2) Dept. of Chemical Engineering, Princeton University, Princeton, NJ; 3) Howard Hughes Medical Institute and Dept. of Molecular Biology, Princeton University, NJ. During Drosophila oogenesis, the EGFR and Dpp signaling pathways specify several subpopulations of the follicle cells which give rise to dorsal eggshell structures. The roof of dorsal appendages is formed by the follicle cells that express Br, a Zn-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. We show that the resulting changes in the Br pattern affect the expression of Dpp receptor thick veins (tkv). By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative feedback loop. We synthesize these observations into a mathematical model, whereby the dynamics of Br expression is driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of br expression, while the feedback loop modulates this pattern in time. Using a computational approach to systematically explore the feasibility and robustness of this mechanism, we show that it can successfully predict the dynamics of the eggshell patterning network in multiple mutant backgrounds.

82 Analysis of synthetic interactions in Drosophila by RNAi. Thomas Horn1, Elin Axelsson2, Wolfgang Huber2, Michael Boutros1. 1) Signaling & Functional Gen, German Cancer Research Ctr, Heidelberg, Baden-Wuerttemberg, Germany; 2) EMBL/EBI - European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SD, UK. RNA interference (RNAi) has been used as a powerful technique to systematically deplete transcripts on a genome-wide scale. Due to the efficiency of RNAi in invertebrates, model organisms such as Drosophila and C. elegans have contributed important insights with the identification of novel components of diverse biological pathways. The transduction and integration of signals within and between pathways are highly complex, often requiring the interaction of multiple genes. Forward and reverse genetics have been a powerful approach to identify non-redundant components of signaling pathways, however, it is likely that redundant factors have escaped identification and will require combinatorial approaches. RNAi provides a powerful tool to probe redundancy in signaling networks and also allows addressing this problem in a systematic manner. In order to probe for functional redundancy in signaling pathways, we generated subsets of functional groups encoded by the Drosophila genome. For example, kinome RNAi libraries with two independent dsRNAs targeting all splice variants of the target transcripts were designed and synthesized. All possible double knock-down combinations were screened for synthetic effects using homogenous and high-content phenotypic readouts in Drosophila cells. This data set is being used to model genetic interaction networks in Drosophila. PLATFORMS: Pattern Formation 113

83 From parts to pattern: deciphering pair rule seven stripe formation with a completed cis-regulatory blueprint. Mark Schroeder, Ulrike Gaul. Rockefeller University, New York, NY. During blastoderm segmentation, the transition from the aperiodic gap gene patterns to the periodic pair rule gene patterns is particularly striking. Previous studies distinguished two steps in this transition: The primary pair rule genes (h, eve, run) establish their 7 stripe pattern piecemeal by interpreting maternal and gap patterns through a set of stripe-specific enhancers, while the secondary pair rule genes (ftz, odd, prd, slp) generate their pattern wholesale by interpreting the patterns of both primary and secondary pair rule genes through a single 7-stripe enhancer. Revisiting this problem with computational and experimental methods, we found that this model required substantive revision. By comparing the temporal evolution of the pair rule patterns with the known enhancers we found that early emerging stripes are driven by maternal/gap input and that, based on this reasoning, stripe-specific elements were missing for 10 stripes. Using the Stubb algorithm and known transcription factor binding preferences, we predicted the missing elements and validated them through lacZ reporter assays. Surprisingly, stripe specific elements generate all 7 ftz and run and 4 odd stripes, revealing pervasive maternal/gap input into 5 of the 7 patterned pair rule genes. With this complete set of pair rule enhancers, we reexamined the regulatory interactions between pair rule genes, which, through strong cross repression, produce two pairs of mutually exclusive 7-stripe patterns and, through weaker differential repression, create gradients and single-cell offsets in the expression patterns. We find that pair rule input is not restricted to 7-stripe elements but also required for proper pattern formation of several stripe-specific elements. Our findings suggest a new network structure which emphasizes the importance of eve, ftz and odd, the pair rule genes whose patterns are in register with the parasegments of the embryo.

84 Reading between the lines: pair-rule regulation of cell adhesion molecules. W. Ray Anderson1,2, Leslie Pick2. 1) Dept. of Cell Biology and Molecular Genetics and; 2) Dept. of Entomology, University of Maryland, College Park, MD 20742, USA. Transcriptional regulation plays a key role in development, yet transcription factors alone cannot build an embryo. Decades of study in the model organism Drosophila melanogaster have identified cascades of selector genes responsible for patterning the body plan in the early embryo, yet few realizator genes have been found. The seven striped expression pattern of the pair-rule genes is among the earliest specification events of the zygotic genome. It has long been known that the pair-rule system establishes the segment polarity gene network along the anterior-posterior axis. The expression of engrailed subdivides each segment into an anterior and posterior compartment. However, it remains an open question: are pair-rule genes, in addition to positioning stripes of engrailed, also “writing between the lines”? Using bioinformatics, RNA expression data, and more recently, transcriptional profiling of tightly staged wild type and ftz factor 1 (ftz- f1) pair-rule mutant embryos, we have identified additional genes in the transcriptional cascade downstream of fushi tarazu (ftz). This microarray analysis identified tartan (trn) as an early target of Ftz-F1. Tartan is a cell surface protein implicated in cell sorting and establishment of compartmental boundaries in the wing disc. Early trn expression overlaps with ftz and is dependent upon ftz and ftz-f1. The cross regulatory nature of the pair-rule system led us to look for genes encoding cell adhesion molecules having complementary transcriptional expression patterns. This identified Toll-6, which is structurally related to Tartan. Further analysis identified a set of structurally related genes implicated in cell adhesion with expression patterns matching additional pair-rule genes. This suggests a pair-rule regulated cell surface code capable of sorting individual cells into appropriate compartments. We are characterizing the expression patterns of these genes in pair-rule mutants as well as the cell sorting properties of combinations of these proteins in embryos and cell culture.

85 Regulation of Ft-Ds signaling by the DHHC transmembrane protein Approximated. Hitoshi Matakatsu, Seth Blair. Zoology, University of Wisconsin-Madison, Madison, WI. The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including planar cell polarity (PCP) and the proximodistal patterning of appendages such as wings and legs. Work from our own and other laboratories indicates that some or all of these effects are mediated by a signaling pathway that is modulated by binding between Ft and Ds. Although recent studies show that Ft regulates growth control via the Hippo signaling pathway, it is unclear what other molecules mediate Ft-Ds signaling. We have therefore been analyzing mutations that show similar phenotypes for proximodistal patterning. approximated (app) mutants have a proximodistal patterning defect, and we have obtained new app mutants by EMS screening that show PCP defects in wing and abdomen. We found that app encodes a multipass transmembrane protein containing a DHHC-CRD (cysteine-rich domain). DHHC proteins have been recently found to add palmitate fatty acids to cytoplasmic proteins, thereby regulating their association with cell membranes. Most DHHC proteins are localized to, and thought to act in, the ER and Golgi. However, we found that App is concentrated at the apical cell cortex, overlapping the region where Ds and Ft are concentrated. We will present data that App regulates the membrane localization of select components of the Ft-Ds signaling signaling pathway. 114 PLATFORMS: Pattern Formation

86 Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis. Dong Yan1,2, Xinhua Lin1,2. 1) Div Developmental Biol, Cincinnati Children’s Hosp, Cincinnati, OH; 2) The Graduate Program in Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, OH. Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila.

87 The homeotic genes labial and Deformed regulate decapentaplegic expression restricted to the peripodial epithelium and required for the formation of the adult head. Brian Stultz1, Mark Mortin2, Deborah Hursh1. 1) DCGT, FDA/CBER, Bethesda, MD; 2) LMG, NICHD/NIH, Bethesda, MD. Expression of decapentaplegic (dpp) in the lateral peripodial epithelium of the eye/antennal disc is necessary for correct morphogenesis of ventral structures of the adult head of the fly. This expression overlaps significantly with the expression of both labial (lab) and Deformed (Dfd) in the peripodial epithelium of the eye/antennal disc. A dpp mutation whose mutant phenotype is limited to the adult head, dpps-hc1, is a 15 bp deletion in the 5’ enhancer region of dpp which removes a HOX consensus binding site, as well as sites predicted to bind the HOX co-factors homothorax (hth) and extradenticle (exd). A 600 bp region around this deletion is rich in putative HOX, hth, and exd binding sites, and produces spatially correct peripodial dpp expression in β-galactosidase reporter constructs. Production of loss of function clones in eye/antennal discs indicates that lab and hth are required for activation of the dpp enhancer, while Dfd negatively regulates lab expression. Putative Lab, Hth, and Exd sites in and adjacent to the 15 bp deletion are required for correct enhancer function in β-galactosidase reporter constructs. Thus the morphogenesis of the adult head from the bilayer eye/antennal disc is regulated by a genetic network of HOX proteins and the dpp signal transduction pathway, restricted to the peripodial epithelium of this structure.

88 The anterior-posterior gradient of microtubule organization in the oocyte depends on Par-1-induced Tau phosphorylation. Ai-Guo Tian, Wu-Min Deng. Dept Biological Sci, Florida State Univ, Tallahassee, FL. Specification of the anterior-posterior (AP) axis in Drosophila oocytes requires posterior enrichment of the serine/threonine kinase Par-1 and proper organization of the microtubule cytoskeleton, but the mechanism by which Par-1 regulates the microtubule cytoskeleton in the oocyte has been unknown. The work reported here demonstrates that it does so through phosphorylation of the microtubule-associated protein Tau. Our results show that excessive overexpression of Par-1 or the Par-1 kinase domain in the germ-line cells disrupts microtubule organization in the oocyte. This phenotype is similar to that of the egg chambers taken from females fed the microtubule-depolymerizing drug colcemid. Interestingly, germ-line clones of tau also showed similar microtubule defects in the oocyte, suggesting a critical role for Tau in oocyte polarity. Using both biochemical and immunocytochemical approaches, we found that Tau is phosphorylated by Par-1 in the oocyte. This phosphorylation prevents active Tau from localizing to the posterior of the oocyte, thus destabilizing the microtubule-organizing center in the posterior and therefore causing the establishment of an AP gradient of microtubule distribution, which is essential for AP axis specification. PLATFORMS: Pattern Formation 115

89 Drosophila EGFR signaling is modulated by differential compartmentalization of Rhomboid intra-membrane proteases. Shaul Yogev, Eyal Schejter, Benny Shilo. Molecular Genetics, Weizmann Institut , Rehovot, Israel. EGF Receptor (EGFR) signaling is repeatedly employed throughout development to coordinate cellular decisions. In Drosophila, the precursor form of the cardinal EGFR ligand, Spitz, associates with the chaperone Star in the ER and is transported to a late compartment of the secretory pathway. There, cleavage by the intra-membrane protease Rhomboid-1 (Rho-1) releases the mature ligand and inactivates the chaperone. How versatility in signaling may arise from such a conserved core machinery is an open question. We now show that two additional Rhomboid proteases, Rho-2 and Rho-3, expressed in the germline and the developing eye, respectively, are localized and active in the ER as well as in the late compartment. While late-compartment cleavage by Rho- 2 and Rho-3 leads to EGFR activation, their ER activity plays an attenuating role, mainly due to premature cleavage and inactivation of the chaperone Star. Thus, differential compartmentalization of Rhomboids is used to modulate the levels of active ligand secretion in developmental settings where a tight control over EGFR activation range is required.

90 Regeneration genes affect the position, time and amount of blastema formation. Anne Sustar1, Kim McClure1,2, Gerold Schubiger1. 1) Dept Biology, Univ Washington, Seattle; 2) Dept Anatomy, UC San Francisco. Regeneration has been a curiosity for millennia and experiments have been performed for over 200 years. However a molecular and genetic understanding of the process is still in its infancy. Drosophila imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing followed by localized proliferation of the regeneration blastema. In many systems the Wingless (Wg) signaling pathway is activated during wound healing and is necessary and sufficient for blastema formation. Progress in this field requires the identification of Wg target genes and their functions in regeneration. Our genome-wide search identified 143 candidate regeneration genes. We focus on candidate genes that are not expressed during leg disc development but are activated at wound sites and in the regeneration blastema by wg expression. In functional tests with this class of genes, we find that all of them dominantly modify regeneration, including: augmenter of liver regeneration (alr, CG12534), regeneration (rgn, CG6014), and Matrix Metalloproteinase 1 (Mmp1). These three genes are conserved in mammalian regeneration. Homozygous Drosophila mutants of rgn and Mmp1 have imaginal discs. We find that mutations in these genes delay, reduce or increase blastema growth, and later modify the discs’ regeneration capacity. We conclude that these genes function in the regeneration process of the organ but not in its normal development. 116 PLATFORMS: Drosophila Models of Human Diseases

91 A prion-like domain in Drosophila fragile X protein is essential for regulating synaptic plasticity. Paromita Banerjee1, Brian P. Schoenfeld2, Sean M.J. McBride2, Thomas C. Dockendorff1. 1) Dept Zoology, Miami Univ, Oxford, OH; 2) Molecular Cardiology, Albert Einstein College of Medicine, Bronx, NY. Fragile X mental retardation proteins regulate translation at synapses and their activity is vital for cognitive processes and synaptic plasticity in all species in which their function has been examined. The Drosophila fragile X protein (dFMR1) has a domain enriched in glutamine and asparagine residues that is similar to prion-like domains described in fungi and Aplysia. The dFMR1 prion-like domain confers protease resistance to green fluorescent protein and induces its aggregation in both yeast cells and Drosophila tissues, indicating that this prion-like domain can modulate the conformation of proteins. We have generated transgenic animals where the sole allele of dfmr1 that is present lacks the ability to code for the prion-like domain. The resulting mutant protein is stable and has no obvious anomalies in spatial expression as judged by Western blotting and whole-mount stains of adult brains. These mutant animals were examined for several neural development and behavior phenotypes that are associated with null alleles of dfmr1. Axon guidance and germline development phenotypes resulting from null alleles of dfmr1 are rescued by the mutant transgene, indicating that dFMR1 protein lacking the prion-like domain has some in vivo function. Notably, the dFMR1 prion-like domain is essential for proper regulation of synapse development at the larval neuromuscular junction, for normal circadian locomotion activity, and for naive courtship behavior and memory after conditioned courtship training. The circadian and courtship anomalies exhibited by the mutants show that the biochemical activity of the prion-like domain establishes the neural circuitry necessary for execution of these behaviors. Our results demonstrate a direct role by a prion-like domain in mediating behavior and synaptic plasticity within an animal model, and underscore the possibility that prion-like domains are vital for regulation of many physiologic processes in metazoans.

92 De novo CoA biosynthesis is required to maintain DNA integrity in a Drosophila model of Pantothenate Kinase-Associated Neurodegeneration. Ody Sibon, Floris Bosveld, Anil Rana, Harm Kampinga. Dept Cell Biol, Univ Groningen, Groningen, Netherlands. In humans, mutations in the PANK2 gene, coding for the first enzyme in the CoA biosynthesis pathway, are associated with Pantothenate Kinase-Associated Neurodegeneration (PKAN). Currently, the pathogenesis of this devastating neurodegenerative disorder is poorly understood. From a forward genetic screen in Drosophila, aimed to identify genes involved in surviving induced DNA damage, we isolated dPPCS (the second enzyme of the CoA biosynthesis pathway) as a novel gene required to maintain DNA integrity. The complete Drosophila CoA synthesis route was dissected, annotated and additional mutants that carry mutations in CoA enzymes were obtained (dPANK/fumble) or generated (dPPAT-DPCK) and used for further investigation. Drosophila CoA mutants are hypersensitive to ionizing radiation, suffer from altered lipid homeostasis, and the larval brains display increased apoptosis and elevated levels of damaged DNA. In addition, disruption of CoA synthesis in general provokes neurodegeneration in adults. Our data provide the first comprehensive analysis of the physiological implications of mutations in the entire CoA biosynthesis route in an animal model system. Surprisingly, our findings reveal a major role of this conserved metabolic pathway in maintaining DNA and cellular integrity during development of the central nervous system, and the data explain how impairment of CoA synthesis during development can lead to neurodegeneration in Drosophila. The presented Drosophila model will be of help to understand the consequences of impaired de novo CoA synthesis in higher eukaryotes and may provide insights in the pathogenesis of PKAN.

93 Using Drosophila to probe the activities of anthrax toxins. Annabel Guichard1, Shauna Mc Gillivray2, Beatriz Cruz-Moreno1, Victor Nizet2, Ehan Bier1. 1) Division of Biological Sciences, UCSD, La Jolla, CA; 2) Division of Pediatric Pharmacology & Drug Discovery UCSD School of Medicine La Jolla, CA. Anthrax is a severe and widely distributed disease caused by Bacillus anthracis, and is still a significant threat in underdeveloped countries. Anthrax primarily affects cattle, and occasionally humans, when contact occurs with sick animals, or, as in the case of the 2001 attack, when weaponized spores are inhaled. B. anthracis achieves infectivity mainly through the secretion of three toxins, PA (Protective antigen), EF (Edema Factor), and LF (Lethal Factor). After binding to surface receptors present on most mammalian cells (TEM8 and CMG2), PA gets endocytosed and permits the entry of EF and LF into the cytoplasm. EF is a potent Calmodulin- dependent Adenylate cyclase, and LF is Zinc metalloprotease that cleaves and inactivates most human Mitogen Activated Protein Kinase Kinases (MAPKK), and possibly unknown targets. B. anthracis is not known to infect insects, which lack homologs to the known receptors necessary for EF and LF entry. However, when expressed intracellularly in transgenic flies, LF and EF induce developmental phenotypes reflective of their established activities. For example, we found that LF inhibits dorsal closure during embryogenesis, and most likely acts at the level of Hemipterous, the Drosophila MAPKK acting in the JNK pathway essential to this process. EF also induces expected effects such as a hedgehog-like phenotype in the wing, consistent with the known role of cAMP- dependent PKA in inhibiting hedgehogh signaling. Here we show that LF and EF induce additional phenotypes suggesting that they cooperate to inhibit signaling pathways not known previously to be affected by these toxins. Importantly, we find that EF and LF act in the same capacity in human endothelial cells, suggesting a novel activity for anthrax toxins relevant to their pathogenicity. PLATFORMS: Drosophila Models of Human Diseases 117

94 Drosophila easily-shocked: phosphatidyl-ethanolamine metabolism and cardiac disorders. Hui-Ying Lim1, Robert J. Wessells2, Rolf Bodmer1. 1) Burnham Inst.for Medical Research, La Jolla, CA; 2) U.Mich,Ann Arbor,MI. We study the role of phosphatidyl-ethanolamine (PE) homeostasis in cardiac function using Drosophila easily-shocked (eas) mutants as a model. Loss-of-eas-function causes the fly to exhibit an increased risk of heart failure under conditions of stress induced by electrical pacing. Conversely, cardiac-specific expression of eas results in fly hearts being more resistant to pacing- induced stress. Moreover, the replenishment of wild-type Eas in the hearts of young eas flies afforded rescue of pacing-induced heart failure to a level close to the wild-type controls, thereby establishing the cardiac phenotypes as a consequence of the eas mutation. The eas mutant harbors a defect in PE metabolism, a fly model in epilepsy. Coincidentally, epileptic patients appear to experience an increased incidence of heart abnormalities that may be significant contributors to causing sudden death. The eas fly serves as an ideal system to explore the molecular basis of PE homeostasis underlying both neuronal and cardiac syndromes. To further examine heart function of eas flies, we analyzed the dynamic properties in semi-intact heart preparations, including heart rate, rhythmicity and contractility. In addition to alteration in cardiac performance, eas mutants displayed myocardial heart tube thinning, which could be rescued with heart-specific expression of eas. Previous studies in S2 cells showed that excess PE feedback inhibits the dSREBP pathway and downregulates the expression of target genes involved in fatty acid and phospholipid synthesis. To determine whether dSREBP is a potential effector of PE signaling involved in cardiac physiology, we expressed a dominant- negative form of dSREBP in eas mutants and found an alleviation of the eas heart phenotypes under both physiological and stressed conditions. Moreover, overexpression of constitutively-active dSREBP partially mimics the cardiac eas phenotype. Collectively, these results suggest that a defect in PE homeostasis of eas mutants may lead to excessive activity of the dSREBP pathway, and thereby resulting in cardiac abnormalities.

95 Regulation of ER stress induced apoptosis by the Unfolded Protein Response. Min-Ji Kang, Hyung Don Ryoo. Department of Cell Biology, NYU, School of Medicine, New York, NY. Proteins that fail to properly fold in the endoplasmic reticulum(ER), activate a signaling network known as the unfolded protein response (UPR), which leads to an increase in the capacity of ER to fold its client proteins or facilitate their degradation. A stress level in the ER beyond a certain threshold leads to apoptosis. Although activation of this cell death pathway has been implicated in certain degenerative disorders, its mechanisms remain controversial and unclear. We found that the UPR pathway involving the endonulease ire-1 and its mRNA target xbp1 is also conserved in Drosophila. The activity of this pathway can be measured through our xbp1-GFP sensor, which in response to ER stress, undergoes splicing and GFP activation. The xbp1-GFP is also activated in the retina of a retinal degeneration disease model caused by mutated Rhodopsin-1 alleles, ninaEG69D/+, and ninaERH27/+ flies. When ninaE is prematurely over expressed during eye disc development, it triggers a strong UPR reaction as evidenced by xbp1-GFP and an eye ablation phenotype due to excessive apoptosis. Mutation in ire-1 further enhances the extent of apoptosis, indicating that the phenotype is associated with ER stress. This has prompted us to examine the link between ER stress and apoptosis. Apoptosis induced by ninaE over expression is suppressed by p35, an effector caspase inhibitor. Unexpectedly, our initial evidence suggests that the initiator caspase Dronc is not involved in ER-stress triggered apoptosis. One of the ER associated degradation (ERAD) factors, sip3 suppresses the eye ablation by lowering the levels of the misfolded protein. These results establish a new model to study ER-stress induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

96 Genetic Interaction between Survival of Motor Neuron Gene (SMN) and BMP Signaling Pathway. Howard Chang1, Yokokura Takakazu1, Dimlich Douglas1, Kankel Mark1, Mukherjee Ashim2, Walker Amy3, Harris Jevede3, Duckworth April1, Hart Anne3, Van Vactor David1, Artavanis-Tsakonas Spyros1. 1) Dept Cell Biology, HMS, Harvard Medical School, Boston, MA; 2) Department of Molecular and Human Genetics Banaras Hindu University Varanasi-221005 India; 3) MGH Cancer Center, Building 149,Charlestown, MA. Spinal Muscular Atrophy (SMA) is a human neurodegenerative disease caused by mutations in the Survival of Motor Neuron (SMN) gene. Loss-of-function SMN leads to clinical manifestation, including neuron degeneration, muscle atrophy and lethality. SMN protein is ubiquitously expressed and is involved in RNA processing. Drosophila However, the tissue specific nature of SMN in neuron and muscle remains elusive. Here, we established a Drosophila model to investigate the SMN genetic circuitry, particularly in neuron and muscle. By using lethality and neuromuscular junction (NMJ) as markers, we found that both neuron and muscle are sensitive to SMN knockdown. Most importantly, the severity of both lethality and NMJ phenotypes correlate to the dosage of SMN in flies. We also investigated the SMN expression pattern at larval NMJ and found SMN is mostly localized at the post-synaptic structure. Finally, we performed a genome-wide genetic screen for SMN modifiers. We identified wit, a component of BMP signaling pathway, enhances SMN dependant lethality and NMJ phenotype. In addition, the BMP signal is reduced in the SMN knockdown background. Moreover, null mutation of dad, an antagonist of BMP signaling pathway, rescued the SMN dependant NMJ phenotypes. We conclude that we have found links between SMN and BMP signaling pathway at neuromuscular junction, which might open new avenues for SMA therapy. 118 PLATFORMS: Drosophila Models of Human Diseases

97 Regulation of phosphoinositide phosphates in Drosophila morphogenesis. Inês Ribeiro, Jared Dennis, Amy Kiger. Div Biological Sciences, Univ California, San Diego, La Jolla, CA. We are investigating the mechanisms that mediate subcellular spatial regulation important for cell shape changes. Phosphoinositide phosphates (PIPs), the phosphorylated forms of phosphatidylinositol, control localized and dynamic cellular processes through the recruitment of specific PIP-binding proteins. The association of mutations in PIP regulators with human diseases further demonstrates that PIP regulation is crucial. However, little is known about the identities and developmental requirements of PIP response in vivo. To address roles for PIPs in morphogenesis, I am taking two approaches. First, I adapted a collection of fluorescently-tagged PIP biosensors for systematic analysis of dynamic PIP subcellular distribution in Drosophila development. The specificities of the PIP biosensors were tested by functional assays in cell cultures, and verified constructs were used to generate transgenic fly lines. I am using these PIP reporter flies to determine the temporal-spatial distribution of specific PIPs during major morphogenetic events of wildtype embryogenesis, including dorsal closure and muscle development. The PIP reporters will be insightful in mutant backgrounds. Secondly, I am using Drosophila genetic mutant analysis to characterize developmental roles for specific PI(3)P regulators implicated in morphogenesis. We previously demonstrated that antagonistic function of the mtm phosphoinositide phosphatase and the class II PI3-kinase, PI3K68D, regulate a PI(3)P-dependent cell shape change. We therefore generated mutant alleles to study mtm and PI3K68D in development revealing that both are essential genes. Importantly, tissue-specific knockdown of mtm suggests its role in adult muscle development, reminiscent of mutations in the highly conserved human MTM1 responsible for X-linked myotubular myopathy. I am currently addressing the underlying role for mtm in muscle morphogenesis, as well as the muscle-requirements for PIP subcellular distribution.

98 Modeling human brain cancer in Drosophila. Renee D. Read, John B. Thomas. Molecular Neurobiology Laboratory. Salk Institute for Biological Studies. San Diego, CA 92037. Gliomas, neoplasms of glial cells and their precursors, are the most common and deadly malignant tumors of the central nervous system (CNS). These tumors diffusely infiltrate the brain and grow rapidly, properties that render them largely incurable. Formation of these tumors is a complex process involving accumulation of mutations in many genes, only some of which are known. In particular, constitutive activation of the EGFR-Ras and PI-3 kinase signaling pathways is a common feature in gliomas and is sufficient to cause glioma in mouse models. Yet, how these pathways specifically regulate glioma pathogenesis is unknown. To understand the molecular basis for this disease, we have developed a novel model in Drosophila for the purpose of carrying out large-scale genetic analyses to identify genes involved in glioma invasion and proliferation. The Drosophila CNS contains multiple glial cell types that are strikingly similar to their vertebrate counterparts in terms of function, development, and gene expression. Using techniques that target gene expression in glia and glial precursors, we have found that co-activation of both the EGFR-Ras and PI-3 kinase pathways in Drosophila glia gives rise to proliferative, invasive cells that create tumor-like growths in the fly brain, mimicking the human disease. We are now performing genetic screens for enhancers and suppressors of the EGFR/Ras-PI-3 kinase phenotype in order to identify new regulators of glial neoplasia. Furthermore, we are performing misexpression screens for additional loci that cause glioma-like phenotypes in the fly brain. The genes already identified in these screens represent excellent candidates for genes directly involved in pathogenesis.

99 Mutations in the gene clueless cause mitochondrial mislocalization and Parkinson-like phenotypes in the Drosophila ovary and muscle. Rachel Cox1,2, Megan Kutzer1, Shelley Paterno1,2, Allan Spradling1,2. 1) Dept Embryology, Carnegie Inst of Washington, Baltimore, MD; 2) HHMI. Mutations in mitochondrial DNA and in nuclearly encoded mitochondrial proteins are responsible for a large number of diseases, both spontaneous and inherited. A hallmark of mitochondrial and aging diseases is neuromuscular degeneration. While many neurodegenerative diseases such as Parkinson’s are associated with decreased mitochondrial function, it is becoming increasingly clear that poorly functioning mitochondria may be a primary cause of the disease, and not simply a result. We found the previously unstudied fly gene clueless (clu) causes severe effects on mitochondria in both muscle and ovarian cells. Mutations in clu are semi- lethal and clu mutants are short-lived. The mutant adults that are able to eclose are very uncoordinated, suggesting neuronal problems. clu also has a striking effect on flight muscle fibers and their mitochondria, causing indistinct sarcomere banding and enlarged, empty mitochondria. The muscle phenotype, lack of coordination, and aging defects are reminiscent of fly mutants in the parkin pathway, the homolog of a gene linked to familial Parkinson’s disease in humans. In the ovary, clu mutants exhibit mislocalized mitochondria clustered to one side of the cell in germline stem cells and germ cells, as well as in a subset of ovarian somatic cells. Mutant females exhibit reduced fertility and males are sterile. Clueless’ molecular function is unknown. The high degree of sequence identity from humans to yeast suggests Clueless is an important conserved mediator of mitochondrial movement and function. Studying clu function offers the opportunity to elucidate the link between mitochondrial mislocalization and function and neuromuscular degeneration. PLATFORMS: Drosophila Models of Human Diseases 119

100 ubiquilin antagonizes presenilin, stabilizes APP and promotes neurodegeneration. Ming Guo, Atish Ganguly, Renny Feldman. Dept Neurology & Pharmacology, Univ California, Los Angeles, Los Angeles, CA. Mutations in the Amyloid Precursor Protein (APP) and Presenilin, the catalytic component of the gamma-secretase complex, mediate familial Alzheimer’s disease (AD). Therefore, identifying regulators of Presenilin and APP is crucial for understanding AD pathogenesis. Recently, a splicing variant, in the ubiquilin 1 gene (UBQLN1) was reported to associate with an increased risk for late-onset AD. Previously, UBQLN1 was found to bind Presenilin in mammalian cells; however, the functional significance of this interaction in vivo remains unclear. Moreover, whether the disease-associated variants in UBQLN1 have altered function is unknown. Drosophila contains a single homolog of UBQLN1, Ubiquilin (Ubqn), which is a protein with a ubiquitin-like (UBL) domain and a ubiquitin associated (UBA) domain. We show that Drosophila Ubqn binds to Drosophila Presenilin (Psn) via its UBA domain, and that loss of ubqn function suppresses phenotypes that arise from loss of psn function during development. Furthermore, overexpression of ubqn in the eye results in adult-onset, age-dependent retinal degeneration, which is at least partially apoptotic in nature. The degeneration associated with ubqn overexpression can also be suppressed by psn overexpression and enhanced by expression of a dominant negative version of Psn. Remarkably, expression of the human AD-associated variant of UBQLN1 leads to significantly more severe and earlier onset of eye degeneration than does comparable expression of the human wildtype UBQLN1. In addition to interact with Psn, we find that Ubqn also physically binds APP via its UBA domain. Loss of ubqn function leads to a decrease in the steady state levels of APP, whereas ubqn overexpression results in an increase in the APP levels. Together, these data identify Ubqn as a regulator of Psn and APP, support an important role for UBQLN1 in AD pathogenesis, and suggest the possibility that expression of a human AD-associated variant can cause neurodegeneration independent of amyloid production. (AG & RF contributed equally.)

101 Genomic regulation of lipid storage. Mathias Beller1,2, Carole Sztalryd1,3, Herbert Jaeckle2, Brian Oliver1. 1) Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda MD 20892, USA; 2) Max-Planck-Institut fuer biophysikalische Chemie, Abt. fuer Molekulare Entwicklungsbiologie, Am Fassberg 11, 37077 Goettingen, Germany; 3) GRECC/Geriatrics , Veterans Affairs Medical Center 10, North Greene Street, Baltimore MD 21201, USA. Lipid droplets are the universal lipid storage organelles and crucial for maintaining organismic as well as cellular lipid and energy homeostasis. Given an emerging importance of lipid droplets in major diseases such as obesity, diabetes and atherosclerosis, the need to understand the regulation and function of this organelle has become urgent. To identify the range of biochemical pathways involved in lipid droplet function as well as specific regulators, we performed a genome-wide RNA interference screen in Drosophila Kc167 cells. Our data reveal that about 3% of the genome is required for cellular lipid droplet deposition or utilization. Most of the few known lipid metabolism regulators were identified. Importantly, the majority of Drosophila genes required for lipid droplet deposition/ utilization were previously not associated with lipid storage. They belong to pathways acting on distinct levels of cell function including gene expression, signaling, oxidative phosphorylation and vesicle-mediated transport. Secondary RNAi screening targeting orthologs of 100 selected Drosophila genes in a mammalian liver cell line showed similar phenotypes and demonstrated evolutionary conservation of function. Our findings suggest a major, evolutionary conserved role of COPI-mediated retrograde vesicle transport and mitochondrial fatty acid beta-oxidation for lipid droplet homeostasis. Further analysis of these and additional identified gene- functions such as signaling components and transcription factors will aid understanding cellular lipid storage regulation and may reveal novel approaches and targets for future therapeutic treatments.

102 Genetic modifiers of MeCP2 function in Drosophila. David Mittelman1, Holly Cukier1, Ann Collins1, Alma Perez1, Zhaolan Zhou1, Huda Zoghbi1,2, Juan Botas1. 1) Departemnt of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Howard Hughes Medical Institute, Baylor College of Medicine, Houston TX. Rett syndrome (RTT) and some cases of autism are caused by a reduction of methyl-CpG-binding protein 2 (MeCP2), while duplication or triplication of the MECP2 locus cause a progressive neurological disorder characterized by mental retardation, seizures, and motor abnormalities. Similar phenotypes are recapitulated in mice that either lack or overexpress MECP2, thus underscoring the importance of properly controling MeCP2 levels. The tight regulation of this protein currently negates gene therapy as a treatment. To identify molecular mechanisms capable of compensating for altered MeCP2 function, we generated transgenic Drosophila producing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. Tissue-specific MeCP2 expression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. Interestingly, these phenotypes are modified by Drosophila homologs of MeCP2 protein interactors Sin3a, REST and N-CoR. Furthermore, we identified novel genetic modifiers of MeCP2 including factors that function in chromatin remodeling. 120 PLATFORMS: Drosophila Models of Human Diseases

103 Generation of Neurotoxic Prion Protein Isoforms and the Role for Hsp70 in Prion Protein Conversion. Sergio Casas-Tinto, Melisa Gomez-Velazquez, Claudio Soto, Pedro Fernandez-Funez, Diego E. Rincon-Limas. Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded and pathogenic isoform (PrPSc) whose unique biochemical and structural properties correlate with disease. In humans, prion disorders such as Creutzfeldt-Jakob disease have typically a sporadic origin, where wild type PrP spontaneously misfolds and aggregates in the brain. Unfortunately, major gaps still exist in the knowledge of how wild type PrP undergoes conformational changes and what are the cellular components involved in this process. To shed light on these issues, we studied the conformational dynamics of wild type PrP from Syrian hamster in transgenic flies. In fly neurons PrP progressively misfolds and acquires biochemical and structural properties of PrPSc, such as insolubility in non-ionic detergents, resistance to denaturing agents and recognition by PrPSc-specific conformational antibodies. The PrP isoform generated in flies is highly neurotoxic as it readily induces spongiform degeneration of brain neurons. Unlike mammalian PrPSc, fly-produced PrP is not resistant to protease digestion, a key feature of infectious PrP. This difference indicates that the neurotoxic conformer in prion diseases might be an intermediary conformer in the process of PrPSc formation. In an attempt to interfere with PrP misfolding in flies, we found that co-expression of PrP and the molecular chaperone Hsp70 protects against PrP-dependent neurodegeneration. Moreover, Hsp70 colocalizes and co- immunoprecipitates with abnormal PrP conformers. Additionally, we also found that Hsp70 inhibits amplification of mammalian PrPSc in a cell-free conversion system in vitro. Therefore, the direct interaction of Hsp70 and PrP prevents PrP misfolding. These results provide new and valuable insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.

104 Rhomboid-7 and Omi are components of the Pink1/Parkin pathway which affects mitochondrial membrane dynamics. Alexander J Whitworth1, Jeffrey Lee2, Angela Poole3, Ruth Thomas3, Venus Ho1, Leo Pallanck3, Angus McQuibban2. 1) Dept of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; 2) Dept of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 3) Department of Genome Sciences, University of Washington, Seattle, USA. Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by prominent loss of dopaminergic neurons in the substantia nigra and the presence of Lewy body inclusions, however, the pathogenic causes remain unclear. Mitochondrial dysfunction has long been implicated in the pathogenesis of PD but has recently been highlighted as a key pathologic mechanism. Genes identified in heritable forms of PD have provided a compelling link to mitochondria, however, the exact function of these genes and the events that lead to mitochondrial dysfunction are unknown. Targeted mutations in the Drosophila homologues of these PD-related genes has begun to reveal important insight into the their biological function. Recently, it was shown in Drosophila that the PD-linked genes pink1 and parkin act in a common pathway that maintains mitochondrial integrity by an unknown mechanism. Here, we identify the mitochondrial rhomboid protease, Rhomboid-7, as a novel upstream regulator in this pathway. We also show the mitochondrial protease Omi/HtrA2, along with Parkin, is a downstream effector of this pathway. Our biochemical data demonstrate a functional molecular link between Rhomboid-7 and both Pink1 and Omi, and genetic epistasis data place these factors in a genetic hierarchy. Furthermore, we present evidence that Pink1 and Parkin genetically interact with components of the mitochondrial fission and fusion machinery. Our findings indicate the Pink1/Parkin pathway likely functions to promote mitochondrial fission. These findings greatly further our understanding of the pathologic mechanisms of PD, firmly linking two new factors in a common pathway that regulates mitochondrial function. Furthermore, they raise the possibility that regulated intra-membrane proteolysis by Rhomboid-7 is a potential therapeutic target for the treatment of PD. PLATFORMS: RNA Biology 121

105 Global analysis of mRNA localization reveals a prominent role in the organization of cellular architecture and function. Eric Lecuyer1, Hideki Yoshida1, Christina Alm1, Neela Parthasarathy1, Tomas Babak1, Pavel Tomancak2, Henry Krause1. 1) Donnelly CCBR, University of Toronto, Toronto, Canada; 2) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. The localization of mRNA molecules is an important regulatory mechanism for targeting proteins to specific cellular compartments, although the overall prevalence and variety of transcript localization events remains unknown. To characterize subcellular mRNA localization dynamics during early Drosophila embryogenesis, we conducted a high-throughput Fluorescent In Situ Hybridization (FISH) screen of over 4,000 distinct mRNAs, and show that the majority of expressed mRNAs (71%) are subcellularly localized. Many novel varieties of subcellular localization patterns were identified, implicating localized mRNAs in the assembly and regulation of diverse cellular modules and processes. Analysis of the localization data, which has been organized within a publicly available database (http://fly-fish.ccbr.utoronto.ca), reveals that transcripts with similar localization dynamics are enriched for specific gene functions and putative regulatory elements. This work establishes mRNA localization as a widespread gene regulatory mechanism and underscores the predictive value of transcript localization phenotypes in assigning gene functions. We have begun dissecting the localization mechanisms and biological functions of various classes of mRNAs and the results of these ongoing studies will be presented.

106 Fragile X Protein controls the efficacy of mRNA transport in neurons. Daniela C. Zarnescu, Patty Estes, Michelle O’Shea. Dept Molecular & Cell Biol, Univ Arizona, Tucson, AZ. Fragile X syndrome (FraX) is the most common form of inherited mental retardation and affects 1/4,000 males. The disease is caused by loss of function for the FMR1 gene, and patients affected by this disorder display cognitive deficits as well as attention deficit and hyperactivity, anxiety, and autism. FMR1 encodes a sequence specific RNA binding protein, FMRP, which is ubiquitously expressed and is thought to function in synaptic plasticity by controlling the localization and translation of target mRNAs in neurons. Using a biochemical purification approach and Affymetryx microarrays we have identified a subset of the Drosophila FMRP associated mRNAs. To determine if FMRP is required for the localization of these target mRNAs, we have developed a genetically encoded system for mRNA tracking in living cells. To visualize FMRP associated mRNAs in Drosophila neurons we are using a GFP tagged MS2 phage protein, which has the ability to bind with very high affinity a specific, stem-loop forming RNA sequence, referred to as the MS2 binding site. We cloned 12 MS2 binding sites downstream of a number of FMRP associated mRNAs, which can now be visualized via the MS2-GFP protein. Using the bipartite GAL4-UAS system we expressed these constructs in primary neuronal cultures and imaged live mRNA trafficking in wild-type and dFmr1 mutant neurons. Using this imaging system we have obtained evidence that mRFP-FMRP colocalizes in motile particles with target mRNAs in living neurons. Quantitative analyses of various trafficking features, including speed, net movement and directionality show that FMRP regulates the efficacy of mRNA transport in neurons. These results are supported by Fluorescent Recovery After Photobleaching experiments, which demonstrate that FMRP controls the dynamic exchange between the cellular mRNA pool and RNA granules. Taken together these data support a model whereby defects in FraX may be due to inefficient mRNA transport to synapses.

107 A genetic screen for asymmetrically localized RNAs in Drosophila tracheal cells. Jayan N Nair1, Maria Leptin1, Paolo Filardo2, Veit Riechmann2, Elizabeth R. Gavis3. 1) Institute for Genetics, University Of Cologne, Cologne, NRW, Germany; 2) Institute for Developmental Biology,University of Cologne,NRW, Germany; 3) Dept. of Molecular Biology, Princeton University, Princeton 08544, USA. Asymmetrical localization of mRNAs and localized protein synthesis have an important role in establishing and maintaining polarity in cells such as neurons or the Drosophila oocyte. Such localized protein synthesis provides a means for the regulation of developmental plasticity. In Drosophila a subset of highly branched cells of the respiratory system exhibits both a high degree of polarity and developmental plasticity. These tracheal cells respond to the need for oxygen in the surrounding tissue by outgrowth of branches, often at sites very distant from the nucleus. On the assumption that some of the proteins required at the site of outgrowth are synthesized locally rather than near the nucleus we have performed a screen for mRNAs with asymmetric subcellular localization. We tagged mRNAs with GFP in vivo and screened the Drosophila genome for asymmetrically localizing RNAs. Briefly, we have combined the MS2-GFP labeling system with the EP transposon technique. A fusion protein of GFP with the RNA binding protein MS2 is used to visualize RNAs carrying MS2 binding sites (RNA stem loop recognized by MS2 protein). The EP transposon, which we have modified by incorporating MS2 binding sites, can be used to generate transgenic lines harboring the EP-MS2 transgene. Each of these lines has a different gene tagged with MS2 binding sites and can be tested in different tissues using tissue specific GAL4-MS2-GFP lines. We have performed a pilot screen in Drosophila tracheal cells, oocytes and neurons and have identified 12 candidates exhibiting specific localization. We are currently analyzing and characterizing these candidates. In a broader perspective these candidates will be useful to understand the organization and function of the signals in mRNAs essential for their asymmetric localization. 122 PLATFORMS: RNA Biology

108 RNA silencing influences gypsy chromatin insulator function and nuclear organization. Elissa P. Lei, Nellie Moshkovich, Patrick J. Boyle. Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD. Chromatin insulators influence gene expression by establishing chromatin domains subject to distinct transcriptional controls, likely through alteration of their spatial organization. Several lines of evidence suggest that insulator proteins bridge distant DNA sequences dispersed throughout the genome, causing looping of the DNA and the creation of a distinct chromatin domain. Nuclear insulator complexes termed insulator bodies are tethered stably to the nuclear matrix possibly forming chromatin loops. Our research provides evidence that RNA silencing, a gene regulation mechanism known to act on the level of chromatin, is involved in gypsy insulator function and higher order chromatin organization. We suggest that RNA may promote the multimerization of insulator complexes and/or their ability to interact with a nuclear scaffold. Several new observations have been documented, which provide a more thorough mechanistic understanding of how RNA silencing contributes to gypsy insulator function. First, Argonaute proteins Piwi and Argonaute2 interact physically with the insulator protein CP190 similarly to the DEAD-box RNA- dependent helicase Rm62 but in an RNA-independent manner. In addition, Piwi can be found to colocalize with insulator bodies. Extensive analysis of known RNA silencing mutants has uncovered a role for two additional helicases, Armitage and Spindle-E. Like Rm62, Armitage and Spindle-E function as negative regulators of gypsy insulator function. Small scale purification of RNAs associated with the gypsy insulator complex has enriched a heterogeneous but specific class of RNA. Our current efforts are focused on thorough cataloguing of this pool of RNAs by deep sequencing and gaining mechanistic insight into this process using both biochemical and genetic approaches.

109 Analysis of the role of splicing and cis-acting elements in oskar mRNA localization in Drosophila oocyte. Sanjay Ghosh, Virginie Marchand, Anne Ephrussi. Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Localization of maternal mRNAs coupled with spatio-temporal control of their translation is a notable feature of early development of many organisms. In Drosophila, oskar mRNA, encoding the posterior determinant of the embryo, is localized in ribonucleoprotein (RNP) complexes, in a multi-step process, to the posterior pole of the oocyte, and its expression is regulated by localization- dependent translation control. Recent studies have demonstrated that splicing of the first intron of oskar and proteins of the exon junction complex (EJC) are required for posterior transport, suggesting interaction(s) between the EJC deposited at the first exon- exon junction upon splicing and trans-acting factor(s) binding to specific cis-acting elements in oskar mRNA. In order to test the sufficiency of splicing at the first intron and to uncover potential cis-acting localization elements in the oskar coding region, we have performed a systematic analysis in vivo involving transgenic deletion and replacement constructs. This study has identified a region within the oskar coding region that, together with splicing, is necessary for posterior localization of the mRNA. Furthermore, by using in vitro splicing and RNase H assays, we are identifying trans-acting factors bound specifically within this region that may affect splicing and/or EJC formation/deposition. The functional significance of the cis-element in formation of localization-competent oskar RNPs will also be discussed.

110 Control of alternative splicing by regulatory networks in Drosophila. Britta Hartmann1, R. Castelo1, S. Boue1, M. Blanchette2, E. Peden3, R. RioSingh3, D. Rio2, J. Valcarcel1. 1) Centre de Regulació Genòmica, Barcelona, Spain; 2) University of California, Berkeley, USA; 3) University of Colorado, Boulder, USA. Alternative splicing (AS) is a major contributor to the complexity of higher eukaryote proteomes. Paradoxically, however, little is known about how regulatory networks influence this process. To address this question we employed whole genome splicing-sensitive microarrays to explore the extent and biological impact of AS in two very different regulatory networks. Sex determination has served as a prime example for AS regulation. In contrast, though signaling pathways have been thoroughly studied in their impact on transcriptional regulation, virtually nothing is known if (and how) they regulate AS. Surprisingly, we identified over 400 genes exhibiting sex-specific AS in adult flies. Subsequent qRT-PCR analysis confirmed our microarray data and uncovered vast differences in isoform levels between sexes. Furthermore, we analyzed the AS pattern of over 40 candidates in the adult body, head and in embryonic cell-lines uncovering interesting tissue-specific patterns of sex-specific AS. For example bicoid possesses a strong sex- biased use of an alternative 3‘splice-site only in the adult head changing the translational activity of the protein. Surprisingly, binding sites of the known sex determinants Tra and Sxl were found only in a small portion of AS events. Overall, these results imply that other regulatory factors are involved in sex-specific splicing regulation. Indeed, RNA binding proteins are overrepresented in our list and could be candidates. The impact of two signaling pathways, Wingless and Insulin, on AS in Drosophila cells was also explored. Activation of both pathways resulted in extensive AS changes. Interestingly, similar functional groups of genes are affected at the level of AS and by gene expression. We observed many signaling components to be regulated suggesting another layer of crosstalk to other pathways. A computational screen among co-regulated AS events revealed new regulatory motifs, which we are currently validating. PLATFORMS: RNA Biology 123

111 Mir-3 and mir-318 regulate Drosophila nautilus gene expression. Anandarao Ravulapalli, Wei Qin, Bruce Paterson. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. The Drosophila MyoD homolog, nautilus, is essential for normal myogenesis since its function is required to seed the correct founder myoblast pattern that prefigures the muscle fiber arrangement during embryonic development (Wei Q, et al., Proc. Natl. Acad. Sci. U.S.A. 104: 5461-6, 2007). The 3‘UTR of the nautilus gene is predicted to bind at least four microRNAs: mir-3, mir-309, mir-318 and mir-306*. To experimentally test this prediction, the nautilus 3‘UTR was sub-cloned directly downstream of the firefly luciferase ORF (F-luc-na-3‘UTR) and was then co-transfected into Drosophila Schneider S2 cells along with individual expression plasmids for the aforementioned miRNAs. Both mir-3 and mir-318 were found to substantially reduce luciferase activity when compared to either the control luciferase ORF without the nautilus 3‘UTR, or to the luciferase ORF with a nautilus 3‘UTR containing mutated microRNA binding sites. To try and understand the role of these microRNAs in vivo, we generated transgenic lines expressing mir- 3 and mir-318 using the gal-4/UAS system. Over expression of mir-3 under the control of the ubiquitously expressed pAct gal-4 driver resulted in a reduced and/or altered nautilus expression pattern and instances of severely disrupted embryonic muscle. However, similar expression of mir-318 did not affect either the embryonic nautilus or muscle pattern, even though mir-3 and mir- 318 are predicted to bind the same site in the nautilus 3’ UTR. This could reflect the fact that mir-3 expression initiates in stage 9 embryos just when nautilus expression is first noted, whereas mir-318 is expressed in the adult so prior site occupation by mir-3 may limit a mir-318 effect. We have initiated a transgene rescue of nauGFP, a nautilus null, using a genomic fragment lacking the mir-3/ 318 binding sites to determine if mir-3/318 regulation of nautilus expression during development is important. The results to date suggest that mir-3 “fine-tunes” nautilus expression in the embryo while mir-318 has a similar role in the larval and adult stages. 124 PLATFORMS: Genome and Chromosome Structure

112 Efficient identification of Drosophila Y-chromosome sequences by short-read sequencing. Bernardo Carvalho1, Andrew Clark2. 1) Dept de Genetica, Univ Fed Rio de Janeiro, Rio de Janeiro, Brazil; 2) Molecular Biology and Genetics, Cornell University, USA. Despite the completion of the genome sequence of 12 species of Drosophila, their Y chromosomes remain poorly known. A major obstacle has been the identification of Y chromosome sequences: due to its high content of repetitive sequences, the Y chromosome has been represented as highly fragmented in most genome projects, hidden within a large number of small, unmapped contigs. Identification of Y-chromosome sequences among these fragments has yielded important insights regarding the origin and evolution of the Y chromosome, but the process of testing Y-linkage remains labor intensive, precluding an exhaustive study of this chromosome. Parallel, short-read sequencing methods hold the promise of revolutionizing the identification of Y-linked contigs. A rapid application of this approach to three available Drosophila genomes (D. melanogaster, D. pseudoobscura and D. virilis), has more than doubled the number of known Y-linked genes in these species.

113 Evolution of nested genes in Drosophila and vertebrates. Fyodor A Kondrashov1, Raquel Rassis2, Alexey Kondrashov2, Eugene Koonin3. 1) Section of Ecology, Behavior & Evolution, UCSD, La Jolla, CA; 2) Center for Computational Medicine and Biology and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109; 3) National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda MD, USA. Genomes with introns ofter contain nested genes, i.e. genes that are coded inside the introns of other genes. In particular, the human genome contains ~150 while the Drosophila melanogaster genome codes more than 800 of functional protein-coding nested genes. Many of these nested genes are functionally characterized, especially those in the human genome, however, the evolution of the nested state has not been considered. We reconstructed the evolution of the simplest and the commonest nested state, where the entire internal gene was contained within a single intron of the external gene. By comparing this gene structure in two sister species and an outgroup we were able to quantify the rate of gain and loss of nested genes. We find that the rate of gain of nested gene is much higher than the rate of loss in both Drosophila and Vertebrate clades, with various types of transpositions being the most common mechanism of acquisition. Thus, genome structure is increasing in complexity by loosing unidimensionality of gene organization through the accumulation of nested genes. Preliminary results find no support to suggest the role of selection in this process of nested gene accumulation.

114 Repetitive Elements and the Rise of Chimeric Genes in the D. melanogaster Subgroup. J Roman Arguello1, Shuang Yang2,3, Xin Li2,3, Yun Ding2,3, Qi Zhou2,3, Ying Chen1, Yue Zhang2, Ruoping Zhao2, Frédéric Brunet4, Lixin Peng2, Manyuan Long1, Wen Wang2. 1) Dept Ecology & Evolution, Univ Chicago, Chicago, IL; 2) Kunming Institute of Zoology, Kunminng, China; 3) Graduate School of Chinese Academy of Science, Beijing, China; 4) Ingénieur de Recherche en Bioinformatique Equipe Génomique Evolutive des Vertébrés IGFL, France. The use of chimeric genes presents a unique system with which to study the origin and evolution of genetic diversity. However, many aspects of their advents, for example the mutational mechanisms generating them, the frequency with which they arise, and the novel functions that they evolve, remain intriguing questions. This uncertainty is in part due to a lack of a data set comprised of experimentally verified young duplicates involved in the formation of chimeras. To address this, we use a battery of methods (expression studies, gene structure studies, sequence analyses) to identify chimeric genes over the D. melanogaster subgroup. In addition, we investigate the flanking regions of the paralog pairs to gain insights about the mutational mechanism giving rise to them. We identify 17 chimeric genes, most of which appear to be functional and have evolved divergent expression profiles. Interestingly, we find that repetitive elements, in particular DNAREP_1, are enriched near or at the boundaries the paralogs, consistent with either models of nonallelic recombination and possibly the shuffling of DNA through the movements of transposable elements. PLATFORMS: Genome and Chromosome Structure 125

115 Highly asymmetrical rates of evolution between paralogs following the duplication of testes expressed genes from neo-X- chromosomes to autosomes. Richard Meisel1, Mira Han2, Matthew Hahn2. 1) Penn State University; 2) Indiana University. Drosophila genomes consist of five major chromosome arms and a dot chromosome. In the ancestral karyotype, four of the major arms are telocentric autosomes, while one arm makes up the telocentric X chromosome. Throughout the evolution of the genus multiple independent fusions of chromosome arms have occurred, including some in which an ancestral autosome was fused to the X chromosome giving rise to a neo-X chromosome. These neo-X chromosomes provide us with a unique opportunity to study how the chromosomal context of a gene influences its function, especially in regards to sex bias and intragenomic conflict between sexes. We identified lineage specific duplicated genes in the sequenced Drosophila genomes for the purpose of comparing patterns of gene duplication between autosomes, ancestral X chromosomes, and neo-X chromosomes. The ancestral X chromosome acts as disproportionate source of inter-arm duplicated genes in some lineages, but not all lineages show this pattern. Two species with neo-X chromosomes (D. pseudoobscura and D. willistoni) have an excess of genes duplicated from the recently X-linked region to other chromosome arms. Many of these duplicated genes arose around the time of the X-autosome fusion event that gave rise to the neo-X chromosomes. Additionally, the derived copies of the neo-X to autosome paralogs tend to have amino acid sequences that evolve at much faster rates than the ancestral copies. Furthermore, the ancestral autosomal copy of many of the genes is expressed in the testis of adult males. We hypothesize that the autosomal derived copies have been specialized for the male functions of the gene, while the neo-X-linked copies have retained the majority of the ancestral suite of functions. The ancestral X chromosome probably also experienced this phenomenon when it became a sex chromosome, but it is currently much closer to equilibrium - in regards to male-biased and female-biased gene content - than the neo-X chromosomes and does not show such a striking pattern of recent duplication of male-expressed genes onto autosomes.

116 Age-related changes in double-strand break repair. William Engels, Christine Preston, Dena Johnson-Schlitz, Carlos Flores. Dept Genetics, Univ Wisconsin, Madison, WI. A recent surprise in the study of how double-strand breaks are repaired in the germline is that the process changes markedly as the organism ages. We monitored three alternative outcomes of I-SceI-induced breaks: SSA: Single-strand annealing NHEJ: Non-homologous end-joining HR-h: Homologous repair using the homolog as template (i.e., gene conversion) Young flies use mostly SSA with NHEJ as a strong second choice and HR-h a distant third. These parameters change steadily with age until HR-h finally becomes the dominant outcome in older flies. In recent work we have broadened these observations in several directions. First, we find that the repair outcomes and the age effect can depend on the genomic location of the break. A particularly oddball location occurs at site 86D where the repair process appears to be NHEJ-phobic in both young and old flies. We also compared the process between the male and female germlines. The dramatic rise of HR-h with age occurs in both sexes, but there are some unexpected differences. Females at all ages had much-reduced usage of NHEJ and an excess of aberrant repair events. We attempt to explain the phenomenon of age-dependent break repair in terms of the “antagonistic pleiotropy” model.

117 Evolutionary genetics of hybrid sterility in Drosophila. Nitin Phadnis, Allen Orr. Dept Biol, Univ Rochester, Rochester, NY. Recent studies in the genetics of speciation have identified several genes that cause intrinsic postzygotic reproductive isolation; almost all of them evolve rapidly. However, the evolutionary forces that drive the rapid changes in these genes remain unclear. The idea that genetic conflict, such as meiotic drive, may cause the evolution of intrinsic postzygotic reproductive isolation is intuitively appealing, but empirical evidence has been scant. Previously we showed that, “sterile” F1 hybrid males between the USA and Bogotá subspecies of Drosophila pseudoobscura become very weakly fertile when aged and produce all daughters, reflecting sex chromosome segregation distortion. Mapping studies showed that the same regions on the Bogotá X-chromosome underlie both hybrid male sterility and hybrid segregation distortion. The critical question is whether the same genes cause both hybrid segregation distortion and hybrid male sterility. We begin by focusing on a region linked to the visible mutation sepia on the D. pseudoobscura XR, which is known to play an essential role in both phenomena. We introduced 200 independent copies of the sepia region from USA into an otherwise Bogotá genome and backcrossed to Bogotá for 28 generations. So far, the genes causing hybrid male sterility from those causing hybrid segregation distortion have proved to be meiotically inseparable. We have further fine-mapped the genes responsible for both hybrid phenomena to an interval containing five genes, including a rapidly evolving candidate gene. Ongoing transgenic experiments may provide the first clear example of genetic conflict driving the evolution of reproductive isolation. 126 PLATFORMS: Genome and Chromosome Structure

118 Drosophila histone variant H2Av localizes to centromeres and regulates normal localization of centromeric histone H3 variant CENP-A/CID. Weiguo Zhang1,2, Gary H. Karpen1,2. 1) the Life Sciences Division, the Lawrence Berkeley National Laboratory, Berkeley, CA; 2) Department of Molecular and Cellular Biology, University of California-Berkeley, Berkeley, CA. CENP-A (CID in Drosophila) is the centromere-specific histone H3 variant and a primary epigenetic regulator for centromere identity and kinetochore formation. Drosophila centromeres are comprised of alternative chromatin domains containing CID or canonical histone H3 nucleosomes. It has been previously indicated that the centromeric H3 nucleosomes have a characteristic pattern of histone modifications distinct from both euchromatin and heterochromatin. Here we identified H2Av, the histone H2A variant in Drosophila, as another important epigenetic regulator of centromeric chromatin. By RNA interference (RNAi) experiments targeting H2Av in Drosophila S2 tissue culture cells, we demonstrated that H2Av is required for normal localization of CID and another essential inner centromeric protein CENP-C. We studied the impact of H2Av on loading of newly synthesized CID versus stability of CID proteins at centromeres. We found that the centromeric protein levels for both CID and CENP-C are significantly reduced after H2Av RNAi. We analyzed the distribution of H2Av in different phases throughout the cell cycle and found that H2Av directly localize to centromeres and that the majority of centromeric H2Av localize to H3 nucleosomes alternative to the CID nucleosomes. Our results provide evidence that the alternative H3 nucleosomes at centromeres are pivotal to normal centromere propagation. This work is supported by the Susan Komen Breast Cancer Foundation Postdoctoral Fellowship to W.Z. and NIH grant GM66272 to G.H.K. PLATFORMS: Techniques and Functional Genomics 127

119 A toolkit for high-throughput gene engineering in flies. Radoslaw K Ejsmont1, Mihail Sarov2, Pavel Tomancak1. 1) Tomancak Lab, MPI-CBG, Dresden, Germany; 2) BAC Facility, MPI-CBG, Dresden, Germany. To study gene regulation in vivo it is necessary to create faithful reporters of gene activity. The randomized nature and insertion bias of transposon mediated ‘gene trapping’ approach prevents systematic genome wide gene tagging. Therefore we developed a reverse genetic strategy to generate gene expression reporters systematically, by high-throughput recombineering of large genomic clones and subsequent P[acman]/phiC31 mediated transgenesis. Our intermediate goal is to establish a collection of live mCherry based gene expression reporters for marking tissues during Drosophila embryogenesis. We introduce mCherry at the N-terminus of selected genes, within large genomic clones, to preserve intact gene regulatory sequences. To allow gene tagging in high-throughput we adapted a recombineering pipeline developed to systematically tag C. elegans proteins. This procedure, based on Red/ET system, performs all recombineering steps in a single bacterial strain, in liquid culture, in 96-well format, within one week. We modified the C. elegans pipeline for fly transgenesis by introducing attB sites and 3xP3-DsRed fly selectable marker. We follow two distinct strategies to achieve high-throughput gene tagging. 1) We replicate the two-step C. elegans approach of first tagging the gene and then subcloning part of the BAC clone. This strategy can be seamlessly applied to any existing BAC library. 2) We generate and sequence a new fosmid library that incorporates attB, 3P3-DsRed and OriV in the backbone. This fosmid library enables highly efficient Red/ET tagging in 96-well liquid culture format and direct phiC31 mediated transgenesis of flies. We will present results of a pilot experiment where we performed tagging of 12 genes expressed during early embryogenesis with different fluorescent proteins (GFP and mCherry) to assess the benefits of different tags for monitoring early zygotic gene expression.

120 PhiC31-mediated cassette exchange in Drosophila. Jack R. Bateman, Anne M. Lee, Lillian Merriam, Laura Stadelmann, C.-ting Wu. Department of Genetics, Harvard Medical School, Boston, MA. We have developed a method for transgenesis that targets constructs to predetermined genomic sites and permits the integration of sequences lacking a phenotypic marker (1). Importantly, this method will facilitate transgenic studies by controlling for undesired position effects from flanking genomic regions and/or from a second transcription unit in the transformation vector. Using the phiC31 integrase system (2) in conjunction with Recombinase Mediated Cassette Exchange (RMCE), we have targeted a variety of donor cassettes, including those that do not carry visible markers, to eight defined loci in the Drosophila genome. Targeting is achieved by exchanging a donor cassette flanked by two attB sites with a previously integrated target cassette carrying the mini-white gene and flanked by two attP sites (also see (3) and (4) for RMCE driven by other recombinases). Because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify successful integrants simply by loss of mini-white eye color. Furthermore, the use of Drosophila lines that carry an endogenous source of the phiC31 integrase (5) can produce integration efficiencies of 50% and greater. We are continuing to develop new reagents to expand the utility of the RMCE system for different types of analyses, and are currently exploring strategies using RMCE to systematically alter a gene of interest at its natural locus. This work was supported by the National Institutes of Health via a fellowship to J.R.B. (1 F32 GM67460) and a grant to C.-t.W. (1 RO1 GM61936), and by Harvard Medical School. 1. Bateman et al., 2006. Genetics 173: 769. 2. Groth et al., 2004. Genetics 166: 1775. 3. Oberstein et al., 2005. Nat. Methods 2: 583. 4. Horn et al., 2005. PNAS 102: 12483. 5. Bischof et al., 2007. PNAS 104: 3312.

121 The Drosophila ORF Collection: a high quality resource for proteomic and functional genomic studies. Mark Stapleton, Charles Yu, Ken Wan, Soo Park, Bhaveen Kapadia, Bayan Parsa, Joseph W Carlson, Susan E Celniker. Genome and Computational Biology, Lawrence Berkeley National Lab, Berkeley, CA. The identification of all expressed genes and the structure(s) of their transcripts are prerequisites for many structural and functional genomic studies. One of the major goals of the Berkeley Drosophila Genome Project is to experimentally define the transcribed portions of the genome by producing a collection of fully sequenced cDNAs. To accomplish this goal, we have produced the Drosophila Gene Collection (DGC) which is the product of sequencing a collection of 263,617 ESTs. The project currently has 15,369 full-insert sequenced clones and contains 7,256 cDNAs whose translation agrees 100% with Release 5.3 FlyBase annotations. This set of clones, the DGC Gold Collection, represents the starting point in generating a high quality resource for functional genomic studies. To facilitate the use of the Gold Collection, we are in the process of cloning open reading frames (ORFs) from 6,662 cDNAs into the CreatorTM (Clontech) universal cloning system. This is a directional PCR cloning system that generates a proteomics-ready, easily transferable set of donor ORFs. The donor vector allows for the subsequent transfer of these ORFs into a wide variety of acceptor expression vectors using Cre recombinase. We are making two types of each ORF that will allow for the expression of three forms of a given protein: the first type of ORF contains its native stop codon which can be used to generate amino-terminal fusion proteins as well as the capacity to express native, untagged proteins; the second type of ORF does not contain its native stop codon which can be used to generate carboxy-terminal fusion proteins. Our goal for the initial phase of the project is to produce a total of 5,000 high-quality donor clones for each of the two types of ORFs. To date, we have made a total of 8,659 high-quality donor clones. This system and a core set of acceptor expression vectors and their uses will be discussed along with preliminary experiments demonstrating the system’s utility. 128 PLATFORMS: Techniques and Functional Genomics

122 Genome-wide mapping and annotation of protein expression and interaction in Drosophila melanogaster, using a hybrid PiggyBac/P-element YFP gene trap system with tandem affinity tags. Ed Ryder1, Helen Spriggs1, Emma Drummond1, Laura Harris1, Jane Webster1, Glynnis Johnson1, John Roote1, Nick Lowe3, Kathryn Lilley2, Svenja Hester2, Julie Howard2, Johanna Rees2, Steve Russell1,2, Daniel St. Johnston3. 1) Dept Genetics, Cambridge Univ, UK; 2) Cambridge Systems Biology Centre, Cambridge Univ, Cambridge, UK; 3) Gurdon Institute, Dept Genetics, Cambridge Univ, Cambridge, UK. We have initiated a screen to generate and characterise protein trap lines in Drosophila using a PiggyBac transposon-based strategy. The ability to generate in vivo tagged proteins has tremendous potential for furthering our understanding of developmental processes by allowing the characterisation of sub-cellular protein localisation and facilitating the isolation of multi-protein complexes. This large project involves collaborations with over thirty UK laboratories. Our Pig/P transposons are tagged with yellow fluorescent protein (YFP) incorporated into endogenous genes via an exon-trapping strategy, thus facilitating the visualization of trapped proteins in living embryos and larvae. As correct incorporation of the fluorescent tag is a rare event we employ an automated embryo sorter to select the insertions. Putative lines are mapped by iPCR and sequencing, and custom software predicts whether the insertion is in the correct frame for a functional YFP fusion. The transposed exons also contain two protein affinity tags that allow the protein to be isolated in its native complex by tandem affinity purification. Complex components are identified by tandem mass-spectrometry with spectra assigned to the fly proteome via the MASCOT search engine, and analysed using ProteinCenter (Proxeon). To aid in the characterisation of YFP-trap lines, we have developed web-based software which allows detailed annotation of protein expression at all stages of development and tissue types (including sub cellular location and spatial descriptors), using GO and FlyBase controlled vocabulary. The system allows multiple groups to work in collaboration and share uploaded images and annotation, whilst still protecting the original data.

123 Identification of compounds that modulate lipid droplet storage in S3 cells using qHTS. Douglas Auld1, Ya-Qin Zhang1, Noel Southall1, Mathias Beller3, James Inglese1, Christopher Austin1, Brian Oliver2. 1) NIH Chemical Genomics Center, NIH, Rockville, MD; 2) National Insitutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda MD, 20892; 3) Max-Planck-Institut for Biophysical Chemistry, Göttingen, Germany. The National Institutes of Health Chemical Genomics Center (NCGC) was formed in 2004 as part of the NIH Roadmap funded Molecular Libraries Initiative. The NCGC aims to enable scientists with useful chemical probes by providing expertise in assay development and industrial scale technologies for HTS of chemical libraries. The NCGC has developed a screening paradigm where libraries >100K in size are screened as seven-point concentration-response (CR) curves (qHTS). This approach has been applied to an assay for lipid droplet accumulation in S3 cells where we used both a fluorescence lipid droplet stain (BODIPY 493/503) and a fluorescent cytoplasmic stain (Invitrogen, CellTracker) to develop a cell-based assay for lipid drop formation following feeding with oleic acid. Laser scanning imaging was use to enumerate the amount of green and red fluorescence for cells seeded in 1,536-well plates enabling the rapid evaluation of potency values for ~10K compounds. The chemical libraries included both known bioactives as well as diverse compounds of unknown function. The types of compounds identified will be discussed. As well, the expansion of this technology platform to enable qHTS of large (>200 K) compound collections will be also be shown.

124 Systems model of ATP-generating metabolic network in Drosophila flight muscle. Jacob D. Feala1, Laurence Coquin2, Andrew D. McCulloch1, Giovanni Paternostro1,2. 1) Dept. of Bioengineering, University of California - San Diego, La Jolla, CA; 2) Burnham Institute for Medical Research. Computational models of biological systems are useful for generating predictions and testing hypotheses in-silico. We have reconstructed the reaction network of ATP-generating central metabolism in Drosophila flight muscle, in a stoichiometric matrix suitable for flux-balance simulations and constraint-based analysis. The network, built previously [1] using the annotated genome and the KEGG database of genes and reactions, was made specific for flight muscle by integrating absolute gene expression data for fly thorax, which is composed mostly of flight muscle. Each enzymatic reaction is included only if validated against the literature, unless required to fill a pathway gap. Boundary conditions were integrated into the model for the special case of steady-state hypoxia, based on metabolite measurements gathered in our lab. Quantitative 1H spectroscopy and biochemical assays were used to measure accumulation of products, substrate depletion, and ATP concentration over 4 hours of hypoxia, for wild-type and several enzyme deletion strains. This refined version of the model can be used to investigate chronic hypoxia in muscle tissue, or the generic model can be similarly tailored to other specific contexts of interest. PLATFORMS: Techniques and Functional Genomics 129

125 A Morphology and Gene Expression Atlas of Drosophila Embryogenesis. Cris L. Luengo Hendriks1, Soile V.E. Keränen1, Pablo Arbelaez2, Gunther H. Weber1, Charless C. Fowlkes3, Clara N. Henriquez1, David W. Kaszuba1, Bernd Hamann4, Jitendra Malik2, Mark Biggin1, David W. Knowles1. 1) Lawrence Berkeley National Laboratory, Berkeley, CA; 2) University of California, Berkeley, CA; 3) University of California, Irvine, CA; 4) University of California, Davis, CA. The Berkeley Drosophila Transcription Network Project (http://bdtnp.lbl.gov) has established a suite of live and fixed embryo imaging and image analysis methods that have provided the first quantitative three-dimensional description of Drosophila blastoderm morphology and gene expression at cellular resolution. This atlas has revealed a wealth of previously undetected biological results and is being used in our system-wide analysis of the early transcript network. During the 10 hours after blastoderm formation, large cell motions and complex patterns of differentiation generate late stage embryos with over 70 cell types and all major larval organs. Using an improved imaging technology that allows an entire embryo to be captured in three dimensions with high fidelity, we are developing computational methods to produce a quantitative, cellular-resolution atlas of all of embryo development. High-resolution fluorescence images of whole, fixed embryos are being acquired and used to develop segmentation methods to locate the individual nuclei and cells. Classification methods, using annotated morphological features and tissue-specific markers, are being developed to recognize specific body-plan and tissue structures. Registration methods are being developed to assemble multiple embryo images into average morphological maps, onto which measured, per-cell mRNA and protein expression levels can be added. To link these stage-specific maps, temporal movements and mitotic events will be tracked using live cell imaging of embryos expressing nuclear, cell membrane and tissue-specific fluorescent proteins. The goal is to produce an expandable computational, morphology and gene expression atlas of Drosophila embryogenesis at cellular resolution. 130 PLATFORMS: Chromatin and Gene Expression

126 Understanding the function of insulators in the Drosophila genome. David J. Marion1, Alexey A. Soshnev2, Kate Appleton3, Xingguo Li3, Misty D. Wehling3, Ryan M. Baxley2, Pamela K. Geyer1,2,3. 1) Genetics Program; 2) Molecular and Cellular Biology Program; 3) Department of Biochemistry, University of Iowa, Iowa City, IA 52242. Eukaryotic chromosomes contain many adjacent genes that show different spatial and temporal regulation. This implies that the long-distance action of transcriptional regulatory elements is restricted in eukaryotic genomes. Insulators block interactions between enhancers, silencers, and promoters in a position-dependent manner, but the mechanisms are poorly defined. A Su(Hw)-dependent insulator, 1A-2, resides in the intergenic region between the yellow and achaete genes, which show differences in temporal and tissue-specific expression. To address whether 1A-2 regulates independent transcription at the endogenous site, this region was deleted by homologous recombination and mRNA levels were assayed by RT-PCR. Our data suggest that the 1A-2 deletion has a minimal impact on yellow and achaete transcription patterns, but surprisingly, has a large effect on the expression of an intergenic non-coding (nc) RNA. Subsequent studies are underway to characterize this ncRNA and to define the mechanism of transcriptional regulation by 1A-2. To gain insights into mechanisms of insulator function, we are using the LacI tethering system developed by Belmont and colleagues, who established multiple transgenic lines that carry a tandem array of lac operator sequences inserted randomly in the genome. Our lab has generated expressor lines carrying insulator proteins fused to the LacI repressor. Tethering these proteins to distinct binding sites will address the following questions. 1) Do insulator proteins direct the formation of chromatin loops between distant sites? 2) Do insulator proteins affect local chromatin condensation and associated histone modifications? These studies permit a comparison of the properties of insulator proteins and will provide insights into how insulators affect chromosome organization.

127 Bhringi, a highly conserved regulator of Twist transcription factor activity. Scott J. Nowak1, Katie Gonzalez2, Mary K. Baylies1. 1) Dept. of Developmental Biology, Sloan-Kettering Institute, New York, NY; 2) Scripps Research Institute, La Jolla, CA. A yeast double interaction screen designed to recover novel Twist interaction partners lead to the identification of CG8580, a gene we have named bhringi (bhr). The phenotypes of bhringi mutants reveal a role for bhr during muscle development: loss of bhr during embryogenesis results in muscle loss, severely altered muscle morphology and defective muscle attachments. bhr encodes a 201 residue protein that is highly conserved across multiple species, from flies to humans. Immunohistochemistry indicates that bhringi is localized to the nucleus and is expressed broadly throughout the embryo during all phases of embryogenesis. Bhringi interacts with Twist both genetically and physically by GST-pulldown and co-immunoprecipitation in vitro. Further, loss of specific Twist- dependent target gene expression is observed in bhr mutants. Bhringi is localized to gene-rich regions of the genome and is also capable of interaction with subunits of the Brahma chromatin remodeling complex. Taken together, these results indicate a mechanism whereby Bhringi interacts with transcription factors and chromatin remodeling machinery to facilitate proper expression of Twist- dependent genes during Drosophila development.

128 Genetic analyses of cofactors that cooperate with the Brahma (SWI/SNF) chromatin remodeling complex in the regulation of target genes. Chhavi Chauhan1, Claudia Zraly1,2, Manuel Diaz1,2, Andrew Dingwall1,2,3. 1) Molecular Biology Program, Loyola University Chicago, Stritch School of Medicine, Maywood, IL; 2) Oncology Institute; 3) Department of Pathology. The Brahma (SWI/SNF) ATP-dependent chromatin remodeling complex uses energy to drive changes in contacts between chromosomal DNA and histones. These changes impact gene expression, either positively or negatively and contribute to the control of cell growth and patterning. We found that the hormone response genes were among the most sensitive to the loss of Brm complex functions during development. To gain insight into the mechanism of target gene regulation, we have focused on identifying additional cellular cofactors important for Brm complex functions on the hormone response genes. A dominant enhancer/suppressor screen was employed to identify these cofactors. One of the candidates, which we named cara mitad (cmi), encodes a protein related to a component of the mammalian MLL2/ALR nuclear receptor coregulator complex. This complex contains a histone methyltransferase activity that specifically modifies histone lysine residues to affect hormone signaling. All of the known components of this complex have orthologs in Drosophila, though few have been well characterized. We have generated null mutations in the cmi gene and we will present our genetic analyses of cmi function during development, as well as genetic interactions with components of the Brm complex. We hypothesize that a conserved nuclear receptor coactivator complex helps to direct Brm complex functions in regulating the hormone response during Drosophila development. PLATFORMS: Chromatin and Gene Expression 131

129 Paused Polymerase in the Drosophila Embryo. Michael S Levine1, Julia Zeitlinger2, Joung-Woo Hong1, Jess Piel1, Dave Hendrix1, Richard A Young3. 1) MCB, UC Berkeley, Berkeley, CA; 2) Stowers Institute for Medical Research, Kansas City, MO; 3) Whithead Institue, MIT, Cambridge, MA. The analysis of ~30 different Dorsal target enhancers suggests that those mediating gene expression in response to high levels of the Dorsal gradient contain a series of disordered low-affinity Dorsal and/or Twist activator binding sites. In contrast, enhancers mediating expression in response to low levels of the gradient (the “type 2” response) contain an ordered arrangement of optimal Dorsal and Twist binding sites. This organization is highly conserved in evolution and is likely to foster cooperative occupancy of linked operator sites. We suggest that type 2 enhancers have some of the properties of a transistor: they amplify weak and unstable signals to produce a constant output of gene activity. The accurate representation of genetic circuit diagrams depends on the designation of this special subset of enhancers. The conventional view of gene activation is that it depends on the recruitment of RNA polymerase II (Pol II) to the core promoter. However, there are a few documented examples of gene activation via Pol II elongation, whereby Pol II is bound, but paused at a fixed site just downstream of the transcription start site. It is not known to what extent this mechanism is used to establish differential patterns of gene expression in development. To investigate this issue, we performed ChIP-chip assays using antibodies directed against Pol II. At least 1,000 genes contain a paused or stalled form of Pol II prior to their activation in the Drosophila embryo. These include many developmental control genes, such as Hox genes and components of the FGF, Wnt, Hedgehog, TGF?, and Notch signaling pathways. Altogether, these results suggest that the regulation of Pol II elongation, not recruitment or initiation, might be a critical mechanism for gene activation in development.

130 Chromatin-remodeling by Kismet in Transcription and Development. Kristel M. Dorighi1, Shrividhya Srinivasan1,2, John W. Tamkun1. 1) MCD Biology, UC Santa Cruz, Santa Cruz, CA; 2) Developmental Biology, Stanford University, Stanford, CA. Factors that regulate chromatin structure play important roles in development. Members of the Polycomb group (PcG) of repressors and their antagonists, the trithorax group (trxG) of activators, act at the level of chromatin to maintain patterns of gene expression and cellular identities in multicellular organisms. Kismet is an ATP-dependent chromatin-remodeling factor of the trxG that facilitates an early step in transcriptional elongation. We are currently investigating how Kismet interacts with other trxG proteins to counteract PcG-mediated transcriptional repression. Using Drosophila melanogaster as a model organism, we found that Kismet promotes the recruitment of two histone H3 lysine 4 methyltransferases of the trxG family - ASH1 and TRX to chromatin. In addition, Kismet opposes the methylation of histone H3 on lysine 27, a repressive histone modification catalyzed by PcG proteins that is required for stem cell self-renewal and the maintenance of pluripotency. Since the function of PcG and trxG proteins has been highly conserved during evolution, these findings suggest the human counterpart of Kismet - CHD7 - regulates stem cell development by counteracting PcG repression. We are currently testing this model in the human embryonic stem cell-like cell line Ntera2. Even if our hypothesis is incorrect, characterization of the function of CHD7 will shed light on the molecular nature of CHARGE syndrome, a serious developmental disorder linked to mutations in human CHD7.

131 Regulation of Myc-induced cell growth by the histone H3K4 demethylase Lid. Julie Secombe, Ling Li, Robert Eisenman. Div Basic Sci, Fred Hutchinson Cancer Res Ctr, Seattle, WA. The Myc family of mammalian transcription factors control cell growth and cell cycle progression and are implicated in the genesis of a wide range of cancers when misregulated. To gain insight into the mechanism by which Myc functions, we are characterizing dMyc, the sole Drosophila ortholog of Myc. dMyc is functionally similar to mammalian Myc proteins and can regulate cell cycle progression and cell growth during Drosophila development. To identify genes required for dMyc-mediated cell growth, we carried out a genetic interaction screen for dose-sensitive enhancement or suppression of a rough eye phenotype generated by overexpression of dMyc. One suppressor identified in this screen was the Trithorax group gene, little imaginal discs (lid). We have shown that Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. In addition, we have demonstrated that Lid is histone H3 lysine 4 demethylase and that Lid’s enzymatic activity is negatively regulated by dMyc, which binds to Lid’s catalytic JmjC domain. We are currently determining the mechanism by which Lid functions in Myc-mediated transcriptional activation and cell growth. 132 PLATFORMS: Chromatin and Gene Expression

132 P element repression by an epigenetic telomeric trans-silencing involving RNA silencing and heterochromatin formation. Stéphane Ronsseray, Thibaut Josse, Laure Teysset, Anne-Laure Todeschini, Augustin de Vanssay, Clara Sidor, Valérie Delmarre, Dominique Anxolabéhère. Dynamique du Génome et Evolution, Inst Jacques Monod, Paris, France. The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, “TAS”) has the capacity to repress in trans, in the female germline, a homologous transposon or transgene located in euchromatin. We will present the properties of this new silencing. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. It displays an epigenetic transmission through meiosis which involves an extrachromosomal maternally- transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding HP1 and Su(var)3-7) and the repeat associated small interfering RNA (rasiRNA) silencing pathway (aubergine, homeless, armitage and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2 which is involved in the canonical RNA interference (siRNA) pathway, nor to a mutation in loquacious which is involved in the micro RNA (miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a rasiRNA pathway linked to heterochromatin formation. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway. We will also present data showing that TSE behaves as a cellular repression mechanism; this mechanism would have been coopted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. PLATFORMS: Physiology and Aging 133

133 The TOR pathway couples nutrition and developmental timing in Drosophila. Pierre Leopold, Sophie Layalle. CNRS/University of Nice, Nice, France. In metazoans, final adult size depends on both the rate and the duration of growth. These two parameters, which together determine the range of animal growth, are modified by nutritional cues. Molecules of the insulin/IGF family control the growth rate, while the duration of juvenile growth is controlled by pulses of steroid hormones. Here we show that in Drosophila, nutrition modifies developmental timing through a sensor tissue called the prothoracic gland (PG) that secretes the moulting hormone ecdysone. When activity levels of the Target of Rapamycin (TOR) kinase, which represents the main nutrient-responsive pathway, are experimentally reduced in the PG, the peak of ecdysone that marks the end of larval development is abrogated. This abrogation extends the duration of larval growth and thereby increases animal size, but without changing the larval growth rate. Conversely, the extension of larval development that normally follows nutrient deprivation can be reversed by specifically reactivating the TOR pathway in PG cells. The sensitivity of the PG to TOR inhibition is temporally restricted to a narrow window occurring towards the end of larval development, defining a nutritional checkpoint that acts before the commitment to maturation. Our data indicate that the PG uses TOR as a molecular sensor to couple nutritional inputs to the production of a hormonal signal that controls the duration of the juvenile growth period.

134 Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary. Lei Pan1,2, Ting Xie1. 1) Stowers Inst, Kansas City, MO; 2) Inst of Biophysics,Chinese Academy of Sciences,Beijing,China. Adult stem cells have the ability to self-renew and generate differentiated cells for replenishing lost cells due to normal ageing, diseases or physical injury. Even though adult stem cells remain in adult tissues throughout an organism’s lifetime, functions of organs and tissues still decline with age. It is widely postulated that ageing could be, at least partially, caused by reduced stem cell number, activity or both. However, it largely remains a mystery as to how stem cell ageing is controlled. Here, we have used Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that both stem cell intrinsic ageing and decline of niche functions contribute to overall stem cell ageing. Partial reduction of niche BMP signals, Dpp and Gbb, speeds up stem cell ageing, while the increase in BMP signaling by overexpression of Gbb in the niche or an activated type I receptor in the GSCs can prolong their lifespan and promote their proliferation, indicating that age-related reduction of BMP signaling contributes to GSC ageing. Interestingly, overexpression of SOD, a gene that is known to help eliminate free oxygen species, in germline and niche cells can prolong GSC lifespan and increase GSC proliferation. Additionally, strengthening interactions between stem cells and the niche by overexpression of the cell adhesion molecule E-cadherin can also prolong GSC lifespan since the cadherin mediate cell adhesion is known to be important for keeping the GSC in the niche. Therefore, this study demonstrates that stem cell ageing is likely caused by a combination of intrinsic and niche ageing.

135 Crosstalk between the Insulin and Toll pathways shifts nutrient metabolism in response to infection. Justin DiAngelo1, Sara Cherry2, Morris Birnbaum1. 1) Institute for Diabetes, Obesity and Metabolism, Univ Pennsylvania/HHMI, Philadelphia, PA; 2) Department of Microbiology and The Penn Genomics Institute, Univ Pennsylvania, Philadelphia, PA. During nutrient abundance, animals increase their size and nutrient stores as regulated by the peptide hormone insulin. However, in response to environmental stresses animals conserve their energy by downregulating insulin signaling, which subsequently decreases animal size and metabolic reserves. One situation where stress-induced energy conservation has been well described is nutrient depletion; however, the effect of a different stress, infection, on energy preservation is less understood. Here, we demonstrate that activating critical mediators of the immune response in the fruit fly suppresses insulin signaling, leading to a decrease in both triglyceride and body size. This interaction is specific for the Toll pathway, as overexpressing the Imd pathway transcription factor relish does not show these phenotypes. These Toll-dependent phenotypic changes are due to decreased insulin signaling activity as the phosphorylation of dAkt, one of the major kinases in the insulin signaling pathway, is decreased in response to Toll activation. Epistasis reveals that Toll antagonizes the insulin signaling pathway at or downstream of PI3-kinase and this involves transcription as overexpressing the NFκB transcription factor dif also decreases dAkt phosphorylation. These genetic data reveal the evolutionarily conserved communication between the innate immune response and insulin action. Moreover, they suggest that this crosstalk evolved as a means to divert energy in times of stress from organismal growth to the acute requirement of combating infection. 134 PLATFORMS: Physiology and Aging

136 Rescue of the flightless phenotype of a Glutathione S-transferase S1 (GstS1) null mutant. Oksana Litvinova1, Sarah Dauback2, Ashis Mondal3, Piotr Zimniak2, Helen Benes1. 1) Neurobiol & Develop Sci; 2) Pharmacol & Toxicol; 3) Internal Medicine, Univ of Arkansas for Med Sci, Little Rock, AR. The products of lipid peroxidation, such as the electrophilic aldehyde 4-hydroxynonenal (4-HNE), may be important mediators of the pathological effects of uncontrolled oxidative stress. Previously, we showed that Drosophila GstS1-1 (DmGstS1-1) is the most abundant enzyme catalyzing 4-HNE conjugation in the fly, with primary localization in the Indirect Flight Muscles (IFMs). Yet, earlier studies of DmGstS1-1 proposed its main role to be a sensor for stretch-activated contraction of the IFMs (Clayton et al., J. Muscle Res. Cell Motil. 19: 117, 1998). Exceptionally among insect GSTs, GstS1-1 has an N-terminal extension which anchors it to tropomyosin-H in the IFMs. To elucidate how GstS1-1 may play a critical role in preventing oxidative toxicity in muscle or in sensing stretch in the contractile apparatus, we isolated mutant alleles of GstS1. Newly eclosed null mutant flies exhibited rapid degeneration of the IFMs, leaving other thoracic muscles intact. We observed no difference in level of tropomyosin-H between wild-type control and null mutant flies, indicating that GstS1-1 is not necessary for the localization of the IFM-specific tropomyosin. We are using the UAS/GAL4 system to determine if the importance of GstS1-1 for IFM preservation or function is due either to its enzymatic activity (in conjugating lipid peroxidation products) or to its structural/mechanical role in the contractile apparatus. Targeting of GstS1-1 to muscle (with the Mhc-Gal4 or Myosin Heavy Chain “driver”) restores flight to null mutant flies, whereas expression of a mouse GST (mGSTA4-4) with particularly high 4-HNE conjugating activity may not be sufficient. Further studies with other “drivers” will determine the role of motoneurons or other tissues in preserving IFM function. Our studies should provide insight into the role of oxidative stress and 4-HNE metabolism in neural and muscular degeneration underlying a number of human diseases.

137 The regulation of Drosophila lifespan by falafel. Brian Sage1, Xi Lou2, Li Qian3, Rolf Bodmer3, Heinrich Jasper2, Marc Tatar1. 1) Dept Ecol & Evol Biol, Brown Univ, Providence, RI; 2) Dept of Biology, Univ of Rochester, Rochester, NY; 3) Center for Neurosciences and Aging, The Burnham Institute, La Jolla, CA. Conserved pathways contribute to the regulation of aging. The C. elegans gene Suppressor of Map Kinase (SMK-1) is required for longevity extension upon reduced insulin signaling. To elucidate how SMK-1 participates in aging regulation we investigated its homolog in D. melanogaster, falafel. Here we show that falafel, a regulatory subunit of the protein phosphatase 4 complex, is a positive regulator of organisimal aging; overexpression of falafel extends lifespan in low nutrient conditions. This interaction between falafel and diet restriction is additive. Furthermore, preliminary data shows that dietary restricted flies with reduced levels of falafel are still long-lived, suggesting that longevity extension mediated by diet restriction doesn’t require falafel. Additional to the organismal aging regulation, flies overexpressing falafel in adult heart are more resistant to cardiac stress and have extended lifespan, indicating that falafel participates in autonomous processes of functional aging and cardiac stress resistance. By genetic epistasis interactions, we show that falafel is required for dFoxo and JNK mediated apoptosis in the eye. We are currently studying if, as in apoptosis, falafel works together with dFoxo regulating longevity. We are also analyzing possible physical interactions between Falafel, dFoxo, JNK, and other proteins that affect longevity. Our current goal is to understand the mechanism by which falafel regulates aging.

138 Genetic and environmental regulation of metabolism, behavior and lifespan by specific nutrient components. Danielle A. Skorupa1,3, Azra Dervisefendic1, Scott D. Pletcher1,2,3. 1) Huffington Center on Aging, Baylor College of Medicine, Houston, TX; 2) Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX; 3) Interdepartmental Program in Cell & Molecular Biology, Baylor College of Medicine, Houston, TX. Systems regulating energy storage and utilization are fundamental to organisms and strongly evolutionarily conserved. Paradoxically, disruption of normal energy intake or its sensing and signaling networks can lead to dramatic extension of organismal lifespan. The practice of dietary restriction, for example, promotes longevity by enacting an adaptive response that increases metabolic efficiency, reduces fecundity and enhances stress resistance. The exact nutrient or environmental cues that signal these beneficial adaptations remains elusive. By dissecting the macronutrients of the diet and altering environmental perception, we have gained a further understanding of the mechanism by which nutrients signal to affect lifespan. We find that particular mutants (i.e., FOXO) are deficient in enacting the appropriate nutrient response. In addition, behavioral (i.e., reproduction and feeding rates) as well as metabolic (i.e., obesity) phenotypes can be induced by the presence or absence of specific nutrients with total caloric intake playing only a minor role in phenotypic determination. Likewise, the mere perception (e.g., taste) of specific nutrients is sufficient to alter Drosophila physiology and behavior regardless of net energy alterations. Taken together, these data suggest that by understanding the specific signals emanating from the environment, lifespan extension as well as desirable metabolic adjustments resulting from dietary restriction regimes can be implemented even in the presence of high overall nutrient consumption while low caloric intake does not guarantee longevity. PLATFORMS: Physiology and Aging 135

139 Regulation of metabolism, organ senescence, and lifespan by the nutrient sensing TOR pathway. Sean Oldham, Claire Davies, Nancy Luong, Ryan Birse, R. J. Wessells, Suzanne Graham, Rolf Bodmer. Burnham Institute for Medical Research, La Jolla, CA, 92037. Drosophila has an evolutionarily conserved insulin/IGF system functionally analogous in the metabolic and growth aspects to the mammalian insulin/IGF system. Drosophila contains functionally conserved components of the insulin/IGF system, including the insulin ligands (Drosophila insulin-like peptides, DILPs). Loss of insulin signaling results in increased lipid levels, increased longevity, female sterility, and stress resistance. The Tuberous Sclerosis Complex (TSC1-2)/Target of Rapamycin (TOR) pathway responds to changes in insulin/IGF levels, amino acid levels, energy charge, lipid status, mitochondrial metabolites, and oxygen tension by adjusting cell growth. Using an allelic series of Drosophila TOR mutants, we show that TOR has additional roles regulating metabolism and lifespan. These phenotypes include lowered glucose and lipid levels, increased longevity, and a block in the age-dependent decline in heart function. We will describe the effects of altering TOR signaling on lifespan, organ senescence, and metabolism.

140 A genetic screen implicates the ER translocon and TRiC/CCT cytoplasmic chaperone complex as novel regulators of autophagy. Andrew M. Arsham, Thomas P. Neufeld. Dept of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN. Autophagy, the starvation-induced process of cellular bulk waste removal and recycling, has recently been implicated in neurodegenerative disorders, cancer, and longevity, and is tightly linked to pathways of cell growth, division, metabolism, and energetics. Autophagy is also thought to suppress cell damage and senescence by eliminating damaged organelles and macromolecules. We have used a mitotic mosaic approach to screen 383 lethal p-element insertions on chromosome 2L for dysregulation of autophagy—to the best of our knowledge, this is the first forward genetic screen for metazoan regulators of autophagy. One class of genes that emerged from the screen is involved in protein quality control and transport. These genes include the Sec61α subunit of the ER translocon and a subunit of the CCT/TRiC cytoplasmic chaperone complex. Subsequent RNAi knockdown experiments confirmed these genes’ involvement in regulating autophagy—knockdown of any single CCT subunit is sufficient to induce autophagy and suggests that defects in cytoplasmic protein quality control activate autophagy as a defense response. These results have important implications both in terms of the basic cell biology of cross regulation between autophagy and protein folding, and for autophagy’s role in suppressing neurotoxic protein aggregates such as polyglutamines in a variety of neurodegenerative diseases. 136 PLATFORMS: Neurogenetics and Neural Development

141 dfezl encodes a novel regulator of neural stem cell self-renewal in Drosophila. Mo Weng1,2, Shufen Situ2, Caitlin Gamble2, Cheng-Yu Lee1,2. 1) Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI 48109; 2) Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109. Asymmetric cell division provides an efficient mechanism for precise regulation of stem cell identity and potential (self-renewal) and generation of cellular diversity (differentiation) during development, maintenance of homeostasis, tissue regeneration and likely tumor suppression. Despite many seminal studies reporting a large number of genes that promote stem cell identity, regulation of stem cell self-renewal remain largely undefined. Drosophila neural stem cells (neuroblasts) divide asymmetrically to self-renew a neuroblast and to generate a differentiating ganglion mother cell, and serve as a model system to study regulation of self-renewal vs. differentiation in the context of development. To better understand regulation of self-renewal, we conducted a genetic screen to identify mutations that resulted in either increased or decreased neuroblast self-renewal. We discovered a novel mutation that showed a dramatic increase in neuroblasts at the expense of neurons. We mapped this mutation to cytological location 22B5-8, and sequence analysis of the mutant allele identifies a single nucleotide change resulting in an amino acid substitution in a previously uncharacterized gene CG31670. CG31670 encodes a novel zinc-finger transcription factor with strong homology in amino acid sequence to mammalian Fez and Fezl (forebrain embryonic zinc-finger and zinc-finger-like) proteins, and is renamed dfezl. Single dfezl mutant neuroblasts generate multiple neuroblast progeny and many post-mitotic neurons. dfezl mutant neuroblasts maintain normal cortical cell polarity. Thus, Dfezl likely functions either downstream or in parallel of the polarity proteins. dfezl appears to represent the most direct known regulator of neuroblast self-renewal. Latest progress on detailed characterization of the dfezl mutants and analysis of the dfezl protein will be discussed.

142 Dephosphorylation of Bazooka by PP2A is required for proper apical-basal polarity in embryonic neuroblasts. Michael P. Krahn, Andreas Wodarz. Department of Stem Cell Biology, University of Goettingen, Germany. Components of the PAR/aPKC (partitioning-defective/atypical protein kinase C) protein complex are essential for the establishment and maintenance of cell polarity in epithelial cells and in neuroblasts (NBs). The underlying mechanisms appear to be highly conserved throughout evolution, from worms to mammals. Research in Drosophila revealed that in the asymmetric division of NBs the determination of the distinct fates of the two daughter cells, the ganglion mother cell (GMC) and the NB, is dependent on asymmetric localization of the PAR/aPKC complex, consisting of Bazooka (Baz; the Drosophila homolog of PAR-3), PAR-6 and aPKC. Phosphorylation of Bazooka by the serine threonine kinases PAR-1 and aPKC is required for the proper localization and function of Baz. In contrast, little is known about the dephosphorylation mechanisms that counteract the activities of PAR-1 and aPKC. We found that PP2A, one of the major ubiquitous protein phosphatases involved in various processes like cell cycle control and cytoskeletal regulation, directly interacts with Baz via its structural A subunit. PP2A dephosphorylates Bazooka at two conserved serine residues and thereby antagonizes the kinase activities of PAR-1 and aPKC. Loss of PP2A phosphatase function leads to complete reversal of polarity in NBs. Proteins localized to the apical NB cortex in wild type, such as Baz, aPKC, Insc, Pins and G?i localize to the basal cortex in PP2A mutants, while Miranda and Prospero, which normally localize to the basal cortex, form a crescent at the apical cortex. We are currently investigating whether the reversed NB polarity is exclusively caused by compromised dephosphorylation of Baz or whether additional targets of PP2A are also relevant in this context.

143 From stem cell to unique neuron: Subdivision of the Castor temporal window by a feedforward loop involving Squeeze, Nab and Collier/Knot. Magnus Baumgardt1, Daniel Karlsson1, Javier Terriente2, Fernando J. Díaz-Benjumea2, Stefan Thor1. 1) Dept. of Clinical and Experimental Medicine, Linköping University Medical School, Linköping, Sweden; 2) Centro de Biología Molecular- Severo Ochoa/C.S.I.C., Universidad Autónoma-Cantoblanco, Madrid, Spain. In the developing Drosophila ventral nerve cord ~100 neurons express the LIM-HD transcription factor Apterous (Ap). The six thoracic Ap clusters consists of 4 Ap neurons of at least three different cells types; the Tvb neuron, that expresses the neuropeptide Nplp1, two ‘generic’ Ap cluster neuron (Tvx), and the Tv neuron, that expresses the neuropeptide FMRFamide. Several genes have been identified that specify Ap cluster neurons. These include the transcription factors ap, col, squeeze and dimmed, the transcriptional co-factors dac, eya and nab, as well as TGFb/BMP retrograde signaling. Genetic analysis reveals that these genes act in two different regulatory cascades to dictate Tvb and Tv cell identity. Both cascades are initially triggered by the expression of col, but importantly, downregulation of col is critical for the Tv cell fate. Previous studies reveal that all Ap cluster neurons are generated from NB 5-6t, and that the initial Ap cluster specification event is col-mediated. But how is this specification event channeled into two distinct cascades, which in turn dictate two distinct terminal cell fates; Tvb/Nplp1 and Tv/FMRFa? We find that the Ap neurons are generated from four consecutive GMCs (Tvb→Tvx→Tvx→Tv) during the last part of a large castor (cas) temporal window. Intriguingly the latter part of this cas window is subdivided into at least two ‘sub-windows’ by a feedforward mechanism, where 1) cas activates col, 2) cas activates sqz, 3) cas/sqz activates nab, and 4) where sqz/nab finally act together to downregulate col, in the last 3 Ap cluster neurons generated. This feedforward-mediated timing mechanism allows for col to perform its critical early postmitotic role - activation of ap and eya - but downregulates col in later born Ap cluster cells, thereby allowing for the specification of the Tv/FMRFa cell fate. PLATFORMS: Neurogenetics and Neural Development 137

144 Temporal transcription factors schedule the end of neurogenesis via cell cycle exit or apoptosis. Louise Cheng, Cèdric Maurange, Alex Gould. national institute for medical research, london, United Kingdom. The developmental stage at which postembryonic neurogenesis in the CNS terminates depends upon anterior-posterior position. In abdominal neuromeres, it is known that neural proliferation shuts down via Hox-dependent neuroblast apoptosis. In most other regions of the CNS, the mechanism by which neuroblasts stop dividing has been unclear. Here we examine the time course of neuroblast disappearance in the central brain and thoracic region during pupal stages. Using time-lapse movies, we observe that the final division of thoracic neuroblasts produces two-equal sized daughters that undergo cell cycle exit. The process executing the switch from size asymmetric-to-symmetric division involves the delocalisation of asymmetric determinants, resulting in the translocation of Prospero from the cell cortex into the neuroblast nucleus. The upstream timing mechanism triggering this Prospero nuclear translocation requires two members of the embryonic temporal transcription factor system, Castor and Seven Up, which are re- expressed in sequential bursts in postembryonic neuroblasts. Several ways of disrupting Castor or Seven Up expression lead to persistent neuroblast divisions in the adult CNS and consequent neural overproliferation. Together, these studies show that the temporal transcription factor system provides a global timing mechanism for shutting down neurogenesis, regardless of whether this is executed via cell cycle exit (thorax) or apoptosis (abdomen). They also provide evidence that the temporal transcription factor system coregulates cell fate and cell proliferation within the CNS.

145 Structural Basis for Robo Receptor Control of Lateral Positioning: An Unexpected Role for Robo Extracellular Domains. Timothy A. Evans1, Barry J. Dickson2, Greg J. Bashaw1. 1) Dept of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA; 2) Research Institute of Molecular Pathology, Vienna, Austria. Drosophila Roundabout (Robo) receptors regulate a number of fundamental axon guidance choices throughout development. Robo and Robo2 cooperate to mediate midline repulsion in response to Slit, while Robo2 and Robo3 specify lateral position of longitudinal axons within the CNS. Although the three Robo receptors share a high degree of structural and sequence similarity throughout their extracellular regions, their cytoplasmic domains are more divergent. Robo2 and Robo3 lack the CC2 and CC3 motifs that mediate interactions with downstream Robo signaling components, suggesting that Robo2 and Robo3 signaling may be qualitatively distinct from Robo. Consistent with this idea, forced expression of Robo2 or Robo3 in medial ipsilateral axons forces them into more lateral positions, while increased Robo expression does not. To investigate the structural basis for this activity, we have analyzed a series of chimeric receptors in which the extracellular and cytoplasmic domains of Robo, Robo2, and Robo3 have been swapped. Remarkably, the extracellular portions of Robo2 or Robo3 are sufficient to shift axons laterally, regardless of which Robo cytoplasmic domain is present. Further, we have determined that the Slit binding region of Robo2 is required for dictating lateral position. Using more restricted domain swaps, we have uncovered a bipartite contribution to lateral shifting activity involving the Slit binding region of Robo2 along with a second extracellular region. These observations suggest that the differential roles of Robo receptors in lateral positioning are caused in part by differential response to Slit, and indicate that multiple ectodomain modules contribute to this response.

146 Down Syndrome Cell Adhesion Molecules as multifunctional Netrin receptors required for midline crossing. Gracie L. Andrews, Thomas Kidd. Biology Department/ms 314, University of Nevada, Reno, NV 89557, USA. A major challenge in the field of axon guidance is to identify extracellular factors and receptors that guide axons. We have identified Down syndrome cell adhesion molecules (Dscams) as novel Netrin receptors necessary for midline axon crossing in the central nervous system (CNS). A reverse genetic screen in Drosophila revealed that mutations in Dscam and Dscam3 affected midline axon crossing, and were spatiotemporally expressed coincident with pioneer commissural axons. Dscam mutants exhibited defects in Bolwig’s nerve projection and salivary gland guidance. The defects in Bolwig’s nerve and the salivary gland were recapitulated in Netrin mutants. frazzled mutants, the previously characterized Drosophila Netrin attractive receptor, exhibit midline crossing defects that are more subtle than those found in netrin deletions, suggesting that there may be at least one more Netrin receptor. Disruption of frazzled and each Dscam (Dscam and Dscam3) in double mutants caused a significant worsening of midline crossing defects. In the Dscam-frazzled double mutant a specific subset of commissural axons (connectin positive) showed guidance defects beyond that of frazzled alone and approaching defects found in netrin deletions. Triple mutants, in which Dscam, Dscam3 and frazzled were mutated, exhibited a commissureless-like phenotype in which most axons were unable to cross the midline. These phenotypes led us to predict that Dscam and Dscam3 encode Netrin receptors. Cell culture binding assays show that Netrin binds to Dscam in evolutionarily conserved association. Kd values of Netrin and Dscam binding are statistically the same as Netrin and its receptor DCC (deleted in colorectal cancer.) We believe Dscam is an entirely attractive receptor. First, Dscam mutations in conjunction with mutations in the frazzled Netrin receptor gene, lead to a dramatic failure of axons to cross the CNS midline. Second, gain of function experiments lead to increased axon crossing of the midline. Finally, Dscam mutants can suppress mutations in the roundabout (robo) pathway that repels axons from the midline. 138 PLATFORMS: Neurogenetics and Neural Development

147 DOUBLESEX establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by directing cell number. Laura Sanders, Michelle Arbeitman. Molecular and Computational Biology, University of Southern California, Los Angeles, CA. doublesex (dsx) encodes sex-specific transcription factors that act at the top of one branch of the somatic sex determination hierarchy. dsx is responsible for directing all aspects of somatic sexual differentiation outside the nervous system. Here we show that the number of DSX-expressing cells in the central nervous system is sexually dimorphic during both pupal and adult stages. The number of DSX-expressing cells is established by both the sex-specific DSX isoform present and the amount of DSX activity in the cell. We demonstrate that in males, DSX is present in a portion of the neural circuitry in which the male-specific product of fruitless (fru) is produced; this circuit underlies all aspects of male courtship behaviors. We show, however, that the number of DSX-expressing cells is established independent of male-specific FRU and that the number of male-specific FRU-expressing cells is established independent of DSX. Additionally, we demonstrate that for one cluster of DSX-expressing cells in the ventral nerve cord, the sexual dimorphism in cell number is due to cell death. Therefore, in addition to its known role in establishing sexual dimorphism outside the CNS, our data demonstrate that DSX establishes sex-specific differences in neural circuitry by regulating the number of neurons.

148 Expression of vestigial in the Drosophila embryonic central nervous system. Kirsten Guss1, Hemlata Mistry1,2, James Skeath2. 1) Dept Biol, Dickinson College, Carlisle, PA; 2) Dept Genetics, Washington University School of Medicine, St. Louis, MO. The Drosophila central nervous system is an excellent model system in which to resolve the genetic and molecular control of neuronal differentiation. Here we show that the wing selector vestigial (vg) is expressed in discrete sets of interneurons. We track the axonal trajectories of a subset of VG-expressing cells in the ventral nerve cord and show that these cells descend from neuroblasts 1-2, 5-1, and 5-6. This work provides the necessary descriptive foundation for functional studies regarding the role of vestigial during interneuron differentiation. PLATFORMS: Cell Division and Growth Control 139

149 Insulin Receptor pathway regulates cell division through miRNAs and p21/dacapo in the Drosophila germ line stem cells. Jenn-Yah Yu, Steve Hatfield, Halyna Shcherbata, Karin Fischer, Hannele Ruohola-Baker. Department of Biochemistry, University of Washington, Seattle, WA. p21/dacapo (dap) is a potential target of miRNAs in regulating cell cycle of germ line stem cells (GSCs) in Drosophila melanogaster. Tissue-extrinsic signals, such as insulin, have also been shown to affect cell division of GSCs. Here, we explored the possibility that insulin receptor (InR) pathway may interact with the miRNA/dap regulatory circuit, therefore control GSC cell division. By using luciferase assay, we demonstrated that dap 3’UTR can be directly targeted by several miRNAs, including miR-7, miR-8, and miR- 309. By using a GFP sensor with dap 3’UTR, we showed in vivo that the dap 3’UTR does not only respond to miRNA activities but also responds to the InR signaling activities in GSCs. These results suggest that the InR activity may regulate dap expression through dap 3’UTR. By using genetic approaches, we demonstrated that the InR and miRNA/dap may act in the same pathway; reduction of dap partially rescued the cell cycle defect of InR deficient GSCs. Furthermore, decrease of InR signaling by restricting protein diet does not affect either dap or dicer-1 mutant GSCs. These data suggest that InR signaling may regulate GSC cell cycle through miRNA/dap.

150 The anaphase promoting complex / cyclosome (APC/C) is required for re-replication control in endoreplication cycles. Norman Zielke1,2, Silvia Querings2, Frank Sprenger2,3. 1) Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA., Select Country; 2) University of Cologne, Institute for Genetics, Zuelpicherstrasse 47, 50674 Koeln, Germany; 3) University of Zuerich, Institute of Zoology, Winterthurerstrasse 190, CH-8057 Zuerich, Switzerland. The endocycle is a cell cycle variant that is applied in various tissues of Drosophila and certain human cell types. Endoreplicating cells undergo multiple rounds of DNA replication that are separated by distinct G-phases, but never enter mitosis. Contrary to the current view of endoreplication, we have found that oscillation of APC/C-Fzr activity is essential for endocycle progression. In salivary gland endocycles, the APC/C-Fzr complex regulates the abundance of the Geminin protein, which is crucial to prevent relicensing in S-phase. Inactivation of the APC/C-Fzr complex in larval salivary glands results in Geminin stabilization and as a consequence DNA replication is inhibited. Geminin only accumulates in cells with high CycE/Cdk2 activity, suggesting that APC/C- Fzr activity is inhibited by CycE/Cdk2. Consistent with this idea we found that Geminin is stabilized upon CycE overexpression. Moreover, we provide evidence that CycE/Cdk2 and E2F1 cooperate in an autoregulaory feedback loop that drives the endocycle. Impaired DNA replication prevents E2F1 turnover in salivary glands and subsequently results in accumulation of E2F1 and its target genes, including CycE. This mechanism ensures that CycE/Cdk2 activity accumulates prior to S-phase and subsequently drops after completion of DNA replication. The peak of CycE/Cdk2 activity simultaneously triggers S-phase and prevents relicensing by Geminin stabilization through inhibition of APC/C-Fzr activity. In the following G-phase, when CycE/Cdk2 activity is low, the APC/C- Fzr complex is released and targets Geminin for proteasomal degradation. This allows licensing of replication origins and prepares for the next round of DNA replication. Thus, we have identified a novel APC/C regulated mechanism that prevents unscheduled DNA licensing in endoreplication cycles.

151 The role of Misato in Drosophila spindle assembly. Violaine Mottier, Giovanni Cenci, Fiammetta Vernì, Maurizio Gatti, Silvia Bonaccorsi. Genetica Biol Molecolare, Univ di Roma La Sapienza, Rome, Italy. The Drosophila misato (mst) gene encodes an evolutionary conserved protein that contains motifs shared among tubulins and myosin. Previous studies have shown that mst mutants die at the larval/pupal boundary and exhibit frequent polyploid cells in larval brains. However, the function of the Mst protein was not well defined. To determine the mitotic role of mst we stained mutant brains squashes for tubulin and Centrosomin (Cnn). We found that most mitotic cells were arrested in metaphase. In addition, 70% of these metaphase figures were monopolar and displayed abnormally long microtubules (MTs) emanating from the two unseparated centrosomes. Recruitment of centrosomal proteins such as DPLP, γ-tubulin, DGrip91, CP190, and D-TACC was not affected in mst mutants, consistent with a normal nucleating ability of the centrosomes. To elucidate the mechanisms underlying monoplar spindle formation we assayed MT regrowth following cold-induced depolymerization. In wild type larval brain cells, release from cold treatment resulted in rapid formation of MTs around both the chromosomes and the centrosomes. In contrast, in mst mutant cells, MTs were nucleated almost exclusively by the centrosomes. Immunostaining with an antibody to Mst showed that mitotic cells exhibit a strong, homogeneous signal that persists from prophase to telophase. Conversely, interphase cells consistently displayed a diffuse and weak Mst staining. Collectively, these results indicate that the mst function is primarily required during mitosis for chromatin-induced MT nucleation, and highlight the importance of this process in mitotic spindle assembly. 140 PLATFORMS: Cell Division and Growth Control

152 FUNCTIONNAL ANALYSIS OF A NOVEL FAMILY OF ERM PROTEIN PARTNERS DURING CELL DIVISION. Sébastien Carreno1,2, Hélène Foussard2, Jérome Miailhe2, Cédric Polesello2, Philippe Valenti2, Pierre Ferrer2, François Payre2. 1) Université de Montréal, IRIC, Montreal, QC, Canada; 2) Centre de Biologie du Développement, CNRS University ToulouseIII, Toulouse France. Cell division involves a stereotyped sequence of changes in cell morphology, regulated by localized cortical acto-myosin contractions. While the microtubule spindle is well known to influence location of the cleavage furrow, how cell shape transformations are coordinated with spindle reorganization throughout mitosis remains largely elusive. ERM (Ezrin, Radixin, Moesin) proteins, which are deregulated in several cancers, are known to link cortical actin to membrane, upon signal-mediated activation. We have previously shown that localized activation of Moesin, the unique drosophila ERM, is required both for cell shape changes and mitotic spindle positioning during cell division. We have isolated GIM a Moesin genetic interactor which defines a novel evolutionarily conserved protein family. Having shown that GIM binds specifically to the Moe protein, we explored a putative function of GIM during cell division. We show that GIM specifically co-localizes with activated Moe, at the cortex in pro/metaphase and at the cleavage furrow in ana/telophase. Interestingly, GIM also localizes at centrosomes and midbody. Similarly to Moe inactivation, depletion of GIM in S2 cells leads to severe defects in mitotic cell shape, with large cytoplasmic bulges that deform the cortex. Phylogenetic analysis allowed identification of 4 GIM orthologs in mammals. We characterized human GIM-like genes and analyzed the distribution of one out the 4 hGim proteins. During cell division, we show that it localizes both at the cleavage furrow and centrosomes/midbody. Taken together, our results show that the GIM family of ERM partners plays an important role in the control of cell division in Drosophila. Its functional conservation in humans may be relevant to understand regulation of ERM proteins during cell division, in normal and pathological situations.

153 Drosophila short neuropeptide F signaling regulates growth by ERK mediated insulin signaling. Kweon Yu1, Kyu-Sun Lee1, Kyung-Jin Min2, Marc Tatar2. 1) Centre for Regenerative Medicine, Korea Res Inst of Bioscience & Biotechnology, Daejeon, Korea; 2) Dept of Ecology & Evolutionary Biology, Brown University, Providence, RI. Insulin/insulin growth factor signaling plays a central role in growth, metabolism, and aging in animals. However, up-stream regulatory signaling for the insulin transcription is not well known. Here we present that Drosophila short neuropeptide F (sNPF) signaling regulates expression of Drosophila insulin like peptides (Dilps) through ERK activation in insulin producing cells and controls growth by regulating insulin receptor/dFOXO signaling in the fat body insulin target tissue. The gain-of-function sNPF or sNPFR1 receptor mutant produced a big body size through ERK mediated Dilps expression. When Drosophila CNS cells or rat pancreatic cells were treated with sNPF or NPY peptide, a sNPF vertebrate ortholog, Dilps or insulin expression was increased by the activation of ERK. In the fat body cells of sNPF mutants, expression of translational inhibitor 4E-BP was increased by the down-regulation of Akt and nuclear localized dFOXO, resulting in the reduction of cell size. Moreover, in sNPF mutants, high glucose levels of larval hemolymph were detected and lifespan was extended. Drosophila sNPF and evolutionary conserved mammalian NPY appear to regulate ERK mediated insulin expression and to thus systemcally modulate growth, metabolism, and lifespan.

154 GTRs - small GTPases implicated as novel regulaTORs of growth. Pankuri Goraksha, Thomas Neufeld. Molecular, Cellular, Developmental Biology & Genetics, University of Minnesota, Minneapolis, MN. Gtr1 & Gtr2 are evolutionary conserved members of a unique subfamily of Ras-like small GTPases. In mammals and S.cerevisiae, Gtr1 & Gtr2 form a stable heterodimeric complex. In S. cerevisiae, the Gtr1-Gtr2 complex is involved in regulation of exit from rapamycin-induced growth arrest and microautophagy1, and general amino acid permease sorting2, a function negatively regulated by TOR activity. In mammalian cell culture, the conserved GTPases Gtr1 and Gtr2 positively affect TOR activity as measured by phosphorylation of its well-known targets S6K and 4EBP3. In a collaborative approach to dissect their significance, we analyzed the requirement of Gtr1 and Gtr2 in the TOR pathway in a higher evolved multicellular organism, namely, Drosophila. We find that both Gtr1 and Gtr2 act as novel positive regulators of TOR pathway in Drosophila by regulating organismal growth, individual cell size, and starvation-induced autophagy. Gtr1 is positively affected by nutrient availability, and expression of its constitutively active form offers cells a growth advantage under nutrient limiting conditions. Gtr-complex activity is independent of Rheb and these effectors act in parallel with each other to enable growth. Thus, our investigation reveals one more novel Ras-like GTPase family complex, in addition to Rheb GTPase, to positively regulate TOR activity, thereby leading to growth and development in Drosophila. 1.Dubouloz F et al., Mol Cell. 2005; 19(1); 15-26 2.Gao M & Kaiser CA, Nat. Cell Biol. 2006; 8(7): 657-67 3.Guan KL (unpublished data). PLATFORMS: Cell Division and Growth Control 141

155 alpha-Endosulfine is a conserved protein required for meiotic maturation that interacts with an E3 ubiquitin ligase and regulates Twine/CDC25 levels. Jessica Von Stetina, Susanne Tranguch, Sudhansu Dey, Ethan Lee, Laura Lee, Daniela Drummond- Barbosa. Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN. Meiosis is tightly coupled to the development of gametes and must be well regulated to prevent chromosome segregation errors. During meiotic maturation, Drosophila oocytes progress from the initial prophase I arrest to an arrest in metaphase I, which is maintained until egg activation. The extrinsic and intrinsic factors controlling oocyte maturation and the metaphase I arrest, however, are poorly understood. Our recent work shows that Drosophila alpha-endosulfine (dendos) plays a key role in this process. dendos mutant oocytes remain in prophase I longer and, instead of progressing into metaphase I, they fail to form a meiotic spindle and their DNA becomes dispersed. This phenotype is remarkably similar to that of twine, the meiotic homolog of cdc25. The CDC25 phosphatase is known to activate cdk1/cyclinB, which is required for meiotic maturation, as shown in other systems. We also find that the levels of a Twine-LacZ fusion protein are reduced in dendos mutants, suggesting that Dendos is required for normal levels of Twine expression and presumably, cdk1/cyclinB activity. Intriguingly, from an in vitro binding screen, we identified the CG17033-encoded E3 ubiquitin ligase as a strong Dendos interactor. We generated null alleles of CG17033 and found that, in these mutants, the transition into metaphase I occurs prematurely. These results are consistent with the model that upon stimulation of the oocyte by a meiotic maturation signal, the rise in cdk1/cyclinB activity is limited by the degradation of Twine, and that Dendos binds to and inhibits this E3 to promote the transition into metaphase I. We are currently testing if overexpression of twine rescues the meiotic phenotype of dendos mutants. Finally, germline-specific expression of either Dendos or human alpha-Endosulfine rescues this defect, and we can also detect alpha-Endosulfine expression in mouse oocytes, which suggests evolutionary conservation of the meiotic function of alpha-Endosulfine.

156 Mutation in the Ubiquitin Activating Enzyme, E1, can cause tissue overgrowth by upregulating Ras pathway activity by both cell-autonomous and non-autonomous means. Hua Yan1, Mei-Ling Chin2, Cathie Pfleger1. 1) Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY; 2) Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, NY. The Ras proteins are important regulators of signal transduction that can promote proliferation, cell survival, and differentiation. Mutations in Ras/Raf are found in 30% of solid tumors and mutations in components of the pathway are found in developmental disorders such as Noonan’s syndrome. In Drosophila, Ras85D represents a single homologue to human K-Ras, H-Ras, and N-Ras. It has recently been demonstrated that H-Ras is constitutively di-ubiquitinated and that a non-ubiquitinatable form of H-Ras demonstrates increased activity in terms of its ability to activate ERK. The biological consequences of H-ras ubiquitination in an organism are still unclear. In this report we demonstrate that cells containing hypomorphic mutation in E1, the most upstream enzyme in the ubiquitin pathway, display an upregulation of Ras/ERK activity and Ras-dependent ectopic division. Complete inactivation of E1 leads to a similar cell-autonomous increase in Ras/ERK activity, and the non-autonomous activation of Ras/ERK in adjacent tissue. Our findings may suggest that loss of Ras ubiquitination itself leads to growth-relevant Ras/ERK activation. Furthermore, different levels of inactivation in E1 lead to cell-autonomous and non-autonomous Ras activation. We propose that maintaining proper levels of E1 function in general, and Ras ubiquitination in particular are crucial to normal development and that the loss of Ras ubiquitination may play a role in developmental disorders and cancer. 142 POSTERS: Cell Division and Growth Control

157A Cell competition and growth control during Drosophila development. Francisco A. Martin, Salvador C. Herrera, Gines Morata. CBMSO (CSIC-UAM), UAM (Cantoblanco), Madrid, Spain. Cell competition is a phenomenon discovered in 1975 by which slow dividing but otherwise viable cells are eliminated by faster dividing cells. The out competed cells were heterozygous (M/+) for Minute mutations, which are defective in ribosomal proteins. We are presently studying the role of cell competition in the process of growth control in the wing imaginal disc. It has been known for long time that clones of fast proliferating (Minute+) cells induced in Minute heterozygous larvae overgrow and frequently fill entire compartments without affecting the final size and morphology of the wing. This suggests the existence of a regulatory process that eliminates the cells outside the clones in order to compensate for the excess of proliferation of the Minute+ clones. It is likely that cell competition would be implicated in this process. We are testing the role of the apoptosis associated with cell competition in this regulatory process by generating Minute+ clones in discs in which apoptosis is prevented or much reduced. We achieve this by overexpressing in compartments containing Minute+ clones the inhibitors of apoptosis DIAP1 and p35. In other experiments the Minute+ clones are induced in discs heterozygous for the deletion (DfH99), which removes the pro-apoptotic genes reaper, grim and hid. The results suggest that apoptosis is not a major factor in the regulatory process: the growth of the Minute+ clones in compartments in which apoptosis is blocked is similar to that in compartments in which apoptosis can be induced.

158B Requirements for cell competition during Drosophila development. Ricardo M. Neto da Silva1, Laura A. Johnston2. 1) Gulbenkian PhD Program in Biomedicine, Instituto Gulbenkian de Ciencia, Oeiras, Portugal; 2) Department of Genetics & Development, Columbia University, 701 West 168th Street, HHSC704, New York, NY 10032. Cell competition describes the ability of some cells to grow at the expense of nearby slower-growing cells. The slower-growing cells, which are viable on their own, are actively eliminated from the tissue by the neighboring cells. Cell competition occurs between wildtype cells and Minute mutant cells, which are impaired in ribosome biogenesis, and between neighboring cells that express different levels of the growth regulator dMyc. We are investigating whether cells must be actively proliferating in order to compete. While early in development all disc cells grow and proliferate, at later stages developmentally defined regions of cells exit the cell cycle and express lower levels of dmyc. In some cases, however, these arrested cells reside next to cells expressing high levels of dMyc. Are non-proliferating cells competed by their proliferating neighbors? Is cell competition restricted to proliferating cells in the disc, or does it also occur between post-mitotic cells? We will present our ongoing experiments to address those questions, which we believe will be helpful in furthering our understanding of the signalling interactions that occur during cell competition.

159C Identification of factors that mediate Myc-induced competition between cells. Nanami Senoo-Matsuda, Laura A. Johnston. Deptartment of Genetics & Development, College of P&S, Columbia University, New York, NY 10032. Our studies have revealed that when neighboring cells in the developing Drosophila wing express different levels of the transcription factor, dMyc, competitive interactions can occur (de la Cova et al., 2004). Cells with more dMyc proliferate and ultimately over- populate the wing, while cells with less dMyc die, thereby preventing wing overgrowth. How cells sense dMyc activity differences, and the nature of the process leading to changes in growth and survival during competition remains unknown. We have developed a cell culture-based assay using Drosophila S2 cells to investigate the mechanism of cell competition. We find that in vitro co-culture of S2 cells that express different levels of dMyc recapitulates many aspects of cell competition in the developing wing. Our data indicate that both cell populations in the co-cultures participate in and are required for the competitive process by releasing soluble factors into the medium. We demonstrate that the response of naïve cells to medium conditioned with competitive co-cultures is dependent upon their potential to express dMyc: cells that can express high levels of dMyc gain a survival advantage and proliferate faster, while cells with lower dMyc levels are instructed to die. We suggest that the ability of cells to perceive and respond to local differences in Myc activity is a cooperative mechanism that could contribute to growth regulation and developmental plasticity in organs and tissues during normal development and during regeneration. We will discuss these results and our efforts to identify genes required for competition to occur using RNAi screens and biochemical purification. POSTERS: Cell Division and Growth Control 143

160A The regulation and function of Drosophila Atg1 in autophagy. Yu-Yun Chang, Thomas Neufeld. Genetics and Cell Development, University of Minnesota, Minneapolis, MN. Autophagy is the major route by which long-lived proteins are degraded, and is induced when cells face nutrition deprivation. Nutrient regulation of autophagy occurs through target of rapamycin (TOR) pathway and involves the products of the ATG genes. Studies in yeast have shown that TOR regulates autophagy in part by controlling the activity of Atg1, a Ser-Thr protein kinase which acts in a multi-protein complex with Atg13 and Atg17 to turn on the autophagic pathway. In Drosophila, loss-of-function mutations in Atg1 fail to induce autophagy; however, how Atg1 is regulated is still unclear. In this report, we are able to show that both the phosphorylation and the protein level of Atg1 are under the control of nutrition and at least two signals, PKA and TOR. Although it is debatable if the kinase activity of yeast Atg1 is required for autophagy, our previous studies show that Atg1 kinase activity is essential for autophagy in Drosophila and the importance of autophosphorylation of Atg1 in autophagy is further investigated here. No Atg1 interacting proteins functioning in autophagy have been reported in fly and other animals. Here we report that Drosophila Atg13 is essential for autophagy but not sufficient to induce autophagy in Drosophila. We will present data demonstrating the interaction of Atg13 and Atg1 in the regulation of autophagy.

161B Drosophila rictor and raptor in cell growth control. Sekyu Choi, Gina Lee, Jongkyeong Chung. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Gu, Taejon, Korea. Target of rapamycin (TOR) has been extensively studied as a critical nutrient-sensitive regulator of cell growth and metabolism. Recently, two different TOR partners, rictor and raptor, have been identified as novel TOR complex organizers. To distinguish the physiological roles of these two TOR-binding proteins, we attempted to compare the in vivo functions of rictor and raptor in Drosophila. In rictor-null mutants, Akt-induced tissue hyperplasia was inhibited and Akt-Ser-505 phosphorylation was decreased. Furthermore, FOXO-dependent apoptosis, which is known to be inhibited by Akt, was enhanced in a rictor-null background, indicating that rictor is essential for the Akt-FOXO signaling module. We found that neither S6K-dependent cell growth nor S6K-Thr-398 phosphorylation was changed in rictor-null mutants. However, the knockdown of another TOR-binding partner, raptor, decreased S6K-Thr-398 phosphorylation and inhibited S6K-induced cell overgrowth. Collectively, our findings demonstrate that rictor and raptor play pivotal roles in TOR-mediated cell growth control by differentially regulating Akt- and S6K-dependent signaling pathways, respectively.

162C ATG1, an autophagy regulator, negatively regulates S6 kinase. Wonho Kim, Sung Bae Lee, Sunhong Kim, Jongkyeong Chung. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Gu , Taejon, Korea. It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. We have characterized the fly mutant of ATG1, an autophagy-regulating kinase, and found ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. In consistent with the fly results, overexpression of ATG1 also reduce S6K activity in mammalian cells. Furthermore, short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K. We found that ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, our genetic and biochemical results indicate the crosstalk between autophagy and cell growth regulation. 144 POSTERS: Cell Division and Growth Control

163A An Essential Role for Gp93, the Endoplasmic Reticulum Hsp90 Chaperone, in Growth Control. Jason C. Maynard1, Eric Spana2, Christopher V. Nicchitta1. 1) Department of Cell Biology,Duke University Medical Center; 2) Department of Biology, Model Systems Genomics, Duke University, Durham, NC. Gp93 (GRP94, gp96), the endoplasmic reticulum(ER) paralog of heat shock protein 90(HSP90), has a very restricted phylogenetic distribution, being found only in metazoans and higher plants; whereas HSP90 is very broadly distributed throughout Eukarya and Eubacteria. In mammalian models, GRP94 is not required for cell viability, but disruption of the GRP94 gene in mouse results in embryonic lethality. Relatively little is know about the identity of the GRP94 proteome - established client proteins include the Toll- like receptor family, IGF-II, and a subset of integrins. The limited phylogenetic distribution of GRP94, combined with emerging information on the identities of its client proteins, suggest an essential role for GRP94 in the cell-cell interaction/communication processes necessary for multicellular life. To investigate the role(s) of Gp93 in metazoan biology, P-element excision mutagenesis was used to make Gp93 deletion alleles. Gp93 null larvae survive to third instar before lethality. These larvae exhibit a profound growth defect in comparison to heterozygote larvae. The observed divergence in growth occurs concurrently with the degradation of maternal Gp93. Faulty endoreplication is not responsible for the growth defect as DNA replication occurs normally in mutants. Analyses of Gp93 null fat body morphology and lipid droplet content, by Nile Red staining, indicate a profound disruption in nutrient accumulation. Biochemical analysis demonstrates highly reduced triglyceride levels in Gp93 null fat body tissue. We used the EGUF/hid system to exclusively generate Gp93 null head tissue and obtained strong pinhead phenotypes, similar to those reported for the insulin receptor(InR) pathway mutations. Because Gp93, as an ER chaperone, regulates secretory/integral membrane protein biogenesis, we hypothesize that Gp93 is required for insulin receptor expression; the loss of GP93 function would thus be predicted to yield a larval growth defect.

164B Vesicle Recognition and Trafficking in Autophagy Regulation. Keith Naps, Thomas Neufeld. Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN. In cells undergoing autophagy, cytoplasmic proteins and organelles are enveloped within a double membrane, forming an autophagosome. Lysosomal fusion facilitates breakdown of cytoplasmic contents into their constitutive amino acids and lipids, which are subsequently recycled. Linked to nutrient sensation, autophagy is induced by starvation, and is required for survival under starvation conditions. While autophagy research has been primarily performed in yeast, new evidence suggests that autophagy regulation may differ in higher eukaryotes. In addition, autophagy has been linked to occurrence of diseases unique to multicellular organisms, such as cancer and neurodegeneration. For this reason, the powerful genetic tools available in Drosophila make it an ideal model for the study of autophagy in higher organisms. Membrane phosphorylation by the class 3 PI3K Vps34, in complex with Atg14 and Beclin1, initiates vesicle nucleation and elongation of the pre-autophagosomal isolation membrane. Although Vps34 and Beclin1 are known to be required for autophagy, little is known about the participation of Atg14 in the complex. Interestingly, Vps34 and Beclin interactions drive other endosomal and vacuolar sorting, leaving Atg14 as the only autophagy-specific member of the complex. RNAi knockdown of atg14 attenuates autophagy in the Drosophila fat body, suggesting atg14 may be required for autophagy. In addition, a Δatg14 allele will allow generation of atg14-null clones. Though preliminary, this work will elucidate the role that Atg14 plays in autophagy. Also intriguing are autophagy regulators downstream of membrane phosphorylation. Specifically, proteins containing FYVE and PX domains are known to bind Vps34-generated PI3P, but many of their genes remain uncharacterized and none have been conclusively shown to be autophagy regulators. RNAi knockdowns of these genes may elucidate proteins that either positively or regulate autophagy. As made possible by fly genetics, putative regulators discovered in the RNAi screen may further be studied through manipulation of p-element insertions.

165C JAK/STAT signaling regulates cellular growth in Drosophila. Aloma Rodrigues, Erika Bach. Pharmacology, NYU School of Medicine, New York, NY. During development organisms achieve reproducible shape and size by balancing proliferation, mass accumulation (also called cellular growth) and apoptosis. An analysis of loss- and gain-of-function mutations in JAK/STAT pathway components suggested that this pathway regulates cellular growth in the Drosophila eye (Bach, 2003, Genetics 165:1149). To define the requirement of the sole Drosophila STAT protein Stat92E in growth control and to extend this analysis to the wing, I induced stat92E clones at precise times in development and measured their size as compared to their sister wild type clones. In the eye and the wing, stat92E clones are substantially smaller than their wild type sister clones. Furthermore, in a Minute background, stat92E clones obtain larger sizes. These data suggest that Stat92E is required for clone survival and that cells lacking stat92E are eliminated, most likely by cell competition. A central regulator of cellular growth and cell competition is dMyc, the Drosophila homolog of mammalian c-Myc. Cells that have higher levels of dMyc are super-competitors and grow at the expense of their (lower dMyc-expressing) neighbors. In contrast, cells with reduced dMyc (called low-dMyc cells) are eliminated by the surrounding wild type cells (de la Cova, 2004, Cell 117:107; Moreno, 2004 Cell 117:117). I assessed whether hyper-activating Stat92E in low-dMyc cells would promote their survival. Indeed, activation of Stat92E in low-dMyc cells significantly rescues them. These data suggest that JAK/STAT signaling promotes a cell’s ability to compete by increasing metabolic activity. This may occur through increasing dMyc levels (possibly by Stat92E regulation of the endogenous dmyc locus, which is intact in low-dMyc cells) or through increasing ribosomal biogenesis targets independently of dMyc. We are currently performing experiments to distinguish between these possibilities. POSTERS: Cell Division and Growth Control 145

166A Structure/Function analysis of the dMyc protein in vivo. Daniela Schwinkendorf, Peter Gallant. Zoological Institute, University of Zürich, Zürich, Switzerland. The transcription factor Myc controls cell proliferation and apoptosis, and it is a potent oncogene in humans. Drosophila contains a single Myc ortholog (dMyc/diminutive) that can partially substitute for vertebrate Myc (and vice versa). Despite this functional conservation, vertebrate and Drosophila Myc only share four conserved motifs: the so-called Myc Box I (MB1) and Myc Box II (MB2, which is essential for Myc function although its molecular role is controversial), a poorly characterized acidic domain (AD), and a Basic Helix-Loop-Helix/Leucine Zipper motif (BHLHZ, required for DNA binding and protein-protein interaction). To determine the roles of these conserved domains in vivo, we used the φC31 system to establish transgenic lines producing dMyc protein mutants for either of these domains. These proteins were expressed in the background of different levels of endogenous dMyc (ranging from wild type to the complete absence of dmyc), as well as in S2 cells, and assayed for their ability to induce growth, apoptosis, and the expression of specific target genes. These experiments showed that a dMyc mutant lacking the N-terminus retains very little activity, even though this region only contains a motif with weak homology to MB1, and its deletion does not affect dMyc protein levels. The AD has a negative effect on dMyc stability, and its deletion allows dMyc protein to accumulate to higher than wild type levels; the resulting protein is not simply more active, but has altered transcriptional activity. Finally, mutation of the MB2 impairs transcriptional activation and biological activities of dMyc. Surprisingly, a dMycΔMB2 mutant retains enough activity that it can rescue flies lacking all endogenous dMyc to adulthood. In conclusion, although MB2 and AD are highly conserved between insect and vertebrate proteins, loss of either of these domains has comparatively mild consequences on Myc activity.

167B Roles for the Drosophila E3 Ligase, Neuralized in Hematopoiesis. Chiyedza Small1, Jemila Caplan1, Vassilis Baoussis2, Christos Delidakis2, Shubha Govind1. 1) Department of Biology, The City College of CUNY, New York, NY, 10031, USA; 2) Institute of Molecular Biology and Biotechnology, FoRTH, P.O. Box 1527, GR 711 10, Heraklion, Crete, Greece. Previous studies have suggested a role for the highly conserved Notch (N) pathway in Drosophila hematopoiesis. Both studies found that N signaling regulate crystal cell lineage specification and number (Duvic et. al. 2002 and Lebestky et. al. 2003). neuralized (neur), a gene in this pathway, encodes an E3 ubiquitin ligase. Neuralized plays an essential role in the emission of an intercellular signal that is critical for its function in cellular differentiation and animal development. neuralized encodes at least two protein isoforms: Neuralized A and Neuralized C. To characterize the roles of the different isoforms in fly hematopoiesis, we initiated complementation analysis using both established and recently identified putative alleles of neurA and neurC. We also misexpressed wild type and mutant forms of Neuralized A and C proteins using the UAS/GAL4 system. We observed lethal effects when the transgenes were ubiquitously expressed. However, when neurC expression was restricted to larval blood cells, we found a significant number to be multinucleate. We also observed changes in crystal cell numbers upon misexpression of wild type and truncated versions of the protein. Antibody staining experiments reveal that Neuralized protein is expressed in cells of the larval hematopoietic system. These results support the idea that Neuralized proteins together with N signaling play critical roles in both proliferation and differentiation of Drosophila hemocytes.

168C Functional studies of the Mitf gene in Drosophila. Tianyi Zhang, Francesca Pignoni. Dept Ophthalmology, Harvard Medical Sch/ MEEI, Boston, MA. The Microphthalmia-related transcription factor (Mitf) encodes a basic Helix-Loop-Helix Zip (bHLH-Zip) protein, and controls the differentiation of many cell types, including melanocytes, osteoblasts, mast cells and retinal pigment epithelium (RPE). Mitf has been shown to regulate cell-cycle genes and control proliferation. The aberrant expression of Mitf is related to cancers, such as melanoma and clear cell carcinoma. However, the roles of Mitf in cell differentiation and proliferation are complex and poorly understood. For example, either high or low concentration of Mitf can inhibit proliferation, and the levels of Mitf in some melanoma are high while in others are low. Here, we use the Drosophila model to study Mitf function in different contexts. Targeted expression of either Drosophila or mouse Mitf (dMitf and mMitf) can suppress eye development by downregulating Eye absent and Eyeless, two retina specification genes. In addition, dMitf or mMitf can suppress proliferation autonomously, but induce proliferation non- autonomously. Lastly, Mitf expressing cells often downregulate the cell polarity gene DLG and move out of the imaginal disc epithelium. Targeted expression of a DNA-binding-impaired form of dMitf (dMitfEA) does not suppress eye identity or cell-autonomous proliferation, not does it disrupt epithelial connections. However, dMitfEA can still enhance cell division non-cell-autonomously. These retinal identity and cell proliferation phenotypes are reminiscent of the effect of Mitf in vertebrates and support Drosophila as a model to dissect the molecular mechanisms of Mitf function in development and cancer. 146 POSTERS: Cell Division and Growth Control

169A DmSGT1 is required to maintain spindle bipolarity through a mechanism that involves Polo stabilization. Torcato J Martins1, Andre F Maia1, Sören Steffensen1, Claudio Sunkel1,2. 1) Molecular Genetics, Institute for molecular and cell biology, Porto, Portugal; 2) ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. Sgt1 is a co-chaperone that has been shown to interact directly with Hsp90. Previous work in Yeast and Humans showed Sgt1 interacts with Hsp90 to form a complex essential for kinetochore assembly. We used DmSgt1 as an entry point to investigate the pathway of kinetochore assembly and its relation to the Spindle Assembly Checkpoint (SAC). Surprisingly, despite all the previous results from Sgt1 in other species we find that mutations in the DmSgt1 gene do not affect kinetochore assembly or checkpoint components. However, we do find that sgt1 mutant cells fail to progress into mitosis normally and when they do arrest mostly in a prometaphase-like state with abnormal spindle morphology and reduced tension between sister kinetochore pairs. This prometaphase arrest is SAC dependent since double mutant sgt1:bub3 cells are able to overcome the mitotic arrest and exit mitosis with precocious sister chromatid separation. Interestingly, analysis of centrosome organization does show significant abnormalities in sgt1 mutant neuroblasts. In these cells centrioles replicate but fail to mature properly and some pericentriolar material components such as gamma-tubulin and DTACC do not localize normally resulting in highly abnormal spindles without asters. This centrosome phenotype was also found in Hsp90 mutant cells and was correlated with a decrease in levels of Polo protein. Indeed, we also observe decreased levels of Polo in sgt1 mutant neuroblasts. Overall, these findings suggest that Sgt1 and Hsp90 are involved in a common pathway to stabilize Polo and promote its function in centrosome maturation.

170B Determinants of CNN targeting and MTOC regulation at centrosomes. Timothy L. Megraw, Jiuli Zhang, Ling-Rong Kao. Green Ctr/Pharmacology, Univ Texas Southwestern Med Ctr, Dallas, TX. Drosophila Centrosomin (CNN) is an essential component of the pericentriolar matrix (PCM) of the centrosome. CNN homologs are found in most eukaryotes, including S. pombe and human. Mutations in the human homolog, CDK5RAP2, cause inherited recessive microcephaly. Mutations in Drosophila cnn disrupt assembly and function of the mitotic centrosome. Important centrosome components fail to assemble into mitotic centrosomes in cnn mutants including γ-tubulin, Aurora A kinase, D-TACC and Msps (XMAP215/Dis1/TOG homolog). Moreover, in dividing cells, no astral microtubules are detected at cnn mutant mitotic centrosomes. cnn mutants are maternal effect lethal and males are infertile due to cytokinesis defects during spermatocyte divisions. In early embryos, cnn null mutant centrosomes do not organize actin into cleavage furrows and embryos ultimately fail to cellularize. Thus, cnn mutant centrosomes are deficient in MTOC and cleavage furrow activities. Two conserved domains are prominent in CNN: Motif 1 near the amino terminus, and Motif 2 at the carboxyl terminus. We have introduced mutations into each Motif to determine their functions in vivo. Motif 2 is a centrosome-targeting domain. Motif 1 mutant (CNNΔ1) localizes to centrosomes and assembles a PCM, however, severe defects consistent with microtubule dysfunction are evident. Live imaging shows that centrosome separation and trafficking of satellite particles are severely impaired in cnnΔ1 embryos. Despite these defects in microtubule-dependent processes, we find that astral microtubules are clearly present at cnnΔ1 mutant centrosomes. However, microtubule growth experiments show that the dynamics of microtubule assembly are impaired at cnnΔ1 centrosomes. Thus, CNN Motif 1 regulates centrosomal microtubule dynamics. Some centrosomal proteins localize properly at cnnΔ1 centrosomes, with the exception of γ-Tubulin, D-TACC and Msps. Surprisingly, in cnnΔ1 embryos cleavage furrow assembly is partially rescued. A candidate partner for Motif 1 has been identified.

171C Developmental Regulation of S-Phase Coupled Destruction of E2F1. Jean M Davidson1, Shu Shibutani1, Robert J Duronio1,2. 1) Department of Biology, University of North Carolina - Chapel Hill, Chapel Hill, NC; 2) Curriculm of Genetics and Molecular Biology, University of North Carolina - Chapel Hill, Chapel Hill, NC. The E2F family of transcription factors both positively and negatively regulates the transcription of genes necessary for DNA synthesis and cell cycle progression. In Drosophila, these genes are regulated by E2f1. During G1 arrest, E2f1 forms a complex with Rbf1 (homologous to the retinoblastoma family of tumor suppressors) to repress transcription of replication factor genes. In response to signals that trigger cell cycle progression, G1 Cyclin dependent kinases (Cdk) phosphorylate and inactivate Rbf1, thereby allowing E2f1 to induce transcription and promote entry into S phase. Once DNA replication has begun, E2f1 is rapidly destroyed and re- accumulates only after the completion of S phase. We are exploring the mechanisms of, and the signals that lead to, S phase- coupled E2F1 destruction, how this is regulated during development, and whether it contributes to cell cycle control. In the early embryo, S phase-coupled E2f1 destruction begins precisely in cell cycle 15, after the initiation of zygotic transcription. We are performing a screen to test the hypothesis that zygotic gene expression provides the mechanism for developmental control of the onset of S phase-coupled E2f1 destruction. POSTERS: Cell Division and Growth Control 147

172A Two microRNAs, bantam and miR-7, play roles in the regulation of Drosophila Germline Stem Cell cell cycle regulation. Steven H. Reynolds, Ellen J. Ward, Jenn-Yah Yu. Biochemistry, University of Washington, Seattle, WA. Previously, our lab has shown that the Dicer-1 dependent microRNA pathway plays an essential role in the regulation of Germline Stem Cell (GSC) cell cycle regulation by down-regulating the expression of the cyclin dependent kinase inhibitor p21/Dacapo. We now show that two microRNAs which were computationally predicted to target Dacapo, bantam and miR-7, can down regulate dacapo 3' UTR in S2-cell Luciferase assays and that each plays a role in GSC cell cycle control. With in vivo sensors we have observed that both miR-7 and bantam are expressed in GSCs and other cells in the anterior of the germarium. Furthermore, we have shown that loss of miR-7 increases the frequency of expression of cell cycle marker CycE but does not change the over all rate of GSC division. Bantam loss of function exhibits a more sever cell cycle phenotype than miR-7, resembling dicer-1 in that it slows GSC division. In order to determine whether the cell cycle effects of bantam and miR-7 are mediated by Dacapo translational repression we will reduce the level of Dacapo with a dap/+ genetic background and evaluate whether this rescues the miR-7 and bantam phenotypes. We will also measure the responsiveness of GFP sensors bearing the dacapo 3’ UTR to bantam and miR-7 loss. In order to evaluate where in the cell cycle bantam and miR-7 are acting we will measure the frequency of expression of cell cycle markers PH3, CycB, and BrdU in bantam and miR-7 clones.

173B Investigating the role of Nuf, a Rab11 effector, in cytokinetic furrow formation. Justin Crest, William Sullivan. Dept MCD Biol, Univ California, Santa Cruz, CA. Animal cytokinesis requires not only the establishment and constriction of an actino-myosin contractile ring but also the addition of membrane via sources such as recycling endosome-derived vesicles. In the early Drosophila embryo, the proper addition of these vesicles to the invaginating furrow requires Nuf, a Rab11 effector. Previously, we have shown that Nuf localizes to the centrosomes in a cell cycle-dependent manner, which correlates with its most phosphorylated isoform. It is unknown how Nuf phosphorylation is regulated and how its phosphorylation is coordinated with its localization. Currently, we seek to address several questions regarding the molecular nature of Nuf, such as is centrosome localization required for Nuf phosphorylation, what kinase(s) is required for its phosphorylation, and what specific residues of the protein are phosphorylated? To address these questions we will use both genetic and biochemical approaches.

174C Cell division requires a direct interaction between microtubule-associated RacGAP and the contractile ring componenent, Anillin. Stephen Gregory1, Saman Ebrahimi1, Joanne Milverton1, Whitney Jones2, Amy Bejsovec2, Robert Saint3. 1) Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, SA, Australia; 2) Department of Biology, Duke University, Durham, NC; 3) Centre for the Molecular Genetics of Development, Research School for Biological Sciences, Australian National University, Canberra, ACT, Australia. The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile ring position. The current model for initiation of Drosophila and mammalian cytokinesis postulates that equatorial localization of the RhoGEF Pebble by a microtubule-associated motor protein complex creates a band of activated RhoA, which subsequently recruits contractile ring components such as actin, myosin and Anillin. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. We report the first direct molecular link between the microtubule spindle and the acto-myosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage, interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring. Both proteins depend on this interaction for their localisation. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex. 148 POSTERS: Cell Division and Growth Control

175A Proteomic analysis of Drosophila Fragile X mutant cleavage stage embryos. Kate Monzo1, Susan R. Dowd2, Jonathan S. Minden2, John C. Sisson1. 1) The Section of MCD Biology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX; 2) The Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA. Reduced Fragile X mental retardation protein (FMRP) activity in brain neurons results in the most common form of heritable mental retardation in humans, Fragile X Syndrome (FXS). FMRP has been implicated in the translational control of specific mRNAs, and the cognitive symptoms of FXS are thought to stem from the aberrant translation of some of these mRNAs, leading to a deterioration of dendrite morphology. We have demonstrated that Drosophila fragile X mental retardation protein (dFMRP) is required in early embryos for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the maternal-to-zygotic transition (MZT). We have also demonstrated that dFMRP regulates the expression of Trailer Hitch, another translational control factor, raising the possibility of an indirect form of dFMRP-dependent translational regulation. In an effort to identify potential targets of dFMRP regulation, we have employed a proteomics-based approach, two-dimensional gel electrophoresis. Proteins in wild type and dfmr1 mutant cleavage stage embryo extracts were labeled with different cyanine dyes, separated on a single 2D gel, and protein spots that showed a difference in expression between the wild type and mutant extracts were removed from gels and analyzed by mass spectrometry (MS). We have discovered forty proteins whose expression differs between control and dfmr1 embryos. Twenty-eight of these proteins have been identified by MS and will be presented. We are using genetic assays to determine if selected candidates interact with dfmr1 and affect cleavage furrow formation and biochemical assays to address whether the identified proteins are direct or indirect targets of dFMRP-dependent translational regulation. Characterization of these candidate targets should provide insight into the mechanisms of dFMRP-dependent regulation of cellular morphogenesis in cleavage stage embryos and the etiology of FXS.

176B Role of DMYPT in Incomplete Cytokinesis During Drosophila Oogenesis. SengKai Ong, Change Tan. Biological Science, University of Missouri-Columbia, Columbia, MO. Actomyosin contractile ring is crucial for the separation of one cell to form two daughter cells during the cytokinesis of cell divisions. Incomplete cytokinesis is necessary for the formation of interconnected germline cell cluster (cyst) during oogenesis by forming the initial ring canals. The ring canals grow alongside with egg chambers and eventually allowing nurse cells dumping of cytoplasm into oocyte to form a bigger, well-nourished oocyte. Expression of DMYPT, the myosin binding subunit of non-muscle myosin II phosphatase binding subunit, is enriched in region where the first incomplete cytokinesis occur during early cyst formation. Mutation in DMYPT produces small ring canals but its role in the incomplete cytokinesis and/or ring canal growth remains mystery. Thus, we perform experiments by exploiting heatshock-promoter induced DMYPT to determine whether DMYPT has a role in incomplete cytokinesis and/or ring canal growth.

177C Enabled and its kinase Abelson provide a secondary input into the Pebble-Rho1-Diaphanous pathway initiating cytokinesis. Sergei Prokopenko, William Chia. Drosophila Development Group, Temasek Lifesciences Laboratory, Singapore. Cytokinesis ensures the successful completion of the cell cycle and the distribution of chromosomes, organelles, and cytoplasm between two daughter cells. It is accomplished by the formation and constriction of an actomyosin contractile ring that drives progression of the cleavage furrow. However, a precise sequence of signaling and cytoskeletal events during contractile ring assembly in late anaphase and its disassembly in telophase remains to be determined. In the course of our mosaic genetic screen in third instar larval brain we identified a new allele of enabled (ena), a known regulator of the actin cytoskeleton, that showed a dramatic cell division phenotype - extremely large clones that occupy a large volume of the brain and consist of hyperploid multinucleate cells. We show that unexpectedly both ena and its negative regulator Abelson (Abl) tyrosine kinase are required for cytokinesis. Similar to ena phenotype in brain clones, mutations in either ena or Abl lead to cytokinesis failure and formation of binucleate interphase cells also during embryonic mitotic cycles. Contractile ring fails to assemble in ena and Abl mutant cells, since Peanut and Scraps (Anillin) fail to localize to the equator of the cell during cytokinesis. In contrast, distribution of components of the centralspindlin complex (Pavarotti and Tumbleweed) and the small G protein effector Diaphanous is not affected. Overexpression of Abelson during early embryogenesis also blocks cytokinesis and this phenotype depends on the kinase activity of Abelson. We propose that Enabled and its kinase Abelson provide a secondary level of regulation of F-actin polymerization via the actin-binding protein profilin. This regulation is likely to be coordinated with the centralspindlin - Pebble - Rho1 - Diaphanous signaling pathway which is known to control levels of actin polymerization via profilin and contractile ring assembly during cytokinesis. Further data showing interaction between the two branches of this pathway converging on profilin will be presented. POSTERS: Cell Division and Growth Control 149

178A Specialized lipids act to couple actomyosin ring to the plasma membrane during meiotic cytokinesis in Drosophila males. Edith Szafer-Glusman, Margaret Fuller. Developmental Biology, Stanford School of Medicine, Stanford, CA. Attachment and stabilization of spatially organized arrangements of actomyosin filament arrays at the plasma membrane are critical for cytokinesis in animal cells. During cytokinesis, animal cells build an actomyosin contractile ring anchored to the plasma membrane at the cell equator. This ring then constricts to pull the cortex inward, separating the two daughter cells. Although critical, the mechanisms that maintain coupling of actomyosin filaments to the plasma membrane during cytokinesis are not yet well understood. Changes in the composition of membrane domains are emerging as important players in regulating assembly and rearrangement of the cytoskeleton. Here we reveal that very long chain fatty acids and their derivative complex lipids are required for coupling and stabilization of the contractile ring to the plasma membrane during cytokinesis in Drosophila spermatocytes. Mutations in the gene bond, which encodes a Drosophila member of the family of Elovl proteins that participate in elongation of very long chain fatty acids, cause abrupt detachment of myosin rings from the cortex during constriction in dividing spermatocytes, and disorganization of microtubule bundles of the central spindle. Organization and tight attachment of cytoskeletal arrays to the membrane is essential to determine membrane mechanical properties and maintain cell integrity during active membrane remodeling in a number of cellular processes, such as migration, phagocytosis and cytokinesis. Our findings from cytokinesis in Drosophila spermatocytes implicate specific lipid composition of the membrane in the organization and stabilization of the cytoskeleton at the cortex.

179B PLC and MLCK regulate actin dynamics during cytokinesis. Raymond Wong1, Lacramioara Fabian2, Julie Brill1,2. 1) Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; 2) Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada. Cytokinesis, the physical separation of a cell’s cytoplasm into two daughter cells, requires the continuous hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). One product of PIP2 hydrolysis is the second messenger 2+ inositol triphosphate (IP3) which binds to IP3 receptors on the ER to mediate Ca + release from internal stores. We have previously shown that inhibiting PLC activity causes cytokinesis failure in Drosophila spermatocytes. Moreover, the addition of Ca2+ ionophores 2+ blocked inhibition of cytokinesis, suggesting that Ca acts downstream of PIP2 hydrolysis. Since treatment of cells with the MLCK inhibitors ML-7 or W-7 blocked furrow ingression in a manner similar to that observed after treatment with PLC inhibitors, we 2+ hypothesized that IP3-mediated Ca release may regulate components of the cytoskeleton during cell division. To identify candidate targets, we examined F-actin and phosphorylated myosin regulatory light chain (phospho-Sqh) during cytokinesis. In control cells, these components localize to the contractile ring. After inhibition of either PLC or MLCK, we found that phospho-Sqh was lost from the contractile ring, followed by F-actin, which then accumulated at the poles. These data show that inhibition of PIP2 hydrolysis and myosin activation have similar effects on actin dynamics in dividing cells. We propose a model in which the PLC-dependent release of Ca2+ activates myosin via MLCK to maintain actin in the contractile ring during cytokinesis.

180C A distinctive basal promoter architecture defines the entire Drosophila Ribosome Biogenesis regulon. Albert Erives1, Seth Brown2, Michael Cole2. 1) Dept. of Biological Sciences, Dartmouth College, Hanover, NH; 2) Dartmouth Medical School, Lebanon, NH. Ribosome biogenesis (RB) is the complex nucleolar process that spans rRNA transcription, assembly, processing, and export from the nucleus. Here we show that a distinctive promoter architecture is associated with the entire growth-critical cohort of RB genes in Drosophila as determined by a whole-genome approach. In addition to showing that these promoters contain a unique compilation and distribution of basal promoter elements, we show that the entire gene cohort is defined in part by the known target site of dMyc, which is encoded by the diminutive (dm) locus and plays an important role in cell growth and proliferation. Myc functions by heterodimerizing with its obligate partner Max to bind the sequence 5’-CACGTG [a CG-core E-box, or E(CG)] and trans-activate target genes. Previous studies have implicated the Myc transcription factor in rRNA transcription, but the possibility that Myc could directly regulate the hundreds of gene products involved in RB, as opposed to a few genes involved in early key steps, has not been investigated. We next show that orthologous RB loci are maintained as E(CG)-bearing core promoters in all holozoan genomes containing Myc. Max, Mad and Mnt, all members of the Myc bHLH superfamily, are all either insufficient or dispensable in explaining the correlation of E(CG) with RB across multiple eukaryotic genomes that differ in their bHLH repertoire. This RB signature also is absent in genomes which diverged prior to the origin of Myc, or which secondarily lost Myc. Thus, in addition to confirmed RB targets of Myc and the observation that dm mutants phenocopy the growth phenotypes of ribosomal protein mutants, these comparative genomic results suggest that the E(CG)-bearing basal promoters are direct Myc targets. These results indicate that a Myc regulon evolved as an inducible growth regime in a stem holozoan ancestor of both animals and choanoflagellates and has largely been conserved ever since then. 150 POSTERS: Cell Division and Growth Control

181A Genetic and biochemical studies of the Mre11/Rad50/Nbs complex in Drosophila. Guanjun Gao, Germana Colazzo, Xiaolin Bi, Cassie Rauser, Yikang Rong. LBMB, National Cancer Institute, NIH, Bethesda, MD. The Mre11, Rad50 and Nbs proteins form a conserved MRN complex, which plays a critical role in DNA repair and recombination, damage response and telomere maintenance. What is not clear about MRN function is their precise role during the development of a multi-cellular organism. Mammalian mrn mutants are early lethal, which have prevented comprehensive studies of MRN during development. We set out to study MRN in drosophila and have selected Nbs as a model since it is the least conserved member of MRN. We reasoned that by studying Nbs in Drosophila, we might uncover novel Nbs(MRN) functions that might be specific for higher eukaryotes. We have taken two approaches. In the first one, we have used a drosophila line with an endogenously tagged nbs gene to biochemically isolate Nbs-interacting proteins, which include Mre11, Rad50 and other novel ones. In the second approach, we have generated a series of nbs point mutations that disrupt specific domains that are conserved among Nbs proteins from high eukaryotes. Some of the mutations displayed developmental stage specific defects. By further characterizing these defects, we hope to shed light on the underlying mechanisms that lead to human Nijmegen Breakage Syndrome, which is caused by mutations in the nbs gene.

182B The role of Drosophila myb in cell proliferation and differentiation. Margritte K. Rovani, Carrie A. Fitzpatrick, Gary Ramsay, Alisa L. Katzen. Department of Biochemistry & Molecular Genetics, University of Illinois at Chicago, Chicago, IL. Drosophila myb is related to the proto-oncogene c-myb, which plays multiple roles in cell proliferation, differentiation, and apoptosis. Studies in our lab have demonstrated the role of Drosophila Myb (DMyb) in regulating the G1/S and G2/M progressions during mitosis. In addition to its role in cell proliferation, however, we have also found that DMyb plays a role in cell differentiation during some developmental processes. Reducing DMyb level in the wing affects cell differentiation processes such as sensory bristle specification at the anterior margin and vein patterning. We found similar phenotypic defects in our genetic interaction studies between myb and components of the Notch signaling pathway that include multiple bristles at the anterior wing margin and thickening of the wing vein. Interestingly, loss of DMyb function also leads to inappropriate neuronal specification at the wing margin. Genetic and molecular studies thus far suggest that DMyb plays a role in differentiation that is largely independent from its role in cell proliferation. These results and current studies investigating the mechanism underlying myb defects in cell differentiation will be discussed.

183C Coordination of larval and imaginal growth in Minutes. Meng-Ping Tu, Laura A. Johnston. Dept Genetics & Development, Columbia Univ, New York, NY. Minutes are a group of haplo-insufficient mutations (~50 loci) sharing a similar phenotype, including delayed development and short, thin bristles. The majority of Minutes carry mutations in genes encoding ribosomal proteins (Rps), which impair ribosome function and protein synthesis. We have followed larval and imaginal growth during development in four Minutes, M(1)15D, M(2)60E, M(3)66D and M(3)95A (encoding RpS5a, RpL19, RpL14 and RpS3, respectively). In general, Minute larvae grow and molt with normal kinetics, suggesting that larval cells are not affected by the Minute mutation and that their endocrine program is intact. However, despite the fact that Minute and wildtype larvae are the same size, imaginal discs in Minute mutants grow and develop very slowly: by L3 wing discs are only half the size of wildtype wing discs. The final size reached by the discs is normal, however, due to a prolonged L3 in which hormone responsiveness is delayed. These data suggest that the continued growth of the imaginal discs inhibits endocrine function in M/+ larvae. We are taking a tissue-specific Gal4-UAS approach to get at the tissues and signals that underlie the mechanisms that coordinate larval and imaginal growth. POSTERS: Cell Division and Growth Control 151

184A Characterization of a novel conserved cyclin in Drosophila. Dongmei Liu. Dept CMMG, Wayne State Univ, Detroit, MI. Cyclins are a conserved family of proteins that interact with and activate Cyclin dependent protein kinases (Cdks). We are studying the function of a Drosophila protein, CG14939, which has a conserved cyclin-like domain. We have shown by yeast two-hybrid assays and by co-affinity purification that CG14939 interacts preferentially with a poorly characterized Cdk called Eip63E. We show that both CG14939 and Eip63E are phospho-proteins and that phosphorylation of CG14939 plays a role in the interaction with Eip63E. To further characterize the function of CG14939, we generated gene knock-out mutant animals by imprecise excision of a nearby P element. The excision we call E8 is homozygous lethal during larval development and metamorphosis. This mutant defect can be fully rescued by a CG14939 genomic transgene or by UAS-CG14939/Tubulin-Gal4 and thereafter this strain was renamed as CG14939E8. We also generated gene knock-down mutant animals by the Gal4/UAS-dsRNA system. We found that CG14939 gene knock-down animals also showed defects during metamorphosis. The specificity of this gene knock-down animal model is being tested. Further characterization of the CG14939/Eip63E regulatory network is ongoing. We expect to identify upstream regulatory genes and downstream targets and hence put this gene into a signaling pathway and elucidate its in vivo biological functions.

185B Regulation of MEI-S332 Centromere Localization. Cristina Nogueira, Hannah Cohen Koyfman, Lisa Deng, Lynn Young, Andrea Page-McCaw, Terry L. Orr-Weaver. Whitehead Institute for Biomedical Research, Cambridge, MA. The Drosophila MEI-S332 protein localizes to centromeres in mitosis and meiosis when sister chromatids are attached. It is essential for the maintenance of centromere cohesion in meiosis until the sister chromatids segregate at anaphase II, and it also contributes to centromere cohesion in mitosis. Centromere localization of MEI-S332 is controlled by phosphorylation, with phosphorylation by Aurora B kinase being necessary for proper localization 1and phosphorylation by POLO kinase being required for MEI-S332 dissociation from the centromere at anaphase2. The regulatory role of these kinases suggests that there are counteracting phosphatases. The PP2A phosphatase has been shown to be needed for centromere localization of the human MEI- S332 homolog, Sgo13, 4. In contrast, PP2A itself requires the human homolog Sgo 2 to localize to the centromere3, 4. We found that a regulatory subunit of the PP2A complex binds to MEI-S332 in two-hybrid experiments. To investigate the hierarchy of relationship between MEI-S332 and PP2A in meiosis and mitosis we have generated deletions for this PP2A subunit and are using these deletions as well as RNAi knock downs to define interactions between these proteins. 1—T. D. Resnick, D. L. Satinover, F. MacIsaac et al., Developmental cell 11 (1), 57 (2006). 2—A. S. Clarke, T. T. Tang, D. L. Ooi et al., Developmental cell 8 (1), 53 (2005). 3—Z. Tang, H. Shu, W. Qi et al., Developmental cell 10 (5), 575 (2006). 4—T. S. Kitajima, T. Sakuno, K. Ishiguro et al., Nature 441 (7089), 46 (2006).

186C Loss of chiasma maintenance with age. Sharon E. Bickel, Vijayalakshmi Subramanian. Dept. of Biological Sciences, Dartmouth College, Hanover, NH. In humans, errors in meiotic chromosome segregation increase dramatically as women age. Although the link between maternal age and meiotic nondisjunction has been recognized for many years, the underlying molecular defects that cause reduced fidelity of chromosome segregation in older oocytes are largely unknown. We have developed an experimental regimen to study age-dependent nondisjunction in Drosophila. When egg laying is suppressed, oocytes arrest at various stages of development and “age” within the abdomen of the female. Such experimentally induced aging of Drosophila oocytes mimics the normal aging process that human oocytes undergo within the ovary during a female’s lifespan. We have used this regimen to test the hypothesis that deterioration of meiotic cohesion with age renders older oocytes susceptible to increased levels of nondisjunction. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to link recombinant chromosomes until anaphase I. If arm cohesion disintegrates over time, recombinant chromosomes that lose their attachments would segregate randomly during the first meiotic division. Our recent experiments demonstrate that when the achiasmate segregation system is compromised (mtrm+/-), reduction of the cohesin subunit SMC1 (smc1+/-) increases age-dependent nondisjunction. Moreover, by determining the recombinational history of missegregating chromosomes, we have found that nondisjunction of recombinant as well as achiasmate homologues increases. Oocytes that undergo aging after the disassembly of the synaptonemal complex are most susceptible to segregation errors and these closely resemble the dictyate stage at which human oocytes remain arrested for years. In summary, our work demonstrates that Drosophila is an ideal model to study the mechanisms underlying age-dependent nondisjunction and argues that deterioration of cohesion with age results in loss of chiasmata and missegregation of recombinant chromosomes. 152 POSTERS: Cell Division and Growth Control

187A Complex genetic interactions control male meiosis in Drosophila. Silvia Bongiorni1, Silvia Volpi1, Giorgio Prantera1, Barbara Wakimoto2. 1) Dept. Agrobiology and Agrochemistry University of Tuscia, Viterbo, Italy; 2) Dept. of Biology University of Washington, Seattle, USA. Drosophila spermatogenesis provides a genetically tractable system for defining components required for the normal progression of meiosis, including those responsible for developmental- or sex-specific regulation of the G2/M transition. Here we describe a set of EMS-induced male sterile mutations, which were recovered from the Zuker Collection and classified as resulting in large spermatid nuclei indicative of skipping one or both meiotic divisions. Our analysis of a subset of the “Z” mutations on Chromosome 2 reveals complex interactions. Genetic complementation tests show that each mutation has a distinctive complementation pattern, with some failing to complement the twine mutation and/ or each other and all alleles complementing the previously described meiotic mutations pelo and boule. We demonstrate that the complex complementation pattern of this “Twine class” of mutations reflects dose-sensitive interactions between at least four genes. Complementation tests using 133 chromosomal deficiencies, which cover 95% of the euchromatin on Chromosome 2, suggest a minimum of ten dose-sensitive regions that interact with one or more of the mutations in the Twine class. To further understand how the Z mutations affect male meiosis, we compared the cytological defects resulting from homozygous, hemizygous, and selected trans-heterozygous combinations of the mutant alleles. The data show visible defects in spindle and centrosome organization with some mutations apparently acting later than twine mutations. Finer level mapping of the mutations is in progress to permit molecular identification of the corresponding genes. Ultimately, the in-depth characterization of this entire group of genes will permit a more comprehensive dissection of the molecular pathway controlling male meiosis.

188B Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation. Jun Cui, Katharine Sackton, Vanessa Horner, Kritika Kumar, Mariana Wolfner. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. “Egg activation” is the process that allows embryogenesis to begin in a fertilized egg. One aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. We show here that wispy (wisp), a gene whose maternal effect mutations block the egg to embryo transition, encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). Wisp is required for poly(A) tail elongation of bicoid (bcd) mRNA upon egg activation. Wisp was previously reported to regulate maternal mRNA stability in Drosophila embryos. We reported here that Smaug (SMG), a major regulator of mRNA destabilization whose translation is activated upon egg activation, is still translated in activated eggs from wisp mutant mothers. We show that wisp mutations cause very early developmental arrest, subsequent to female meiosis. Activated eggs laid by wisp mutant females are capable of completing female meiosis and forming male and female pronuclei. However, pronuclear migration does not occur in wisp embryos.

189C A conserved pachytene checkpoint is linked to meiotic crossover formation in Drosophila. Eric F. Joyce, Kim S. McKim. Waksman Inst, Rutgers Univ, Piscataway, NJ. During meiosis, recombination is initiated with programmed DNA double-strand breaks (DSBs) whose repair yields either crossovers or noncrossovers. Crossovers mature into chiasmata which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. It is not surprising; therefore, that crossover formation is a tightly regulated process. The total number of recombination events is usually in significant excess to the number of crossovers, suggesting meiotic DSB repair is biased toward producing noncrossovers. Thus, the cell must undergo a decision process as to which subset of DSBs will become crossovers. We show that Drosophila mutants in genes required for processing DSBs into crossovers exhibit delay phenotypes consistent with the activation of a checkpoint. This delay is suppressed in mutants of genes required to promote crossover formation but, surprisingly, is independent of DSBs. The delay in pachytene progression also depends on the Drosophila homolog of pch2, which in nematodes and budding yeast meiosis is required for a DSB-independent checkpoint pathway. These findings suggest that this surveillance mechanism exists in Drosophila meiotic prophase and may monitor events leading to the generation of crossovers. We propose a model where the conditions to promote crossovers are established independent of DSB formation early in meiotic pachytene. POSTERS: Cell Division and Growth Control 153

190A Z2-4034 Identifies a Gene Required for Chromosome Segregation. Apple G. Long, Sarah J. Radford, Kim S. McKim. Dept. of Genetics, Waksman Institute, Rutgers University, Piscataway, NJ. During meiosis in female Drosophila, chiasmata formation leads to segregation of homologous chromosomes at meiosis I. Nondisjunction (NDJ), the failure to separate homologous chromosomes, can occur when crossing over or spindle assembly defects take place. We have identified a mutation, Z2-4034 , which exhibits high X-chromosome NDJ (~20%), but normal crossing over. This phenotype suggests a role for this gene in late meiosis, possibly in bipolar spindle assembly and maintenance. These functions are often required for mitotic cell division, so genes required late in meiosis also often play a role in mitotic chromosome segregation. We have mapped this mutation through three-point recombination and deficiency mapping to an interval between 54C1-54C10 cytological units. Sequencing of a candidate gene in that area, Kinesin-like protein (Klp)54D, in the Z2-4034 mutant revealed a G-to- A transition mutation that results in an amino acid change from glycine to serine. We will report further work to conclusively identify the gene affected in Z2-4034 mutants, including rescue experiments with a Klp54D transgene and the creation and characterization of additional mutant alleles. We are also investigating the phenotype of the mutant through cytology methods. Preliminary results in larve brain cells have shown that Inner centromere protein (INCENP), a major passenger protein required for cell division, fails to localize to the midzone at anaphase and telophase in Z2-4034 mutants, as it would in wild-type cells. This phenotype suggests that Klp54D may be required for INCENP translocation during the metaphase to anaphase progression. To further investigate KLP54D’s function, I will also examine the localization of other major passenger proteins and kinesins involved in cell division, to characterize their relationship with KLP54D.

191B MCM related genes in the precondition class promote crossover repair early in meiotic prophase. Shree N Tanneti, Eric F Joyce, Kim S McKim. Waksman Inst, Rutgers Univ, Piscataway, NJ. Meiosis, the process in which gametes are produced, involves an orchestrated interaction between many genes in the double strand break (DSB) repair pathway. This process promotes recombination and the formation of crossovers between homologous chromosomes, providing stability to the homologs and ensuring their proper segregation at anaphase I. Our preliminary evidence suggests that some crossover genes such as those in the precondition class may function prior to DSB formation. Mutations in the precondition class of genes affect both the levels of meiotic crossing over as well as the location of the residual crossovers. The mechanism that controls crossover site selection is affected in these mutants, suggesting precondition gene products influence the process that decides which recombination sites will become crossovers. Interestingly, all three known precondition genes (mei-218, rec and mcm5) encode MCM family members. An unexpected result was that mei-218 and recmutants suppressed DSB-independent pachytene delays caused by DSB repair-defective mutants, suggesting precondition genes and the decision to produce a crossover are earlier than the first steps of DSB repair. Although it is not known when DSBs are committed to the crossover pathway, it has been proposed in S. cerevisiae that the decision to produce a crossover is made very early, at or prior to the appearance of the first stable strand exchange. Even more striking, precondition mutants strongly reduce MEI-P22 staining, a protein required for DSB formation. We propose an “early decision” model in which precondition gene products promote crossover repair by predisposing DSBs to follow the crossover repair pathway or by suppressing alternative repair pathways.

192C Analysis of dRING function during Drosophila oogenesis. Pei Zhou, Sharon Bickel. Department of Biological Sciences, Dartmouth College, Hanover, NH. Previous work in our laboratory identified the PcG protein dRING as a yeast two-hybrid interactor of ORD, a Drosophila protein required for meiotic sister-chromatid cohesion in both males and females (Balicky et al., 2004). ORD activity also is required for normal levels of homologous recombination in oocytes (Webber et al., 2005). We have begun an analysis of dRING function in the female germ-line to test the hypothesis that dRING is involved in regulating sister-chromatid cohesion and/or chromosome segregation during Drosophila meiosis. dRING is an essential protein encoded by by Polycomb Group gene, Sex combs extra (Fritsch et al., 2003). The Sce1 mutation results in a truncated dRING protein that is missing the C-terminal domain required for ORD interaction. Using a FLP/FRT strategy, we have generated Sce1 germ-line clones in the ovary and monitored meiotic chromosome morphology in germaria by examining the localization of sister-chromatid cohesion and synaptonemal complex proteins. Thread-like SMC1 and C(3)G staining appears normal in Sce1 clones during early pachytene suggesting that that temporal and spatial regulation of synaptonemal complex assembly does not require the function of dRING. However, in contrast to wild type, mutant Sce1 cysts in region 3 of the germarium often contain two nuclei with extensive C(3)G thread-like signal. In addition, Sce1 mutant cysts at more posterior locations in the ovariole are often dramatically smaller than normal and in some cases the oocyte is mis-positioned within the cyst. Together, these preliminary data suggest an unexpected role for dRING protein in oocyte determination and/or development. 154 POSTERS: Cell Division and Growth Control

193A Analysis of the dynamics of the condensin subunit Cap-G. Sabine Herzog, Sonal Nagarkar, Stefan Heidmann. Genetics, University of Bayreuth, Bayreuth, Germany. Chromosomes undergo many structural changes during the cell cycle. At onset of mitosis, the two sister chromatids are compacted into two cytologically distinguishable cylindrical structures. This condensation process is essential for faithful segregation of sister chromatids in anaphase. Condensin, a complex composed of the five subunits SMC2, SMC4, Cap-D2, Cap-G, and Cap-H is implicated in this process. We are investigating the dynamic behaviour of fully functional EGFP-tagged Drosophila Cap-G during early embryonic divisions. We found that Cap-G is nuclear enriched during interphase, it starts to associate with chromatin in early prophase and loading is complete before the nuclear envelope breaks down. Moreover, Cap-G shows dynamic association with chromatin in metaphase as determined by fluorescence recovery after photobleaching (FRAP) experiments. Taken together, the dynamics of Cap-G during the cell cycle is similar, but not identical, to the behaviour of Drosophila Cap-H/Barren. To delineate the parts of Cap- G that are responsible for nuclear localization and/or chromatin association we are currently performing a domain analysis using EGFP-tagged Cap-G fragments. Preliminary data suggest the existence of two independent domains that mediate Cap-G chromatin association and of a C-terminal NLS that is dispensable for Cap-G function.

194B Differential requirements for mitotic sister-chromatid cohesion in the soma and in the germ-line. Ana Marques1, Rui Tostões1, Thomas Marty2, 2R Screen Team2, Rui Martinho1. 1) Instituto Gulbenkian da Ciência, Oeiras, Portugal; 2) Skirball Institute, NYU- Medical Center, USA. Drosophila embryonic development begins with a series of mitotic divisions without cytokinesis. During blastoderm cellularization these nuclei are enclosed in individual cells resulting in the formation of a polarized epithelium. From a maternal screen previously done in the laboratory of Dr. Ruth Lehmann (NYU, USA), we isolated a complementation group whose embryos show abnormal blastoderm cellularization due to syncytial nuclei division defects. Although they have normal centrosomes, normal mitotic spindles, and are able to activate the mitotic checkpoint, they show chromosome segregation defects (with lagging chromosomes and chromosome bridges). We called these mutants atado (atado means tight-up in Portuguese). We cloned atado mutation and concluded that it is allelic to the gene separation anxiety (san). san is an acetyltransferase and is important for mitotic sister-chromatid cohesion in Drosophila and in humans [1, 2]. Consistent with previous reports [1], we observed that larvae mutant for atado contain smaller and morphological abnormal brains and the neuroblasts show mitotic defects. We also observed that imaginal disc clones mutant for atado are significantly smaller than the twin-spot wild-type clones, which suggests that atado mutant cells have proliferation defects. Surprisingly, and although the isolated atado mutants are loss-of-function alleles of san, we did not observe any obvious phenotype during the mitotic divisions of the germ-line stem cell. Furthermore, egg-chambers mutant for atado developed normally with a condensed karyosome and without any spindle phenotype. The fact that atado/san is important for somatic but not germ-line cells suggests that differences are likely to exist between the processes that regulate mitotic sister-chromatid cohesion in these cell types. [1] Byron C.W. et al. (2003), [2] Hou F. et al. (2007).

195C Defining the cell cycle roles of fly Sgt1. Lucia Mentelová1, Gonçalo Costa1, Fátima Pereira1, Claudia Florindo1, Álvaro Tavares1,2. 1) Cell Division Group , Inst. Gulbenkian de Ciência, Oeiras, Portugal; 2) Chemical Eng. Inst. Superior Tecnico, Lisboa, Portugal. The budding yeast gene Sgt1p was shown to be required for kinetochore assembly by activation of the CBF3 complex, cAMP signalling and for cell cycle advance. At least some of these functions seem to be conserved in the mammalian homologue, which appears to be an essential protein and critical factor for kinetochore assembly. In order to study kinetochore assembly in Drosophila, we have cloned and characterized Drosophila homologue of Sgt1 and identified Sgt11 mutant allele resulting from P-element insertion as a null. Homozygous individuals are dying either at the late 3rd instar larval or early pupal stages. Brains from homozygous 3rd instar larvae are smaller compared to the wild-type and imaginal discs are greatly reduced in size. The mitotic index in these tissues is very low and DNA hypercondensation, fragmentation of chromosomes and aneuploidy is frequently observed together with mitotic spindle defects and with multiple centrosomes. Depletion of DmSgt1 protein from S2 cells leads to similar mitotic defects. In addition, we show that Sgt1 co-precipitates with SkpA protein, homologue of the essential budding yeast protein Skp1p required for the formation of kinetochore complex. Depletion of DmSgt1 results in a mislocalisation of SkpA from the mitotic spindle. Altogether, our results indicate that Sgt1 is an essential gene for proper spindle assembly and chromosome segregation. POSTERS: Cell Division and Growth Control 155

196A Drosophila no poles encodes an E3 ubiquitin ligase required for genomic stability in the early embryo. Julie Merkle, Laura Lee. Department of Cell & Developmental Biology, Vanderbilt University Medical Center, Nashville, TN. In a screen for cell-cycle regulators, we identified a new maternal effect-lethal mutant that we have named “no poles” (nopo). Embryos from nopo females undergo mitotic arrest with a high frequency of acentrosomal spindles (hence the name no poles), misaligned chromosomes, and multipolar spindles. Our genetic studies show that Checkpoint kinase 2 (product of the mnk gene) is activated in nopo mutants, suggesting that nopo plays a role in maintenance of genomic integrity. We have identified CG5140, the Drosophila homolog of the human TRIP gene (TRIP=TRAF-interacting protein, TRAF=TNF-α receptor-associated factor), as the nopo gene. Human TRIP has been shown to physically interact with Traf proteins, but its role in TNF signaling is unclear. TRIP-deficient mice have recently been shown by others to die during early embryonic development due to proliferation defects and excessive cell death, suggesting an evolutionarily conserved role for NOPO/TRIP in cell-cycle regulation. NOPO/TRIP contains a RING domain at its amino terminus; a conserved residue within this domain is mutated in our EMS allele. The RING domain of NOPO resembles that of known E3 ubiquitin ligases, and in vitro studies by others have shown enzymatic E3 ligase activity of human TRIP. Our phenotypic analyses and yeast two-hybrid results indicate that NOPO and BEN/UEV1A, a heterodimeric E2 conjugating enzyme (Ubc13/Uev1A homologs), form an E2-E3 ubiquitination complex required for genomic stability during embryogenesis. Consistent with this model, our preliminary data suggest that NOPO localizes to the nuclei of syncytial embryos. Future goals include identifying NOPO targets and interactors so as to elucidate the mechanism by which NOPO promotes genomic stability.

197B Cyclin J is required for normal oocyte development. Govindaraja Atikukke1, Russell L Finley, Jr.1,2. 1) Dept Biochem & Molec Biol, Wayne State Univ, Detroit, MI; 2) Center for Molecular Medicine and Genetics, Wayne State Univ, Detroit, MI. Cyclin J is a poorly characterized cyclin originally identified in Drosophila and conserved in metazoans including human, mouse, and mosquito. In Drosophila, cyclin J is found exclusively in the female germ line and early embryo, suggesting that it may play a role during oogenesis or in the unique division cycles of early embryogenesis, which lack gap phases and certain checkpoints. Consistent with this possibility, we previously showed that injection of embryos with cyclin J inhibitory aptamers or antibodies results in cell cycle defects [Kolonin and Finley, Developmental Biology 227 (2000)]. To further explore the role of cyclin J we have taken two independent approaches to create loss of function alleles. Using FLP-mediated recombination between two FRT-bearing transposon insertion alleles around the Cyclin J genomic region, we have created a deficiency strain that lacks cyclin J and the two neighboring genes, armitage and CG14971. The resulting animals are complete male and female sterile. In females, ovaries contain the normal number of ovarioles but the oocytes fail to develop beyond stage 6. Providing cyclin J from a transgene in the 3-gene deletion background promotes oocyte development up to stage 11. Currently we are working on determining the specific process that requires cyclin J. We are also using a P-element inserted close to cyclin J to create imprecise excision strains disrupting cyclin J. We have generated a few cyclin J loss of function alleles that have oocyte development abnormality. Finally, we are using a protein interaction map centered on cyclin J as a further guide to identifying the genetic regulatory networks to which cyclin J may belong.

198C A genetic system to study compensatory growth in the imaginal discs of Drosophila melanogaster. Abigail Gerhold, Adrian Halme, Iswar Hariharan. Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA. To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. This process is often referred to as compensatory or regenerative growth. During Drosophila larval development, imaginal discs show remarkable regenerative capacity. Damage to imaginal discs, either by irradiation or genetic ablation, results in the compensatory growth of surviving cells, producing normally sized and patterned adult organs. While the mechanisms of apoptosis are relatively well understood, much less is known about how surrounding cells change their developmental program to compensate for the loss of their neighbors. We have developed a genetic system that allows us to conditionally ablate significant portions of developing imaginal discs. Using this system, we can test candidate genes and conduct an unbiased screen for novel genes involved in compensatory growth. We have identified the gene cropped, the Drosophila homologue of the human transcription factor AP-4, as required for robust compensatory growth following tissue ablation. Work is ongoing to characterize the role of cropped in compensatory growth and to identify additional factors involved in this process. 156 POSTERS: Cell Division and Growth Control

199A Functional and Regulatory Analysis of SUBITO in Drosophila melanogaster. Jeffry Cesario, Kim McKim. Waksman Inst, Rutgers Univ, Piscataway, NJ. Subito is a Drosophila homolog of the human Mitotic Kinesin-Like Protein 2 and a member of the Kinesin 6 family that assembles the central spindle by localizing to the antiparallel overlap of microtubules. Mutants in this protein have spindle assembly defects in both meiotically and mitotically dividing cells at both metaphase and anaphase. Nevertheless, the mechanism that localizes SUBITO to the microtubules has yet to be ascertained. We are investigating the regulation of SUBITO by taking advantage of the known synthetic lethal interaction between subito and such genes as Incenp, ncd, and aurora B. These interactions can be used to characterize new genes involved in spindle assembly. We are expanding this list of genes by performing a synthetic lethal screen with deficiencies on the third chromosomes and using EMS to create mutants on the second chromosome. Currently, this screen has discovered two areas on interest: 61A-61D and 64C-65C. Furthermore, we are also investigating interactions that affect meiosis. Mutants in the motor domain of subito strongly interact with lower dosages of SUBITO. Dominate negative mutants of Incenp have been created to investigate its interaction with SUBITO. These transgenes, when expressed with heterozygous subito, showed increased levels of nondisjunction. This suggests a pivotal relationship between INCENP and SUBITO during meiosis. We will also report studies investigating the RAN-GTP as a possible regulator of SUBITO.

200B Understanding the mechanisms involved in the activation of the Hippo pathway by the tumor suppressor fat. Caroline Badouel1, Laura Gardano2, Helen McNeill1. 1) Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada; 2) Wellcome Trust Centre for Cell Biology, Edinburgh, Scotland. The Hippo kinase signaling pathway plays a key role in restricting organ size during development. Mutation in any member of this pathway leads to a dramatic overgrowth of epithelial tissues. Recently, it has been shown that the transmembrane proto-cadherin Fat activates the Hippo kinase pathway via the cytoskeletal protein Expanded, constituting the first evidence of an extracellular input on this pathway. Both Fat and Expanded co-localize in epithelial junctions and loss of Fat results in a reduction of Expanded at the junction. But how Hippo activation occurs downstream of Expanded and Fat is still unknown. We are using biochemical and molecular analysis of Fat and Expanded to try to understand their function in the Hippo pathway activation. We are currently analyzing protein interactions by mass spectrometry and GST-pull down and conducting a structure-function analysis to determine essential domains for pathway activation.

201C Local and systemic responses to ionizing irradiation in Drosophila larvae. Adrian Halme, Abigail Gerhold, Iswar Hariharan. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA. Drosophila melanogaster imaginal discs have a remarkable capacity to repair damage that may occur during their growth within the larvae. This is acheived through two mechanisms: 1) There is a burst of compensatory growth in radiation-damaged imaginal discs. 2) Tissue repair produces a delay in developmental timing through the inhibition of endocrine signaling. This extended larval period accommodates the compensatory growth required to replace damaged cells within the imaginal discs. We have used novel genetic screens to identify pathways that are important for both the local and systemic responses to tissue damage. We have identified the gene cropped, encoding the Drosophila homologue of the human AP-4 transcription factor (dAP-4), as a regulator of tissue repair after radiation damage. We are currently determining the role of dAP-4 in compensatory growth. Additionally, we have demonstrated that the activation of the Jun-N-terminal Kinase (JNK) pathway is important for producing the consequent developmental delay. We are characterizing several mutants that may help us to define the molecular pathways downstream of JNK that are responsible for the communication of imaginal disc repair to the endocrine system. POSTERS: Cell Division and Growth Control 157

202A The E1-ubiquitin-activating enzyme uba1 in Drosophila controls apoptosis autonomously and tissue growth non- autonomously. Tom Lee, Tian Ding, Zhihong Chen, Vani Rajendran, Heather Scherr, Melinda Lackey, Clare Buldoc, Andreas Bergmann. Biochemisty & Molecular Biology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin- conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of uba1, the only E1 in Drosophila. We found that weak and strong uba1 alleles behave genetically differently with sometimes opposing phenotypes. While weak uba1 alleles protect cells from cell death, clones of strong uba1 alleles are highly apoptotic. Strong uba1 alleles cause cell cycle arrest which correlates with failure to reduce Cyclin levels. Surprisingly, clones of strong uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to down-regulate Notch signaling in uba1 mutant clones. In summary, the phenotypic analysis of uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate uba1 as a tumor suppressor gene.

203B To Specify or to Proliferate: That is the Question in the Early Fly Retina. Shera Lesly, Justin Kumar. Dept Biol, Indiana University, Bloomington, IN. During the development of complex tissues there is a constant balancing act between cell proliferation and specification. A developing tissue needs to calculate and generate the appropriate number of required cells. As cells are generated they in turn must differentiate into their respective specific cell types. The number and type of differentiating cells as well must be accurately counted and adjustments to the rate of cell proliferation must be made constantly throughout development. Early in the development of any tissue the ratio of undifferentiated to differentiated cells favors the undifferentiated state. In other words cell proliferation is an actively promoted process. However, as the number of differentiated cells approaches the requirements for a tissue the ratio inverts and the number of newly generated cells decreases and eventually cell proliferation stops. If this check and balance on cell proliferation is altered then tumorigenesis and cancer may result. The developing compound eye is an excellent model system for elucidating the molecular links between cell proliferation and tissue specification. Mutations within members of the eye specification network lead to a block in retinal development. If the entire eye is mutant then the developing eye disc is very small due to a reduction in cell proliferation and an increase in cell death. However, an interesting phenomenon is observed when only a portion of the eye disc (such as a mosaic clone) is mutant for any one of the retinal determination genes. In certain circumstances, such as clones of loss- of-function dachshund alleles, the mutant tissue will over proliferate instead of degenerating. In other situations, such as clones of eyes absent mutants, the neighboring wild type cells will be induced to over proliferate. These results suggest that communication between mutant and normal cells in the early eye disc is crucial to balancing the rates of cell proliferation and tissue determination. Our results also indicate that the Notch pathway mediates this balancing act.

204C Multitasking: How Dpp manages to regulate patterning and growth. Gerald Schwank, Simon Restrepo, Konrad Basler. Institute of Molecular Biology, University of Zuerich, Zuerich, Zuerich, Switzerland. Organ growth is regulated by a variety of factors, which are in part extrinsic to the organ and in part intrinsic. Several experiments place the intrinsic growth programs downstream of patterning. The conceptual and molecular details of this connection remain unresolved. The Dpp gradient in Drosophila wing disc development is the key factor in patterning of the AP axis, and functions as a growth modulator. Thus it provides an excellent model system to expand our understanding about the connection between patterning and growth. Our results suggest, that Dpp uses different strategies to regulate patterning and growth. These data will be presented. 158 POSTERS: Cell Division and Growth Control

205A The influence of nutrition on growth and insulin signalling in Drosophila melanogaster. Gerhard Seisenbacher, Hugo Stocker, Ernst Hafen. Institute of Molecular Systems Biology, ETH Zurich, Switzerland. Growth of multi-cellular organisms has to be tightly regulated to allow survival under different conditions and to prevent diseases. Single cells, organs and the whole organism have to sense their environment constantly and respond to changes and adapt to novel and possibly harmful or beneficial situations. A variety of genetic networks integrate environmental signals to control proliferation and growth. In this context, metabolism is a highly interactive node between the organismal pathways (sensing, uptake, conversion) and environmental signals (source, composition, accessibility). Disturbances in signalling pathways that govern those processes and changes in nutrition compositions result in complex diseases such as the metabolic syndrome, chronic inflammation, and cancer. Using the fruit fly Drosophila melanogaster as a model system we want to study how the insulin signalling pathway assures normal growth and proliferation under various nutritional conditions. Here we present how different nutritional conditions influence the growth properties of wild-type flies and mutants for insulin signalling components. In greater detail we analyse the effect of nutritional influence on PTEN mutant flies and PTEN mutant clones. We show, that although PTEN mutant flies are more sensitive to starvation, PTEN mutant clones possess a growth advantage compared to the wild-type cells. Similarly altered growth properties are observed under different stress conditions. We present a detailed analysis of how different environmental conditions modulate the growth properties of insulin signalling mutants on a cellular and systemic level. Especially for PTEN, a tumor suppressor, we hope to gain inside into how growth advantages of a few cells arise depending on the environmental situation.

206B Insulin-signaling and the developmental regulation of allometry in Drosophila. Alexander Shingleton. Department of Zoology, Michigan State University, East Lansing, MI. Among all organisms, the size of each organ scales with overall body size, a phenomenon called allometry. Although the regulation of allometry is a fundamental developmental process, the mechanisms that underlie this regulation remain virtually unknown. Here we examine the developmental regulation of allometries in Drosophila. We show that the male genitals show a different allometric relationship with body size than other organs, and demonstrate that this difference reflects how genital size responds to changes in developmental nutrition compared to other organs. The insulin-signaling pathway regulates growth with respect to nutrition in all animals, and suppression of the pathway’s activity has a different effect on the size of the genitals than on the other organs. Thus, variation among organs in their sensitivity to changes in insulin-signaling appears to underlie variation in their scaling relationships with each other and with body size. We present data indicating that it is the organ-specific expression or activity of insulin-signaling genes that regulates insulin sensitivity, and show that altering the expression of these genes in a single organ is sufficient to alter that organ’s allometric relationships. These data clearly implicate the insulin-signaling pathway as a key regulator of relative organ size, and provide one of the first examples of genes that control allometry.

207C Imaginal disc growth regulates critical size for metamorphosis in Drosophila. Alexander Shingleton, Bradley Stieper, Michael Driscoll. Department of Zoology, Michigan State University, East Lansing, MI. The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects, growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Attainment of critical size initiates a hormone cascade that eventually ends in metamorphosis. Previous research has suggested that growth of the imaginal discs influences the timing of metamorphosis in Drosophila. However, whether this is a consequence of imaginal disc growth affecting critical size is unknown, particularly because critical size has not yet been measured directly in Drosophila. Here we measure critical size in Drosophila and show that damage to the imaginal discs using a low does of X-irradiation retards metamorphosis both by increasing critical size and by extending the period between attainment of critical size and metamorphosis. Complete removal of imaginal tissue using a high dose of X-irradiation also influences the period between critical size and metamorphosis but does not alter critical size itself. These data suggest that both attainment of critical size and the timely onset of metamorphosis are regulated by organ growth in Drosophila. POSTERS: Cell Division and Growth Control 159

208A Regulation of regenerative growth in imaginal discs. Rachel K. Smith-Bolton, Melanie Worley, Hiroshi Kanda, Iswar Hariharan. Molecular and Cell Biology, University of California, Berkeley, CA. Drosophila imaginal discs are capable of undergoing regenerative growth to replace tissue that has been surgically removed. We have developed a new genetic system that can induce tissue ablation and allow regeneration consistently and efficiently in a large number of animals. This system uses a Gal4 driver to target imaginal disc tissues, a cell death promoting gene to induce ablation, and Gal80ts to allow temporal control over ablation and regeneration. We have used this system to observe changes in the expression levels and patterns of morphogens and growth regulators throughout the regenerative process, and have found that Wingless and Myc are expressed at elevated levels in non-canonical patterns and may be important for promoting and directing new growth in response to tissue ablation. We have also defined a developmental window during which imaginal discs are competent for regeneration; discs that have aged beyond this window do not regenerate. In order to understand what is preventing regenerative growth in older discs, we have examined differences in the expression of important genes in younger and older damaged discs. The amount of regenerative growth that occurs in this system can be altered by manipulating the genetic background. Therefore, we are testing known growth regulators and signaling pathways as well as screening for novel factors that are required for regenerative growth. In addition, we are screening for mutations and overexpression transgenes that allow regeneration to occur in older discs that are normally incapable of replacing ablated tissue. In summary, we have developed a genetic system to induce and manipulate regenerative growth efficiently and consistently. We will present our analysis of the roles of Wg, Myc, and other regulators during imaginal disc regeneration.

209B The role of dMyc in pattern-directed growth during development of the Drosophila wing. Christine Wu1, Laura A. Johnston2. 1) Biological Sciences, Columbia University, New York, NY; 2) Genetics & Development, Columbia University, New York, NY. Myc is a conserved protein that is essential for growth in most animals. In both mice and Drosophila, hypomorphic alleles of myc result in animals with a smaller body size. Growth in the Drosophila wing imaginal disc is especially sensitive to expression levels of dmyc: even two-fold differences between neighboring cells can result in cell competition, where cells with more dmyc outgrow and induce apotosis of neighboring cells with less dmyc. However, within the wing, dmyc is expressed regionally at strikingly different levels during development, and it is unknown how wing disc growth responds to this dynamic expression pattern. Our goal is to determine whether the patterned expression of dmyc translates into a regional growth requirement that is important in the formation of an adult wing of correct size and proportion. Initial studies using clonal analysis of dmyc mutant cells indicate temporal and regional requirements for dmyc that correlate with its dynamic expression pattern. In addition, we find that cell competition has temporal and regional specificity. We will discuss these and other studies that aim to elucidate how dMyc-directed growth affects wing size and shape at the end of development.

210C p53-independent apoptosis limits aneuploidy following irradiation. Laura M McNamee, Michael H Brodsky. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA. A prominent therapy for cancer is to induce cell death with DNA damaging agents, such as ionizing irradiation. In normal cells, the p53 tumor suppressor induces cell death after damage. However, approximately 50% of all human cancers are mutant for p53; in these cells, irradiation can still induce an attenuated response to DNA damage. Given the role of DNA damage-induced responses in the genesis and treatment of cancer, it is critical to understand what mechanisms are used to regulate apoptosis in the absence of p53. Unprotected telomeres or irradiation induces p53-independent apoptosis in Drosophila. Here, we demonstrate that p53- independent apoptosis in Drosophila is mediated by the JNK pathway and acts to limit aneuploidy following ionizing radiation (IR). Previous studies have demonstrated that IR induces aneuploidy, resulting in the Minute mutant phenotype due to haploinsufficiency of one of 65 ribosomal protein genes. Clones of Minute mutant cells are eliminated by JNK and hid-dependent apoptosis. We demonstrate that IR induces both ectopic expression of brk, which is sufficient to induce JNK signaling, and increased JNK signaling and hid expression, which are required for induction of apoptosis. p53, hid double mutant animals exhibit increased numbers of Minute cells following IR, indicating that p53-independent apoptosis acts to eliminate aneuploid cells. Mutations in the checkpoint kinase grp and the JNK phosphatase, puc, strongly sensitize p53 mutant cells to IR-induced apoptosis. Our data indicates that induction of JNK-dependent apoptosis represents a novel, p53-independent mechanism to eliminate aneuploid cells following DNA damage. The roles of JNK and cell cycle checkpoint pathways in this response may have therapeutic implications for treating cancer cells deficient for p53. 160 POSTERS: Cell Division and Growth Control

211A Identification of novel tumor suppressor genes through DCP-1 with GMR-GAL4 mediated modifier screening. JuHyun Shin, OokJoon Yoo. Dept Life Sci, KAIST, DaeJeon, Korea. We tried to find out the regulators of cell death in vivo by DCP-1 with GMR-GAL4 mediated modifier screening. Modifier screening reveals novel regulators of cell death. Using a modifier screening, we have screened 39 genes. These genes are kinase family and transcription factors. Among these genes, we selected 16 genes which have high degree of homology compared to human genes. Tumor suppressor genes induce cell death to tumor cells and killed tumor cells. So, we performed cell death assay to prove cell death inducing ability of novel candidate genes and find out several genes expressing cell death inducing ability. We try to connect effector caspase dcp-1 mediated cell death pathway and tumor suppression.

212B The Rab11 GTPase controls somatic cell fate decisions in the Drosophila ovary and behaves as a neoplastic tumor suppressor in follicle epithelial cells. Jiang Xu, Lan Lan, Nicholas Bogard, Robert S. Cohen. Department of Molecular Biosciences, University of Kansas, Lawrence, KS. The egg chamber is the basic unit of Drosophila oogenesis and provides an excellent system in which to study the specification and differentiation of cell fates. Here we show that the Drosophila Rab11 GTPase, a key component of both endocytic and exocytic membrane trafficking pathways, plays multiple roles in the specification and differentiation of polar, stalk and epithelial cells, which comprise the somatic components of the egg chamber. We show, for example, that rab11-null follicle stem cells (FSCs) give rise to too many polar/stalk cell precursors at the expense of epithelial cell precursors. We further show, through depleting Rab11 later in the follicle cell lineage, that Rab11 is required for epithelial cell differentiation. Strikingly, the rab11-null epithelial cells arrest differentiation, lose all signs of polarity, delaminate from the epithelium, and invade neighboring tissues. This invasion phenotype can be rescued by a wildtype rab11 transgene, but not by mutant rab11 transgene that encodes a protein unable to bind to the Rab11 effector dRip11. Based on these observations, we propose that rab11 be classified as a neoplastic tumor suppressor gene (tsg). Unlike cells carrying mutations in previously characterized Drosophila tsgs, the rab11-null cells sever all contacts with the epithelium, reminiscent of the metastatic tumor cells of higher animals. Our findings highlight the important role of membrane trafficking in cell fate determination, differentiation, and neoplastic growth. POSTERS: Cytoskeleton and Cellular Biology 161

213C The cadherin Fat2 is required for planar cell polarity and organ shape in Drosophila. Christian Dahmann, Tina König, Ivana Viktorinova. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Planar cell polarity is a fundamental property of epithelia in animals and plants. In the Drosophila ovary, epithelial follicle cells display planar cell polarity as evident by the stereotyped arrangement of actin filaments perpendicular to the long axis of the developing egg. kugelei mutants are known to display defects in actin filament organization that correlate with the production of misshaped (spherical) eggs, indicating a close link between planar cell polarity and organ shape. However, the gene, which is defective in kugelei mutants, has not been identified and, therefore, the molecular mechanisms underlying this link are elusive. Here, we have generated mutations in fat2, a gene closely related to the planar cell polarity gene fat. We show that Fat2 is required in follicle cells for polarized actin filament formation and normal egg shape. Moreover, we find that fat2 is allelic to kugelei. Sequencing of the fat2 gene in seven different kugelei mutants identified premature stop codons. F-actin organization and egg shape were only affected in large, but not small fat2 mutant follicle cell clones, indicating that Fat2 acts globally to orchestrate planar cell polarity and egg shape. Finally, we provide evidence that genes of the known planar cell polarity pathway (dsh, dgo, pk, stbm) are not required for proper egg shape. Our results suggest that follicle cells use a novel, Fat2-dependent pathway to orchestrate planar cell polarity and organ shape.

214A Characterization of α-catenin using a structure-function approach. Ridhdhi Desai, Milena Pellikka, Ritu Sarpal, Ulrich Tepass. Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada. During animal morphogenesis, intercellular adhesion is essential for maintaining the proper architecture of epithelial tissues. DE- cadherin, a member of the classic cadherin family, is expressed in Drosophila epithelial tissues and is a core component of adherens junctions (AJs). The conserved cytoplasmic region of DE-cadherin binds to Armadillo, which then binds α-catenin to form a cadherin- catenin complex (CCC). α-catenin is thought to be an essential component of the CCC that associates the complex with the actin cytoskeleton. Toward deciphering the molecular mechanisms of how α-catenin supports cadherin activity during development, we use Drosophila oogenesis as a model to carry out a detailed structure-function analysis of α-catenin. α-catenin is structurally related to Vinculin and shares 21-33% sequence identity at three regions previously characterized as Vinculin Homology (VH) regions 1, 2 and 3. Based on its sequence similarity with Vinculin and a limited amount of structural information from mammalian α-catenin fragments, we have generated tagged deletion constructs that remove several conserved regions within α-catenin. Preliminary characterization of transgenic flies bearing these constructs show that the tagged full-length α-catenin localizes to the AJs and rescues the α-catenin mutant phenotype. Additionally, we also show that while all three VH regions are essential for α-catenin function, only VH1 and VH2 are required for the recruitment of α-catenin to the AJs.

215B Cdc42 promotes adherens junction integrity by negatively regulating apical endocytosis. Kathryn Harris, Ulrich Tepass. Cell & Systems Biology, University of Toronto, Toronto, ON, Canada. Cdc42, a member of the Rho family of small GTPases, is part of the intricate network of factors that establish and maintain epithelial cell polarity. Cdc42 binds to Par6, which subsequently promotes the kinase activity of atypical protein kinase C (aPKC), which contributes to the formation of the apical membrane. Here we report that adherens junctions (AJs) are lost from the neuroectodermal epithelium when Cdc42 function is compromised in Cdc42 mutants or through expression of a dominant negative form of Cdc42. AJ integrity is severely affected in the ventral neuroectoderm when neuroblasts ingress but not in other parts of the ectoderm. The defects that result from disrupting Cdc42 function can be rescued by suppressing neuroblast formation, suggesting that Cdc42 has a specific function in supporting the integrity of dynamic AJs as they breakdown and reform, a process required during neuroblast ingression. Further analysis showed that in addition to the complete loss of AJ markers, all examined components of the apical membrane, including Crumbs, Notch and the Par proteins Par6, Bazooka and aPKC, are lost from the apical membrane, whereas basolateral proteins are unaffected in Cdc42 compromised embryos. Apical proteins but not AJ proteins are found in large puncta in the cytoplasm that we have identified as late endosomes. Cdc42 has been described as both a positive and negative regulator of endocytosis, and recent evidence suggests that polarity factors in general may play a key role in controlling membrane traffic. We therefore propose that Cdc42 negatively regulates apical endocytosis during Drosophila embryogenesis, and that perturbing Cdc42 activity in morphogenetically active tissues can deplete the apical membrane of important polarity determinants, resulting in a loss of AJ integrity. We will continue to explore how endocytic traffic is regulated by Cdc42, as well as other polarity factors in Drosophila, and how this regulation contributes to normal epithelial morphogenesis. 162 POSTERS: Cytoskeleton and Cellular Biology

216C Characterization of lumen morphogenesis in the Drosophila retina. Nicole Husain1, Milena Pellikka1, Kwang-Min Choe2, Thomas R. Clandinin2, Ulrich Tepass1. 1) Cell & Systems Biology, University of Toronto, Toronto, ON, Canada; 2) Department of Neurobiology, Stanford University, Stanford, CA. Creating intercellular space within an epithelium is an important and fundamental morphogenetic process, however, very little is known about how a luminal space is shaped and maintained. Within each ommatidium of the Drosophila retina is an epithelial lumen, the interrhabdomeral space. The formation of this space is critical for the development of a functional fly retina, as it optically isolates the light sensing rhabdomeres of individual photoreceptor cells. We have identified and characterized eyes shut (eys), a novel gene that is essential for the formation of the interrhabdomeral space. Eys is a component of the apical extracellular matrix secreted by photoreceptor cells. In the absence of Eys function, photoreceptor cell apical membranes remain attached to each other and no lumen forms. Except for the lack of interrhabdomeral space, eys mutants photoreceptors appear to undergo normal differentiation. Eys is a proteoglycan related to Agrin and Perlecan and is heavily glycosylated with several consensus sequence sites for glycosaminoglycan chains. Proteoglycans are molecules with long glycosaminoglycan chains attached to a core protein and can influence a variety of cellular and physiological activities. In addition, glycosaminoglycan chains were shown to be important in many protein-protein interactions. To determine whether Eys contributes to lumen formation through its core protein, or through its glycosaminoglycan chains a structure-function analysis is being performed. Also, potential Eys interactors are being characterized to further explore the mechanisms contributing to epithelial lumen formation.

217A Interactions between the FERM protein Yurt and septate junction proteins define a novel basolateral epithelial polarity pathway. Patrick Laprise1, Sarah Paul3, Kathryn Harris2, Greg Beitel3, Ulrich Tepass2. 1) CRC-HDQ, Laval University, Quebec, QC, Canada; 2) Department of Cell and Systems Biology, University of Toronto, Ontario, ON, Canada; 3) Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evaneston, IL, USA. The transmembrane protein Crumbs (Crb) is an essential regulator of epithelial polarization where it promotes apical membrane growth. We have recently shown that Crb recruits the FERM-domain protein Yurt (Yrt) to the apical membrane of epithelial cells. This interaction is involved in a local negative regulatory feedback loop in which Yrt antagonizes Crb activity to regulate apical membrane size. Here we show that Yrt also acts as a basolateral polarity protein. Yrt co-localizes and interacts functionally with components of the septate junction (SJ). Mutations in yrt and several genes encoding SJ components have a profound impact on tracheal tube morphogenesis, and cause an elongated dorsal tracheal trunk as a result of an increase in apical membrane size. yrt shows strong genetic interactions with mutations in genes encoding SJ proteins such as the α subunit of the Na,K ATPase (Atpα), suggesting that Yrt cooperates with SJ proteins in negatively regulating apical membrane formation. Moreover, Crb overactivation is responsible for tube elongation in the absence of Yrt or Atpα as reduction of Crb rescues tube size defects in both mutants. SJs appears normal in yrt mutants and Yrt and the Atpα regulate epithelial polarity prior to SJ formation. Together, our data suggest that Yrt and components of the SJs form a novel basolateral polarity pathway that acts during organogenesis to control apical morphogenesis of trachea and other epithelia.

218B Dynein-mediated apical localization of crumbs transcripts is required for effective Crb activity in epithelial polarity. Zhouhua Li1, Liwei Wang1, Thomas S Hays2, William Chia1,3, Yu Cai1,3. 1) Germ Cell Development Group, Temasek Life Sciences Laboratory,National University of Singapore, Singapore; 2) Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 3) Department of Biological Science, National University of Singapore, Singapore. Asymmetric localization of transcripts coupled with localized translation constitutes an important mechanism widely deployed to regulate gene activity in a spatial manner. The conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. However, it remains unclear how it is targeted to the apical domain. Here, using Drosophila follicular epithelia as a model, we show that the cytoplasmic dynein complex transports both Crb protein and transcripts to the apical domain of follicular cells (FCs). The crb 3’UTR is necessary and sufficient for the apical localization of its transcript and this apical transcript localization is crucial forcrbfunction. In crbmutant FCs, Crb protein derived from transgenes lacking the 3’UTR does not effectively localize to the apical domain and does not effectively restore normal epithelial polarity. We propose that dynein-mediated mRNA transport coupled with a localized translation mechanism is involved in localizing Crb to the apical domain to mediate epithelial apico-basal (A/B) polarity and this mechanism might be widely used to regulate cellular polarity. POSTERS: Cytoskeleton and Cellular Biology 163

219C muscleblind gene regulates microtubule organization and epithelial integrity in follicle cells. Gouthami Nallamothu, Tien Hsu. Dept Pathology & Lab Medicine, Medical Univ South Carolina, Charleston, SC. Our laboratory previously showed that the human homolog of Drosophila Muscleblind (Mbl) protein, Mlp1 is involved in RNA dependent protein localization of integrin α3 and actin (Adereth et al., 2005, Nat. Cell Biol.12:1240). More recent results indicate that Mlp1 regulates RNA transport by directly regulating the organization of microtubules. Drosophila mbl has been shown to regulate differentiation of photoreceptors and muscle cells. Based on its potential role in regulating microtubules, we examined whether mbl is also involved in epithelial morphogenesis. Drosophila ovary is a good model to study the epithelial integrity. We studied the effect of loss of mbl during Drosophila oogenesis and observed that loss of mbl results in abnormal cell division as seen by the cells with large nuclei, indicating defects in microtubule organization and cell division and an occasional pilling up of epithelium, suggestive of tumorigenesis. mbl mutant follicular epithelium showed loss of tubulin expression on the apical and lateral sides indicating changes in cytoskeletal organization. Over expression of Mbl-GFP in the anterior follicular epithelium showed most of the GFP expression is cytoplasmic and tubulin is more concentrated to the lateral sides indicating its role in establishment of polarity. Based on these experiments and our previous work we propose that mbl might regulate the cytoskeleton organization and epithelial polarity by regulating the integrin localization (during establishment of basement membrane) and through its association with actin and tubulin, establish the cytoskeleton to maintain the integrity of the follicular epithelium.

220A The role of the oocyte nucleus in grk mRNA localization during oogenesis. Amanda Norvell, Carolyn Gray, Joshua Schoenfeld, Jing-jing Feng. Dept Biol, Col of New Jersey, Ewing, NJ. The Drosophila oocyte is a highly polarized cell with multiple asymmetries. Several well-characterized features of the polarized oocyte are the microtubule network, the cytoplasmic position of the oocyte nucleus and the cytoplasmic distribution of specific mRNAs and proteins. One such asymmetrically distributed transcript is gurken (grk) mRNA. grk mRNA localization patterns during oogenesis are dynamic, but during the later stages it is found in the dorsal-anterior(D-V) corner of the oocyte in close association with the oocyte nucleus. Several nuclear factors required for the D-V localization of grk mRNA, including the RNA binding protein Squid (Sqd), fs(1)K10 and nuclear lamin, have been identified. Furthermore, there is ample evidence that grk mRNA localization and the position of the oocyte nucleus are also dependent on the microtubule network. However, the relationship between the oocyte nucleus, its position and structure, in the cytoplasmc localization of grk mRNA is less clear. To investigate these issues we have taken two approaches. Firstly, to assess the relationship between nuclear structure and the cytoplasmic localization of grk mRNA we are currently testing whether mutations in genes encoding nuclear structure proteins (lamin and BAF) interact with mutations in sqd and K10. Secondly, we have disrupted the microtubule network within the oocyte by treatment with colchicine. In late stage egg chambers from drug treated females, the oocyte nucleus is frequently mispositioned. In preliminary results, we have found that grk mRNA remains associated with the mispositioned nuclei, except in egg chambers from females mutant for sqd or K10. These initial findings suggest that the perinuclear localization of grk mRNA is independent of microtubule integrity, while it does require Sqd and K10 protein function. The molecular basis for their requirement is not yet known, however we are investigating whether these proteins interact with known components of the nuclear structure, such as Lamin or BAF.

221B Function of Drosophila α-catenin in adherens junction formation. Ritu Sarpal, Milena Pellikka, Ulrich Tepass. Cell and Systems Biology, University of Toronto, Toronto, ON., Canada. α-catenin is a critical component of the cadherin catenin complex and its loss compromises cadherin-based adhesion. However, its mechanism of action remains controversial. We have isolated excision alleles of Drosophila α-catenin to understand its role in cadherin dependent morphogenetic processes. α-catenin zygotic null mutants are embryonically lethal and show defects in head morphogenesis similar to weak DE-cadherin alleles. Preliminary clonal analysis during oogenesis revealed that α-catenin is required for follicular epithelium integrity. To uncover how α-catenin supports cadherin activity during development, we plan to further characterize the α-catenin mutant phenotype and to use fusion protein between DE-cadherin and α-catenin to rescue morphogenetic defects in α-catenin mutants. Two competing models suggest that α-catenin may either act as a physical linker between the cadherin and the actin cytoskeleton or, alternatively, may not be a physical linker but promote adherens junction formation by locally stabilizing the actin cytoskeleton. In order to address this issue, we have generated transgenic flies carrying DEcad::α-catenin chimeric proteins. A DEcad::α-catenin fusion of full length proteins rescues the embryonic phenotypes of α-catenin zygotic null mutants suggesting that the fusion protein can replace endogenous α-catenin. In contrast, a fusion protein that lacks the domain of α-catenin responsible for Armadillo binding and dimerization does not rescue the embryonic lethality of α-catenin mutants indicating that the α-catenin dimerization domain (VH1) is required for the fusion protein to interact with the actin cytoskeleton. We will extend this analysis to further investigate if these fusion proteins can substitute for endogenous α-catenin during morphogenesis. 164 POSTERS: Cytoskeleton and Cellular Biology

222C Awd, the Homologue of the Human Metastasis Suppressor Gene, Regulates Epithelial Integrity of Follicle Cells. Julie Woolworth1, Tien Hsu2. 1) Dept Molecular & Cellular Biol, Medical Unif South Carolina, Charleston, SC; 2) Dept Pathology & laboratory Med, Medical Unif South Carolina, Charleston, SC. One of the most life-threatening aspects of cancer is metastasis. The origin of metastasis is still under debate, but the emergence of potential metastasis suppressor genes has provided hope for understanding the origin of metastasis and the design of effective therapies. Nm23 was the first metastasis suppressor gene discovered and encodes a nucleoside diphosphate kinase that is potentially a GTP supplier. However, like other metastasis suppressor genes its precise function remains elusive. In addition, clinical data regarding Nm23 correlation with increased tumorigenesis is conflicting. In the same tumor type both up-regulation and downregulation of Nm23 has been shown to correlate with aggressive tumor progression and unfavorable outcome. During our studies in the follicular epithelium of Drosophila melanogaster, we have noted the function of awd, the Drosophila homologue of Nm23H1/H2, in establishing and maintaining epithelial integrity. First, in normal epithelium, Nm23/awd is critical for down-regulating membrane accumulation of adherens junction (AJ) components (β-catenin and α-spectrin). Second, the epithelial function of Nm23/awd depends on both its expression level and its subcellular localization. Third, both up-regulation and down-regulation can disrupt the epithelium contributing to different aspects of tumorigenesis. Lastly, we found that sequential combination of up-regulation of Nm23/awd followed by down-regulation leads to a more invasive/metastatic phenotype in the FCs. The novel Nm23/Awd function discovered here is the first report of the physiological role of the metastasis suppressor and can shed light on its role in tumorigenesis.

223A Local regulation of F-actin during the first step of Drosophila myoblast fusion. Christine Dottermusch, Gritt Schaefer, Renate Renkawitz-Pohl, Susanne-Filiz Oenel. Biology, Philipps-University, Marburg, Germany. The somatic body wall musculature of Drosophila melanogaster consists like the vertebrate skeletal musculature of multinucleated muscle fibers. In Drosophila, the process of somatic muscle formation is a dynamic relationship between two different myoblast types, founder cells (FCs) and fusion competent myoblasts (FCMs). FCs serve as seeds for distinct muscles and fuse with FCMs in a two step process. During the first fusion step, a tri-nucleated precursor cell is formed, which undergoes subsequent fusion events till a mature muscle fiber is formed. On molecular level both myoblast populations express cell type specific transmembrane molecules Duf/Kirre and Sns ensuring the recognition and adhesion between FCs and FCMs and the formation of a Fusion-Restricted Myogenic- Adhesive Structure (FuRMAS, Kesper et al., 2007). Here F-actin plugs are characteristic at the opposing membranes. After this initial step, the signal from the membrane becomes transferred into the cell. This finally results in the activation of the Arp2/3 complex, which produces branching F-actin at the site of cell-cell contact points. During our work we have gained evidence that the formation of branching F-actin is required for the integration of FCMs into a growing myoblast. Our work further suggests that the formation of F-actin is differently controlled during the first and the second fusion step. This indicates that different signaling cascades operate during both fusion steps to transduce the signal. We therefore aim to identify further fusion-relevant components and have started to investigate the first fusion step in more detail.

224B Control of nuclei positioning in contractile muscle cells. Hadas Elhanany, Talila Volk. Molecular Genetics, Weizmann, Rehovot, Israel. The position of nuclei in eukaryotic cells is crucial for many biological processes such as cell division, cell migration, fertilization and synaptic maturation. During somatic muscle development, myoblasts fuse to form multinucleated myotubes which then mature into large syncytial muscle fibers. The mechanism underlying nuclear positioning in syncytial muscle fibers as well as in contractile cardioblasts is yet to be elucidated. We followed the position of muscle nuclei in different stages of embryonic development. At stage 13, syncytial muscle nuclei were observed at the cell center while at stage 16 these nuclei are organized at the periphery of the muscle cells in a typical pattern. The KASH domain protein, MSP-300, has been implicated in nuclear anchoring in nurse cells during oogenesis. At stage 13-14 of embryonic development MSP-300 appeared in a pattern of scattered dots in the muscle cytoplasm. However, at stage 16 it is observed in a fiber like structures and at the muscle tendon junction. The change in MSP-300 distribution parallels with nuclear reorganization. Elimination of MSP-300 in homozygous mutants was accompanied by nuclear mislocalization in both somatic and the heart cardioblasts. Furthermore, Klaroid mutant embryo (deficient of the SUN domain protein Klaroid) exhibited an even more pronounced nuclear mislocalization phenotype. Interestingly, MSP-300 displays three putative isoforms whose expression appears to be developmentally regulated. In a search for a mechanism controlling the developmental appearance of MSP-300 isoforms, we found that how mutant embryos displayed a major change in the expression profile of MSP- 300 splice variants. Moreover, in these embryos nuclear organization within muscles was abnormal and aberrant subcellular distribution of MSP-300 in the heart and somatic muscles was observed. Theses results suggest the essential contribution of MSP-300 and Klaroid for nuclear positioning within muscles and provide a mechanism in which a developmentally regulated splicing of MSP-300 promoted by the RNA binding protein HOW, controls this process. POSTERS: Cytoskeleton and Cellular Biology 165

225C Rap1’s role in Drosophila morphogenesis. Nathan Harris, Jessica Sawyer, Mark Peifer. Biology, UNC - Chapel Hill, Chapel Hill, NC. Adherens junctions (AJ) help coordinate proper cell-cell interactions during animal development. Specifically, these junctions mediate cadherin-based adhesion and help organize the cortical actin cytoskeleton within cells. While cadherins and catenins are accepted as core components at these junctions, other proteins are recruited to AJs. Investigation of Canoe (Cno), the Drosophila homolog of mammalian Afadin/AF-6, indicates roles in morphogenesis. Cno has a number of binding partners, among which is the small GTPase Rap1. Mammalian Rap1 has been proposed to regulate integrin adhesion, while Drosophila Rap1 is suggested to regulate cadherin localization in imaginal discs. In Xenopus and zebrafish, Rap1 has also been shown to have functions during gastrulation. I am using Drosophila as a model to understand how Rap1 regulates cell adhesion and the actin cytoskeleton during morphogenesis. When I inactivated maternal and zygotic Rap1, I observed embryos that have twisted gastrulation and a failure to invaginate their mesoderm properly, indicating an important role for Rap1 in apical cell constriction. How Rap1 functions to regulate adhesion and the cytoskeleton during gastrulation and other events during development is not well understood. Currently, I’m continuing to investigate Rap1’s roles by further analyzing maternal/zygotic mutants, examining Rap1’s localization throughout embryogenesis, and looking for possible genetic interactions with other proteins that localize to the AJ or are predicted to bind Rap1 based on work in other organisms. This work has indicated relationships between Rap1 and E-Cadherin, the fog/cta pathway, and Cno.

226A Roles of Spectrins in Drosophila photoreceptor morphogenesis. Sang-Chul Nam1, Tony Chen1, Kwang-Wook Choi2. 1) Department of Biology, Baylor University, Waco, TX; 2) Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Spectrins are major proteins in the cytoskeletal network of most cells. Drosophila has three spectrins: α-Spectrin, β-Spectrin and βHeavy-spectrin (Karst). βHeavy-spectrin is known to play a role in photoreceptor morphogenesis by forming a protein complex with Crumbs. Here we analyzed the effects of α- and β-spectrin mutations on developing eye to determine their roles in photoreceptor morphogenesis. We found that α-Spectrin is not essential for retinal differentiation in eye imaginal discs during larval stage. However, α-spectrin mutant photoreceptors display mislocalization of Crumbs during pupal eye development, suggesting that α-Spectrin is required for Crb localization during photoreceptor morphogenesis. The eye phenotype of β-spectrin mutant is similar to that of α- spectrin mutant. α-Spectrin is undetectable in β-spectrin mutant photoreceptors whereas the β-Spectrin localization is not dependent on α-Spectrin. This result indicates that β-Spectrin plays a primary role for α-Spectrin localization. Eye phenotype of βHeavy- spectrin mutant was relatively weaker than that of α- or β-spectrin mutant.

227B Enabled and Capping Protein Regulation of the Actin Cytoskeleton in Drosophila Development. Stephanie Nowotarski1, Julie Gates2, Mark Peifer1. 1) Biology, University of North Carolina, Chapel Hill, NC; 2) Biology, Bucknell University, 701 Moore Avenue, Lewisburg, PA 17837. Rearrangement of the actin cytoskeleton mediates changes in cell shape, migration and subsequently cell behavior during morphogenesis, wound healing and homeostasis of adult tissue. This regulation is facilitated by an array of proteins that function in actin nucleation, polymerization, capping, bundling and severing. The decision to continue filament elongation or to terminate is a critical decision in the context of the cell. Capping protein (CP) binds to the actin barbed end to terminate elongation, while the Enabled (Ena)/VASP family proteins function antagonistically, acting at barbed ends to promote continued elongation. We previously analyzed the role of Ena during embryogenesis revealing that Ena plays a role in subset of morphogenetic processes: germ band retraction, segment groove formation, head involution, and dorsal closure. Work in mammalian cell culture has provided support for an antagonistic relationship between CP and Ena/VASP. We are currently extending this analysis, teasing apart the antagonistic relationship between CP and Ena by studying its affect on dynamic actin during oogenesis. We are also starting to explore how Ena’s multi-domain structure affects its function. Studies of motility of cultured cells of the intracellular bacterium Listeria revealed that the EVH2 domain is sufficient for motility and anticapping function. The EVH1 domain is required for localization to sites of active actin turnover, while a proline rich domain is known to bind profilin. This data suggests that different Ena domains may be responsible for different morphogenetic processes in Drosophila. We are currently testing this hypothesis. 166 POSTERS: Cytoskeleton and Cellular Biology

228C The function of the PDZ-GEF Dizzy in ventral furrow formation. Alice Ott1, Martina Rembold2, Sam J. Mathews2, Maria Leptin2, Rolf Reuter1. 1) Division of Animal Genetics, University of Tübingen, Tübingen, B-Württemberg, Germany; 2) Inst. for Genetics, Developmental Genetics Unit, University of Cologne, Cologne, Germany. Gastrulation is the first morphogenetic movement of the Drosophila embryo, through which the monolayered embryo folds into a multilayered one. In previous studies we identified the PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy (Dzy), which acts via the small GTPase Rap1, as an essential component for proper cell shape and motility in embryonic macrophages. Since other GEFs have pleiotropic functions in development, we wanted to investigate the early function of dzy in the embryo. For this purpose we generated germline clones by means of the FLP-FRT system. Gastrulation is affected in dzy germline clones, independent of the zygotic contribution of a mutant copy of dzy (dzymz) or a dzy+ (dzym) from the father. During ventral furrow formation, the initial apical constriction of the ventralmost cells does occur in dzy germline clones, but the furrow is formed too slowly and partially not deep enough. The concerted fast cell shape changes of the ventralmost cells might be affected, such that a proper invagination of the ventral furrow is not achieved. In later development most of the dzym embryos can compensate the early abnormalities and hatch, while dzymz embryos are strictly associated with embryonic lethality. In cuticle preparations anterior, ventral and dorsal scabs and holes are visible. We are currently characterizing the cellular processes that are affected in embryos lacking dzy function, in order to understand its role in ventral furrow formation.

229A Second-site noncomplementation screen to identify genes that interact with Rho1. Kistie Patch, Shannon Stewart, Aaron Welch, Robert Ward. Dept Molecular Biosciences, University of Kansas, Lawrence, KS. The Rho pathway is highly conserved among animals and is responsible for a wide range of morphogenetic events. For example, regulation of this pathway is critical for proper gastrulation, neurulation, and heart formation in vertebrates. Although aspects of the Rho pathway have been well characterized, very little is known about how its activity is regulated. We have investigated the regulation of Rho signaling during morphogenesis using leg imaginal disk development. In Drosophila, certain heterozygous Rho1 mutants show a low penetrance malformed leg phenotype. We are conducting a second-site noncomplementation screen with Rho1E(br)246 to identify novel genes that interact with Rho1 during imaginal disc morphogenesis. For the primary screen we tested over 500 Exelixis deficiency stocks that collectively uncovered ~50% of the euchromatic genome. After the primary screen, 18 intervals were identified as containing putative Rho1-interacting genes. Ongoing secondary screening using additional Rho1 alleles and overlapping Bloomington deficiency stocks is 75% complete. At this point we have good evidence that four intervals contain novel Rho1-interacting genes, and we are still investigating seven additional intervals. Finally we will attempt to elucidate the single genes responsible for these interactions.

230B Microtubule-driven transformations in ER morphology during oogenesis. Nancy Pokrywka1, Anna Payne-Tobin1, Tulle Hazelrigg2. 1) Dept Biology, Vassar College, Poughkeepsie, NY; 2) Dept Biol Sci, Columbia Univ, New York, NY. The egg chamber is a rare example of a syncytium that contains two dramatically different cell types: the nurse cells and the oocyte. Although many proteins and organelles are found in both nurse cells and the oocyte, their functions are likely to vary in a context-specific manner. As a means of testing this model, we chose to monitor the ER, due to its role in intracellular (and in this case, intercellular) trafficking. Three GFP-tagged ER markers (PDI, Sec61? and Rtnl1) were used to monitor ER morphology in both cell types during mid-oogenesis. Time lapse imaging of living egg chambers combined with observation of fixed material suggests that the ER has a highly dynamic but characteristic distribution in nurse cells, and is continuous across the nurse cell/oocyte boundary. However, upon entry into the oocyte, the ER undergoes substantial spatial reorganization. Differences in the behavior of each GFP-tagged marker suggest an additional level of organization within the ER. Since ER organization is microtubule-dependent in other systems, we also investigated the effects of alterations in microtubule dynamics on the structure of the ER in nurse cells and the oocyte. Mutants of msps (an anti-pause factor) were used to dampen microtubule dynamics and were compared with drug- induced microtubule inhibition. Both the msps mutation and treatment with colchicine resulted in an alteration in ER in egg chambers. We also find that microtubule associations mediate the movement of ER into the oocyte where contact with the ooplasm results in rearrangements of both the microtubules and ER. Based on this observation, one mechanism of transport into the oocyte may be via association with the ER. We tested this by analyzing the transport of Exu protein, an RNP component required for the transport of bcd RNA into the oocyte. We find that Exu is associated with a sub-compartment of the ER in nurse cells and that this association is also lost upon entry into the ooplasm. Our data point to the existence of oocyte-specific factors that remodel both microtubules and ER during mid-oogenesis. POSTERS: Cytoskeleton and Cellular Biology 167

231C The roles of Abelson Kinase in cytoskeletal regulation during embryonic morphogenesis in Drosophila. Edward M. Rogers1, Donald T. Fox2, Mark Peifer1. 1) Biology Department, UNC-Chapel Hill, Chapel Hill, NC; 2) Carnegie Institute, Baltimore, MD. Chromosomal translocations that fuse the BCR and Abelson Kinase (abl ) genes trigger most cases of Chronic Myelogenous Leukemia (CML) and play roles in other leukemias. Both normal Abl and Bcr-Abl regulate cytoskeletal dynamics. To fully understand Bcr-Abl’s oncogenic properties it is necessary to understand Abl’s normal functions. I hypothesize that Abl coordinates cellular signals with cytoskeletal rearrangements during morphogenesis. Our previous work and that of other labs revealed that Abl regulates many processes during embryonic development, including cellularization, coordinated apical constriction, germband retraction, dorsal closure and axon pathfinding. We hypothesize that Abl influences these events both via its kinase activity and also by interacting with the actin cytoskeleton, cytoskeletal regulators like Enabled, and potentially the microtubule cytoskeleton. To test these hypotheses, we are studying Abl function in Drosophila embryos, in which powerful genetic and cell biological tools can be used for studying regulation of cytoskeletal dynamics in the whole animal. To complement these studies, I am also doing parallel experiments in S2 cells and other Drosophila derived cell lines allowing me to correlate morphological phenotypes in individual cells to phenotypes in the whole animal due to the coordinated movements and shape changes of many cells. To distinguish between functions of Abl that depend on it’s kinase activity and/or it’s direct association with the cytoskeleton I have created, by site directed mutagenesis, a series of GFP-tagged Abl transgenes with point mutations or deletions in protein domains that putatively regulate Abl’s kinase activity, cytoskeletal interactions and protein-protein interactions. Preliminary data suggests that Abl’s direct interaction with the actin cytoskeleton may be important for it’s function during normal development.

232A WASP and SCAR/WAVE play distinct roles in activating the Arp2/3 complex during Drosophila myoblast fusion. Gritt Schäfer1, Susanne Berger1, Anne Holz2, Lothar Beck1, Renate Renkawitz-Pohl1, Susanne-Filiz Önel1. 1) Dept. for Biology, Philipps-University Marburg, Germany; 2) Institute for Allgemeine und Spezielle Zoologie, Justus-Liebig-University Giessen, Germany. Cell-cell fusion is essential for somatic muscle formation in Drosophila and mammals. The larval body wall musculature of Drosophila arises from two populations of myoblasts - founder cells (FCs) and fusion-competent myoblasts (FCMs). The FCs control the identity of the muscles and fuse with the greater population of FCMs in a two-step process. During the first fusion step a trinucleated precursor cell is formed, which recruits additional FCMs in a second fusion step, giving rise to a mature muscle fiber. The initial recognition and adhesion between FCs and FCMs is mediated through the Ig domain containing transmembrane molecules Duf/ Kirre and SNS. At cell-cell contact points both molecules form a ring like adhesion structure possessing an actin-rich core in its center. We have termed this structure Fusion-Restricted Myogenic-Adhesive Structure (FuRMAS; Kesper et al., 2007). The formation of new actin filaments is regulated by the actin-related protein (Arp)2/3 complex, which consists of seven subunits including Arp2 and Arp3. The Arp2/3 complex produces branched filaments and becomes activated by so-called nucleation promoting factors such as WASP and SCAR/WAVE. Here we present the identification of a new wasp and Arp3 allele. Both mutants show severe defects in myoblast fusion. Electron microscopy analyses suggest that WASP is required for the formation of a fusion pore between a FC/ growing myotube and FCMs. However, in the absence of Arp3, the FCMs fail to integrate into the growing myotube suggesting that the FCM is pulled into the growing myotube by the force of actin. To further investigate the role of actin regulation during myoblast fusion we have generated double mutants with wasp and Arp3 and scar/wave and verprolin 1. Interestingly, these experiments indicate that WASP and SCAR/WAVE control different fusion steps during myogenesis. Based on these findings we propose a new model of actin regulation during the first and the second fusion step.

233B Scar Coordinates with Solitary to Regulate Actin Cytoskeletal Dynamics During Myoblast Fusion. Kristin Sens, Elizabeth Chen. MBG, Johns Hopkins Sch Medicine, Baltimore, MD. Previous studies of myoblast fusion in Drosophila have demonstrated that myoblast fusion requires signal transduction from transmembrane receptors to the actin cytoskeleton. In wild-type embryos, interactions between cell-specific fusion receptors results in formation of actin enriched foci at sites of fusion in both founder cells and fusion competent cells. We have previously demonstrated that Solitary/dWIP is required for actin foci formation in fusion competent cells. It is not known, however, what regulates the formation of actin foci in founder cells. Here we show that Scar, the Drosophila homologue of WAVE, is required for myoblast fusion. Loss of function alleles of scar exhibit a lack of fusion phenotype as well as a persistence of actin foci to late stages of embryonic development. Interestingly, unlike solitary mutant embryos, these enlarged actin foci appear to be primarily accumulated in fusion competent cells in scar mutant embryos. Moreover, elimination of both solitary and scar results in a dramatic reduction in actin accumulation in founder and fusion competent cells, supporting the hypothesis that Scar is required for the accumulation of actin at the site of fusion in founder cells. Although both are positive regulators of actin polymerization, Solitary and Scar are not functionally interchangeable, suggesting distinct signaling cascades target these proteins. These data suggests that Scar coordinates with Sltr to regulate actin cytoskeletal dynamics in distinct populations of myoblasts during the fusion process. 168 POSTERS: Cytoskeleton and Cellular Biology

234C Rho-Kinase is required for epithelial morphogenesis in the Drosophila embryo. Robert Simone, Stephen DiNardo. Dept Cell & Developmental Biol, Univ Pennsylvania, Philadelphia, PA. Proper control and direction of epithelial morphogenesis is vital to the reproducible generation of functional tissues. Because it is both genetically manipulable and easily visualized, the epidermis of the Drosophila embryo is an excellent model to study tissue morphogenesis. Initially, the cells of the ventral epidermis are packed into an array of roughly equilateral polygons. Over the course of several hours, they undergo reproducible changes in shape and orientation and, specifically, they align their anterior and posterior edges into parallel rows. Components of actomyosin contractility become enriched in the aligning epidermal domains as alignment proceeds. We hypothesized that actomyosin contractility may be required for proper cell alignment. Here we also show that this alignment process is dependant on Myosin Heavy Chain/Zipper (MHC/Zip), as it is disrupted in MHC/Zip mutants. Previous studies in the Drosophila embryo have implicated Rho-kinase (Rok) in the activation of morphogenetic actomyosin contractility. We implicate Rok in normal cell alignment because cells fail to align when Rok function is inhibited. We suggest Myosin Regulatory Light Chain/ Spaghetti Squash (MRLC/Sqh) as the relevant Rok substrate in alignment because expression of a phosphomimetic allele of MRLC/Sqh suppresses the alignment defect seen after Rok inhibition. Overall, we show that actomyosin contractility — mediated by Rok, through MRLC/Sqh and MHC/Zip — is required for normal cell alignment in the Drosophila ventral embryonic epidermis.

235A Characterization of phosphatidylinositol 4-kinase IIIα during Drosophila development. Julie Tan1,2, David Hipfner3, Julie Brill1,2. 1) Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada; 2) Dept of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 3) Dept of Epithelial Cell Biology, Institut de Recherches Cliniques de Montreal, Montreal, QC, Canada. PI4Ks modify phosphatidylinositol (PI) to produce PI 4-phosphate (PI4P), one of seven PI lipids that reside within cellular membranes.

PI4P and its phosphorylated derivatives PI(4,5)P2 and PI(3,4,5)P3 act as address tags to recruit and activate proteins involved in signaling, cytoskeletal organization and cell proliferation. PI4KIIIα/Stt4p, one of two essential PI4Ks in budding yeast, is required for actin organization, vacuole morphology and cell wall integrity. To examine the role of PI4KIIIα in the development of a multicellular organism, I generated a deletion in the Drosophila melanogaster PI4KIIIα. This deletion is lethal, demonstrating that PI4KIIIα is essential, and lethality is rescued by the introduction of a PI4KIIIα transgene. Oogenesis is disrupted in PI4KIIIα female germline clones, with mutant egg chambers displaying aberrant actin organization and nurse cell nuclei in the ooplasm. To examine roles in other tissues, I expressed a PI4KIIIα RNAi construct in the eye using the ey-Gal4 driver. PI4KIIIα knock-down eyes have smaller eye fields, and ommatidia that are tetragonal rather than hexagonal. These unusal phenotypes implicate PI4KIIIα in actin organization, and in maintaining cell morphology in both germline cysts and the developing eye.

236B Cloning and characterization of uninflatable, a gene required for tracheal inflation. Liang Zhang, Aaron Olson, Robert Ward. Dept Molecular Biosciences, Univ Kansas, Lawrence, KS. Our lab is interested in understanding the mechanisms of hormone dependent morphogenesis and we have been using leg eversion during metamorphosis as a model. In a screen for dominant modifiers of the malformed leg phenotype associated with br1, we identified mutations in at least 15 different genes. One of these mutations, originally identified as Enhancer of broad 155 (E(br)155), shows complete embryonic lethality, with poorly-differentiated cuticle and defects in dorsal closure and head involution. P element based meiotic mapping indicated that the original mutation contained two closely linked mutations, one in 26 and the other in 27C- D. After recombining the mutations apart we discovered that both mutations are required for the embryonic phenotypes and for the interactions with br1Genomic sequencing indicated that the mutation in 27C-D is in the gene CG9138. CG9138 is predicted to encode a single transmembrane protein consisting of 3557 amino acids. The extracellular domain of the protein contains multiple EGF-like, CCP, CUB, HYR, CLECT, FA58C, Lamini-G, and GCC2-GCC3 domains. The intracellular domain of the protein contains ~118 amino acids without conserved domains. This protein is conserved in insect species but there apparently is no close homologue in vertebrates. Animals heterozygous for two different mutant alleles of CG9138 are primarily early larval lethal and have partially inflated tracheae, thus we named the gene uninflatable (uif). In situ hybridization indicated that uif is expressed in ectodermally derived epithelial cells beginning at stage 5 of embryogenesis and is strongly expressed in the tracheal system. Antibodies against the intracellular domain and extracellular domain of UIF are being raised and more detailed characterization of uif will be presented. POSTERS: Cytoskeleton and Cellular Biology 169

237C Investigating the mechanisms of the cortical localization of APC2. Meng-Ning Zhou, Andrea Blitzer, Brooke McCartney. Dept Biological Sci, Carnegie Mellon Univ, Pittsburgh, PA. Adenomatous Polyposis Coli (APC) proteins function to negatively regulate Wnt signal transduction and to organize the microtubule and actin cytoskeletons. APC proteins reside in various locations within the cell including microtubule plus ends, the cell cortex and the nucleus. These pools of APC likely carry out different cellular functions. The molecular mechanisms which affect the cortical localization of APC proteins are not well understood. We have shown that Drosophila APC2 localizes to the cell cortex in Drosophila embryos as well as in S2 cells, and to microtubule plus ends in some cellular contexts. Furthermore, we have demonstrated in embryos and in S2 cells that both the N-terminal and the C-terminal domains of APC2 are required for its cortical localization. To understand what protein partners are important for the cortical localization of APC2, we are using RNAi to knock down candidates in S2 cells. In addition, we are examining the dynamic behavior of APC2 with these candidates relative to cortical actin in live embryos.

238A The endosomal SM protein dVps45 is necessary for cell signaling and proliferation. Mohammed Akbar, Sanchali Ray, Helmut Kramer. Dept of Neuroscience , Univ Texas SW Medical Ctr, Dallas, TX. During endocytic trafficking, the fusion of vesicles with target membranes is a specific and tightly regulated process. One contribution to the specificity originates from the interactions of the HOPS complex, a multi-subunit tether factor, with the Rab GTPases and SNARE proteins. The HOPS subunit Carnation is one of three members of the Sec1/Munc18 (SM) protein family that act in the endocytic pathway. We found that another SM protein, dVps45, also associates with HOPS components to form a distinct HOPS-like complex. Loss of dVps45 function is lethal. Analysis of flies mosaic for dVps45 cells points to a function of this complex in early endosomes. Loss of dVps45 function causes endosomal accumulation of signaling molecules such as Notch or Delta. Unlike carnation cells with similar appearing accumulations, dVps45 cells exhibit changes in cell fate and proliferation. We are focusing on understanding the mechanism by which changes in early endocytic trafficking modulate cell fate and control cell divisions.

239B Trafficking of the Drosophila vesicular monoamine transporter is required for a subset of amine-dependent behaviors. Anna Grygoruk, David E. Krantz. Psychiatry & Biobehavioral Sciences, School of Medicine, Brain Research Institute, UCLA, Los Angeles, CA. Membrane trafficking regulates the function of neurotransmitter transporters in vitro, but its effects on synaptic transmission and behavior remain unclear. The Drosophila vesicular monoamine transporter (DVMAT) is required for the exocytotic release of serotonin, dopamine and octopamine, and endocytosis at the plasma membrane is thought to be required for its localization to synaptic vesicles. We have previously shown that deletion of C-terminus of DVMAT dramatically reduces endocytosis in vitro, and we now show that a mutation of a tyrosine-based motif in the C-terminus replicates this effect. To explore the importance of neurotransmitter transporter trafficking in vivo, we have generated a series of transgenic flies expressing mutations in the DVMAT C-terminus. Using both wild type and DVMAT trafficking mutants to rescue a mutation in the endogenous dVMAT gene, we show that the C-terminus is required for the localization of DVMAT to synaptic vesicles in vivo. To test the potential affects of altered DVMAT trafficking on amine- dependent behaviors, we compared the ability of wild type and trafficking mutant transgenes to rescue a mutation in the endogenous dVMAT gene. Interestingly, the DVMAT- C-terminal deletion mutants show a specific defect in the rescue of female sterility, a process thought to involve both dopamine and octopamine. These data indicate that subsets of aminergic circuits are preferentially sensitive to changes in monoamine release and transporter trafficking. Further experiments will determine the identity of the relevant circuits, and more precisely determine the role of specific trafficking motifs contained within the DVMAT C-terminus. 170 POSTERS: Cytoskeleton and Cellular Biology

240C Rabenosyn and Vps45 regulate cell polarity and early endosomal entry. Holly A. Morrison1, Heather Dionne1, Tor Erik Rusten2, Andreas Brech2, Bill Fisher3, Barret Pfeiffer3, Susan Celniker3, Harald Stenmark2, David Bilder1. 1) Dept of Molecular & Cell Biology, University of California, Berkeley, CA; 2) Centre for Cancer Biomedicine, University of Oslo, and Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway; 3) Genome Sciences Department, Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, CA. Targeted vesicular trafficking steps are vital for maintaining the proper segregation of intracellular compartments and their protein cargoes. Here we have identified and characterized two important regulators of one of these trafficking steps, Rabenosyn (Rbsn) and Vps45. Rbsn and Vps45 were isolated in a genetic screen for mutations affecting the eye imaginal disc. Cells lacking either of these gene products lose apicobasal polarity and overproliferate, eventually killing the animal. Live trafficking assays demonstrate that endocytosis is defective in these mutant discs; rbsn and Vps45 therefore represent novel endocytic neoplastic tumor suppressor genes. Rbsn localizes to early endosomes and binds to the activated small GTPase Rab5, while Vps45 binds to Rbsn. Vps45 genetically interacts with the syntaxin Avl, suggesting that the target of this protein in Drosophila is a trans-SNARE complex containing Avl. Moreover, TEM analysis indicates that rbsn and vps45 mutant cells have endocytic defects which strongly resemble those seen in avl or rab5 mutants. Our data support a model in which Rbsn localizes to the early endosome via binding to Rab5, recruiting Vps45; Vps45 can then promote vesicle fusion into the early endosome through an interaction with an Avl-containing trans-SNARE complex. This work therefore suggests that Rbsn and Vps45 are part of a tethering complex on early endosomes that links activation of the Rab GTPase to the SNARE fusion machinery.

241A Phosphatidylinositol 4-kinase and Sac1 regulate trafficking to lysosome-related organelles in Drosophila. Jason Burgess1,2, Ho-Chun Wei1, Helmut Kramer3, Julie Brill1,2. 1) Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Rm 13-401L, 101 College St, TMDT East Tower, Toronto, ON, Canada; 2) Department of Molecular Genetics, University of Toronto; 3) Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA. The lipid phosphatidylinositol 4-phosphate (PI4P) is synthesized by PI 4-kinases (PI4Ks) and dephosphorylated by Sac1, a PI4P phosphatase. PI4P plays a crucial role in post-Golgi trafficking through the secretory pathway, as well as trafficking of hydrolytic enzymes to late endosomes/lysosomes. PI4P acts by recruiting specific complexes required for protein sorting and vesicle biogenesis. Consistent with a role in trafficking to the lysosome, Drosophila type II PI4K (PI4KII) partially co-localizes with the lysosomal marker GFP-LAMP. To further investigate the function of PI4KII, we used P element mutagenesis to generate deletions that remove the entire CG2929 (PI4KII) gene. Immunoblotting with an antibody specific for PI4KII indicates these deletions are protein null. PI4KII mutants are viable and male sterile. In flies bearing a copy of a mini-white transgene in a w1118 background, PI4KII homozygous mutants exhibit lighter eye color than heterozygous controls, suggesting defective trafficking to lysosome-related pigment granules. Indeed, PI4KII mutants exhibit synthetic genetic interactions with the pigment granule mutant dor1. PI4KII dor1 double mutants exhibit lighter eye color than dor1 at 21oC, and are pupal lethal at 25oC, supporting a more generalized role for PI4KII in trafficking to late endocytic compartments. Remarkably, a hypomorphic allele of the PI4P phosphatase sac1 also results in an eye pigmentation defect, which can be suppressed by null alleles of PI4KII. This strongly suggests that PI4P levels must be precisely regulated to ensure proper trafficking to lysosome-related organelles such as the pigment granule.

242B A unified biophysical model for the dynamics of antero-posterior and terminal systems: diffusion and nucleocytoplasmic shuttling in the syncytium. Matthieu Coppey1,2, Alistair N. Boettiger3, Yoosik Kim1,2, Alexander M. Berezhkovskii4, Stanislav Y. Shvartsman1,2. 1) Carl Ichan Lab, Princeton Univ, Princeton, NJ; 2) Department of Chemical Engineering,Princeton Univ, Princeton, NJ; 3) Department of Molecular and Cell Biology, University of California, Berkeley, USA; 4) Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, USA. We developed an assay to quantify the terminal patterning system in the embryo. The patterning of the terminal regions of the embryo relies on the spatially restricted activation of MAPK signaling. Using image processing routines and statistical methods, the graded activation of the MAPK cascade has been shown to exhibit a dynamical evolution during the transient time course of torso activation. We explain these dynamics using a simple biophysical model, which is based the nucleocytoplasmic shuttling of activated MAPK. We then show that changing the chemistry of our model allows us to reproduce most of the recent experimental data on the Bicoid gradient. We argue that the dynamics of maternal gradients in pre-cellular embryo can be described by a unique simple biophysical framework based on nucleocytoplasmic shuttling, which links the establishment of the gradients with the dynamics of the syncytial nuclear density. POSTERS: Cytoskeleton and Cellular Biology 171

243C In vivo measurement of kinesin-1 and dynein stalling forces: how does motor number affect transport? Steven P Gross1, George Shubeita1, Susan Tran2, Jing Xu1, Michael Welte2. 1) Dev. and Cell Biology, UC Irvine, Irvine, CA; 2) Department of Biology, University of Rochester, Rochester, NY 14627. Motor-driven transport is essential for cellular processes, but little is known about its regulation. In-vitro studies indicate that cargo motion changes dramatically when the number of motors per cargo is altered. In vivo, however, it is not established how many motors move cargos, how this is determined, and whether motor number modulates transport. In Drosophila embryos, lipid-droplets move bidirectionally along microtubules, switching frequently between plus- and minus-end motion. Dynein is the minus-end motor; using biochemistry, genetics, and antibody inhibition, we show that Kinesin-1 is the plus-end motor. To determine the number of motors moving droplets, we developed an optical trap capable of measuring stalling forces on individual droplets in vivo. The stalling-force distributions allowed us to determine how many motors move each droplet. Reducing kinesin (khc) dosage resulted in the expected two-fold decrease in the embryonic pool of Khc protein (as determined by Western analysis) as well as a roughly 50% decrease in droplet-bound active motors (determined by stall measurements). These observations show that the number of active motors per cargo depends on bulk availability of Khc. Surprisingly, numbers of active dyneins per cargo decreased also, indicating that opposing motors are either attached to the cargo together or are activated in a coordinated manner. How do these changes in the number of engaged motors affect cargo motion in vivo? Using nanometer-level particle tracking and analysis, we quantified droplet motion in the two genotypes and found that the factor of two decrease in the engaged plus-end motors resulted in a surprisingly modest change in transport, suggesting that motor number alteration may not be a primary pathway to regulate transport. Our measurements reveal an advantage of bi-directional transport: by linking the function of opposite motors, variations in protein levels cancel and structural robustness is achieved.

244A Vegetable, a GPI mannosyltransferase II, is required by rhodopsin and for photoreceptor viability. Suraiya Haroon, Erica E. Rosenbaum, Katie L. Caillouette, Nansi Jo Colley. Dept. of Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin, Madison, WI. GPI mannosyltransferase II (PIG-V) is a family of resident endoplasmic reticulum (ER) membrane proteins that are involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors. The GPI anchor plays an essential role in attaching a diverse array of proteins to the membrane. GPI anchors are acquired by proteins in the ER during their biosynthesis and not only function to tether proteins to the membrane, but also direct the protein to its correct location in the cell. The Drosophila vegetable (veg) gene encodes a GPI mannosyltransferase II that displays 32% identity with human GPI mannosyltransferase II. It has been previously shown that severe mutations in veg result in defects in the development of the peripheral nervous system and are lethal. We have identified a homozygous viable allele of veg that harbors a point mutation leading to an amino acid substitution from an alanine to a valine in a highly conserved region. We have identified a second allele that contains a transposable P-element insertion in the coding sequence and is homozygous lethal. Flies with the P-element carried over the point mutation are viable and were used in our studies. We have shown that rhodopsin (Rh1) is reduced in both veg mutants. Mutations in veg also lead to retinal degeneration and result in abnormal electroretinograms. Our findings demonstrate that a Drosophila GPI mannosyltransferase II is required in the eye for Rh1 biosynthesis, photoreceptor cell function and photoreceptor cell survival.

245B Identification and characterization of Drosophila UNC-76 binding proteins. Rebecca Josowitz, Jordan Cox, Monica Zapata, Joseph Gindhart. Department of Biology, University of Richmond, Richmond, VA. The microtubule motor kinesin-1 is essential for anterograde transport of intracellular cargos. Kinesin contains a motile head domain, a stalk domain, and a tail domain responsible for interacting with cargo and regulator molecules. UNC-76 was identified in a yeast two-hybrid screen as a binding partner of the kinesin tail domain. UNC-76 is a neuronal protein that is the homolog of C. elegans Unc-76 and mammalian FEZ1. UNC-76 mutants display defects in axonal transport similar to those observed in larvae lacking the kinesin heavy chain or the kinesin light chain, suggesting that UNC-76 and kinesin work together in vivo. Whether UNC- 76 regulates kinesin-cargo interactions or is involved directly in the binding of kinesin to cargos is not well understood. To test these models of UNC-76 function, we are using a split-ubiquitin DUALmembrane yeast two-hybrid assay to screen a Drosophila embryonic cDNA library for novel binding partners of UNC-76. We are now using molecular and biochemical approaches to characterize the observed interactions. By identifying the proteins that bind UNC-76 in vivo, we will gain further insights into the role of UNC-76 in kinesin-1 transport pathways. 172 POSTERS: Cytoskeleton and Cellular Biology

246C The dynamics of nanos mRNA incorporation into nascent pole cells. Dorothy A. Lerit, Timothy T. Weil, Elizabeth R. Gavis. Department of Molecular Biology, Princeton University, Princeton, NJ. At the posterior of the Drosophila embryo, a specialized cytoplasm is required for axial patterning and germ cell specification. This pole plasm is selectively incorporated within the germline precursor cells, or pole cells, upon their formation. nanos (nos) mRNA is localized to the pole plasm where its translation is required for abdomen formation. The subsequent inclusion of nos within the pole cells is required to maintain transcriptional and mitotic quiescence, ensure their proper migration through the body anlarge, and suppress apoptosis. It is currently unknown how nos localizes within pole cells as they bud out from the embryo. Preliminary in vivo imaging of fluorescently tagged nos mRNA suggests nos transitions from a stably anchored state to an active transport pathway, resulting in translocation into the nascent pole cell. Further studies of nos particle movement will clarify the mechanism of this transport and provide insight into germ cell formation.

247A A role for the RNA-binding protein Lark in oskar RNA localization to the posterior pole during oogenesis. Gerard McNeil, Sheryl Purrier, Manpreet Kaur, Ruth Kang. Department of Biology, York College/CUNY, Jamaica, NY. Pattern formation during early Drosophila development is governed by maternally-inherited genetic factors, which determine the organization and polarity of the two major embryonic axes. This process begins prior to fertilization during oogenesis and the developmental functions of many of some of these maternally-expressed genes have been studied in detail. Here, consideration is given to an essential, maternally-acting gene called lark, which encodes an RNA-binding protein essential during oogenesis. Elimination of the lark+ maternal component results in female sterility due to defects in cytoplasmic transport during oogenesis resulting from disruption of the actin cytoskeleton. This phenotype is also observed in transgenic flies expressing Lark containing mutations in the RNA-binding domains. We present recent results that show Lark expression during oogenesis is required for the stability of oskar mRNA localization to the posterior pole during late oogenesis.

248B Sec23-IP is required for rhodopsin transport, lipid metabolism and photoreceptor viability. Anne C. Muller, Erica E. Rosenbaum, Katie L. Caillouette, Nansi Jo Colley. Dept. of Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin, Madison, WI. Photoreceptor cell survival depends on successful biosynthesis and transport processes. Here, we describe a novel Drosophila Sec23-interacting protein (Sec23-IP) that displays 49% identity with the human Sec23-IP (p125). Human Sec23-IP has been shown to interact with Sec23. Sec23 is a constituent of the coat protein complex, COPII, which is essential for the budding of vesicles at the exit sites of the endoplasmic reticulum (ER) membrane. COPII assembly is initiated by Sec12 (guanine nucleotide exchange factor), which recruits Sar1-GDP to the ER membrane where GDP is replaced by GTP. Sar1-GTP subsequently recruits the Sec23/Sec24 complex to the site of vesicle budding, followed by the recruitment of the Sec13/Sec31 complex. In addition to Sec23’s role as a coat component, it acts as a GTPase Activating Protein (GAP) on Sar1-GTP to produce Sar1-GDP, promoting the disassembly of COPII after vesicle budding. The Drosophila and human Sec23-IP both contain a proline-rich domain that promotes protein-protein interactions for vesicle budding. Unlike the human p125, Drosophila Sec23-IP also contains a WWE (tryptophan and glutamate) domain that may be involved in protein ubiquitination and/or ADP ribosylation. Finally, the Sec23-IP contains a DDHD (aspartate and histamine) domain, a signature of the Nir/rdgB family of proteins that are known to cause retinal degeneration. RDGB is a phosphatidylinositol (PI) transfer protein. Sec23-IP contains a phospholipase A1 (PLA1) domain within its DDHD domain, implicating it in lipid metabolism. Five mutant alleles of sec23-IP were identified and all alleles display a severe reduction in rhodopsin (Rh1) levels. In addition, mutations in sec23-IP lead to an accumulation of Rh1 protein throughout the secretory pathway, accumulation of smooth membranes and severe retinal degeneration. Here, we demonstrate a role for the Sec23-IP in the vesicular trafficking of Rh1 during its biosynthesis in Drosophila photoreceptor cells. POSTERS: Cytoskeleton and Cellular Biology 173

249C A cellular basis for Wolbachia transmission in the maternal germline. Laura Serbus, William Sullivan. MCD Biology, University of California, Santa Cruz, CA. Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. In arthropods, this maternal transmission is accomplished via incorporation of Wolbachia into germline precursor cells known as pole cells. Some Wolbachia strains concentrate at the posterior of host oocytes, thus promoting Wolbachia incorporation into posterior pole cells during embryogenesis. The asymmetrical distribution of Wolbachia in oocytes suggests involvement of an active localization mechanism, however the molecular basis for this is unknown. Here we report a stepwise mechanism for posterior Wolbachia localization in Drosophila oogenesis involving both host and Wolbachia-based factors. Function-disruption experiments in D. melanogaster indicate that posterior Wolbachia localization relies on directed transport by kinesin-1 and posterior pole plasm assembly in oogenesis. Ectopic pole plasm tests suggest that kinesin-1 and pole plasm contribute independently to posterior Wolbachia concentration. These data suggest a two- step mechanism for posterior Wolbachia enrichment; kinesin-1-mediated transport of Wolbachia toward the oocyte posterior, followed by pole plasm-mediated anchorage of Wolbachia to the posterior cortex. We have additionally tested whether Wolbachia contribute to their posterior localization pattern using a trans-infection approach. This work revealed that the wMel Wolbachia strain endogenous to D. melanogaster also exhibits posterior localization in D. simulans oocytes, unlike the wRi Wolbachia strain endogenous to D. simulans. This indicates that factors intrinsic to Wolbachia are also important to drive posterior concentration of Wolbachia in the oocyte. This distinction between posteriorly concentrating and evenly dispersed Wolbachia strains may be due to different abilities of those strains to interact with posterior pole plasm.

250A Protein Phosphatase 2A negatively regulates Smoothened protein trafficking to modulate Hedgehog signaling outcomes. Ying Su, Jason Ospina, Andrew Michelson, Alan Zhu. Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH. Hedgehog (Hh) signal transduction is critically important in regulating cell proliferation, differentiation, and cell fate decisions during animal development. Inappropriate Hh signaling has been implicated in a variety of developmental defects and many forms of cancer, including basal cell carcinoma (the most common human cancer) and medulloblastoma (the most common childhood brain tumor). Hh signaling is mediated by two transmembrane proteins, the Hh receptor Patched (Ptc) and an essential activator Smoothened (Smo). We have studied the cell biology of Smo protein, and demonstrated that Drosophila Smo protein localization to the cell surface is a pre-requisite for Hh target gene activation in vivo. Similarly, mouse Smo becomes localized to the primary cilia, a specialized membrane structure, in response to Hh activation. Hyper-activation of Smo results in tumor formation; fly versions of the oncogenic forms of Smo are constitutively present at the cell surface even in the absence of Hh activity. Thus, Smo protein trafficking must be tightly coordinated for appropriate Hh signaling. In searching for key factors that regulate Smo trafficking, we found that reducing protein phosphatase 2A (PP2A) activity is sufficient to promote Smo protein surface localization in cultured Drosophila cells. Hh signaling is activated in PP2A loss of function clones in the wing imaginal disc. Conversely, heightened PP2A expression reduces the Hh target gene activity. Furthermore, we demonstrated that elevated Hh signaling by adding Hh protein or by reducing ptc activity leads to reduced PP2A expression and activity in vitro. We are in the process to investigate the mechanism of how PP2A functions in the Hh signaling pathway in vivo. We believe that increased PP2A expression restricts Hh signaling by preventing Smo from localizing to the cell surface, and Hh signaling in turn controls PP2A activity through Ptc to ensure a proper Hh signaling outcome.

251B Myotubularin Phosphoinositide Phosphatase Regulation of Lysosomal Fusion and Cellular Morphogenesis. Michaella Velichkova, Inês Ribeiro, Amy Kiger. Div Biological Sci, Univ California, San Diego, La Jolla, CA. The accumulation of specific phosphoinositide phosphates (PIPs) at distinct membrane domains provides spatial control for diverse cellular processes, including membrane trafficking, protein sorting and cytoskeletal organization. Although mutations in PIP regulators are frequently associated with human disease, little is known about the in vivo regulation and normal developmental roles for PIPs and their cellular responses. Myotubularins (MTMs) encode a large family of predicted PI(3)P and PI(3,5)P2 phosphatases, with specific members associated with human myopathy and neuropathy disorders. We found that Drosophila myotubularin (mtm) is important for adult fly development, including a specific function in muscles. Using RNAi in cell cultures we characterized a requirement for mtm in an induced cell shape change, coincident with defects in late endosome-lysosome homeostasis and F-actin remodeling. We established that both the giant lysosome and morphogenesis phenotypes in mtm depleted cells were mediated by inappropriate regulation of specific PI(3)P pools. In vivo labeling of phosphatidylinositols revealed elevated levels of PI(3)P in mutant mtm cells. Immunofluorescence detection of the PI(3)P biosensor, Cherry-2xFYVE, indicated that PI(3)P accumulation localized to Rab7- positive late endosomes. The giant lysosomes in mtm depleted cells contained abnormal multiple vesicles and excess membrane outfolds, as shown by ultrastructural analysis. To identify potential protein interactions and molecular mechanisms regulated by Mtm, we conducted RNAi screens for genetic modifiers of mtm phenotypes. Knockdown of specific genes involved in the biogenesis and function of late endosomes and in late endosome-lysosome fusion suppressed the giant lysosome phenotype caused by mtm RNAi. Together, these data suggest a possible regulatory role for Mtm at the stage of late endosome-lysosome fusion. Currently we are investigating whether Mtm requirements for membrane homeostasis and intracellular transport are linked to its role in adult muscle formation and development. 174 POSTERS: Cytoskeleton and Cellular Biology

252C GSK3/Shaggy is required for axonal transport. Carole Weaver, Lawrence S. B. Goldstein. Department of Cellular & Molecular Medicine, University of California at San Diego, La Jolla, CA. Kinesin-mediated cargo transport in the axon is critical for synaptic function and neuronal survival, and failure of cargo transport has been implicated in neurodegenerative diseases. Nevertheless, relatively few molecules have been identified that regulate this complex biological process. Because exogenous Glycogen Synthase Kinase 3 (GSK3/Shaggy) is capable of inhibiting anterograde cargo movement in squid axoplasm and causing dissociation of kinesin-1 from vesicles, it has been proposed to regulate axonal transport; but the in vivo requirement for GSK3/Sgg remains to be examined. Here, we show that sgg loss-of-function mutants have severe axonal transport defects. Furthermore, genetic interactions between sgg and kinesin heavy chain support the model that Sgg acts as a negative regulator of kinesin-1. Surprisingly, we find that Sgg inhibits both anterograde and retrograde transport of a known kinesin-1 cargo that is involved in Alzheimer’s disease, the Amyloid Precursor Protein (APP). We are currently using genetic, biochemical, and live imaging approaches in Drosophila larvae to determine the molecular mechanism by which Sgg regulates axonal transport. Our initial model is that Sgg influences both cargo-motor association and microtubule stability. As GSK3 is likely a major player in both bipolar disorder and Alzheimer’s disease, its role as a transport regulator in neurons may be connected to how GSK3 dysfunction contributes to disease in the brain.

253A Rab-regulated vesicle trafficking in Development. Jun Zhang, Matthew Scott. Dept. of Developmental Biology, HHMI, Stanford University, Stanford, CA. Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, more than 75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. To investigate Rab functions in vivo, we have begun a systematic genetic study of the 33 Rab genes in Drosophila. We generated fluorescent protein-tagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful toolkit for investigating Rab functions in vivo and marking the intracellular compartments. Here we are going to report basic characterizations of these lines and some studies with a few Rabs that we have pursued further investigation.

254B Bcr-Abl interacts with Rho1 to alter cell migration during Drosophila development. Nicholas Artabazon, Sara Tittermary, Traci Stevens. Biol Dept, Randolph-Macon College, Ashland, VA. Bcr-Abl is an activated fusion protein linked to leukemia in humans. Bcr-Abl results from a reciprocal translocation between chromosomes 9 and 22 that fuses most of the abl gene to the bcr gene. Normal, cellular Abl (c-Abl) is a tyrosine kinase that regulates cell migration through direct interactions with actin and indirectly through the phosphorylation of proteins that regulate actin dynamics. Compared to c-Abl, Bcr-Abl has increased tyrosine kinase activity, and studies in both cell culture and Drosophila suggest that Bcr-Abl alters actin dynamics and cell migration. The goal of our research is to gain insight into the Bcr-Abl signaling pathways. Rho GTP-binding proteins are a family of actin regulators required for cell migration in Drosophila, and studies in cell culture suggest that Rho proteins may play a role in Bcr-Abl signaling pathways. To address the role of Rho1 in the effects of Bcr-Abl on cell migration, we examined the phenotypes of double mutants that express Bcr-Abl and are heterozygous for a Rho1 mutation. We found that loss of one copy of Rho1 modified the embryonic phenotypes associated with Bcr-Abl expression. Surprisingly, loss of Rho1 had different effects when combined with two different isoforms of Bcr-Abl, p185 and p210. These two isoforms contain the same region of Abl, but differ in the amount of Bcr sequence, and are associated with different types of leukemia in humans. In embryos that express p185 Bcr-Abl, loss of one copy of Rho1 enhanced the defects in cell migration. In contrast, a Rho1 mutation suppressed the defects associated with p210 Bcr-Abl. Furthermore, in order to characterize the interaction between Rho1 and Bcr- Abl, we examined the effects of dominant-negative Rho1 on Bcr-Abl signaling. We found that both active Abl kinase and Ena, a target of Bcr-Abl kinase, are mis-localized in cells expressing this mutant version of Rho1. Taken together, these studies suggest that at least some of the effects of Bcr-Abl on cell migration may be through Rho1, but that Rho1 may play different roles in the p185 and p210 Bcr-Abl signaling pathways. POSTERS: Cytoskeleton and Cellular Biology 175

255C Two Studies of Transcriptional Control of Development. Adam Bousum, Jesse Hogan, Hilary Price, Thomas Kidd. University of Nevada, Reno, Reno, NV. During development, an organism transitions from a single cell to a vast and complicated network of interacting cells. Cells move great distances in the maturing organism and specialize themselves for specific tasks. Transcription factors play critical roles in these processes. Recently, we have been working with two projects that explore the migration and differentiation process within the developing Drosophila embryo. The first of these is our effort to characterize the Hog gene, which is located in an intron of the Netrin B gene. Hog is maternally deposited, and is required for transcription of the bHLH transcription factor twist. Twist is critically required for mesoderm formation in Drosophila. In hog mutants, a very few random cells fail to express twist. Interestingly, these cells remain undifferentiated and fail to migrate appropriately during gastrulation. Hog has a forkhead associated domain suggesting it cooperates with forkhead transcription factors during development. The second project centers around Ret, the Drosophila homologue of the human RET proto-oncogene which is mutated in Hirschsprung Disease. The expression pattern of the fly gene shares remarkable conservation with the human gene, being dynamically expressed in the developing enteric nervous system. By examining regions in Ret’s introns that are highly conserved across different Drosophila species, we hope to uncover the enhancers that drive the gene’s expression. Candidate enhancers will be tested transgenically in the fly and thereby yield new insights into the developmental and cell migration processes.

256A Domain Analysis to Dissect the Role of Drosophila PINCH in Epithelial Adhesion and Migration. Maria C. Elias1,3, Julie L. Kadrmas1,3, Mary C. Beckerle1,2,3. 1) Department of Oncological Sciences, University of Utah, Salt Lake City, UT; 2) Department of Biology, University of Utah, Salt Lake City, UT; 3) Huntsman Cancer Institute, University of Utah, Salt Lake City, UT. The molecular scaffold PINCH is an essential 5 LIM domain protein that, together with its binding partners, is an important regulator of cell adhesion and migration. LIM1 of PINCH binds directly to Integrin Linked Kinase (ILK) providing a link to integrins, the extracellular matrix, and the actin cytoskeleton, while LIM5 of PINCH binds to Ras Suppressor-1 (RSU-1) to regulate the Jun N- terminal Kinase cascade. This protein complex plays a role in all of the well characterized integrin dependent processes in the fly including dorsal closure, muscle integrity, and wing adhesion. In order to understand the mechanism of action for PINCH, we have engineered transgenic flies carrying mutant forms of PINCH fused to GFP. PINCH Q38A transgenes specifically disrupt the interaction with ILK and fully rescue the lethality of PINCH null mutations. This indicates that while PINCH and ILK are both required for viability their physical interaction is not necessary. Interestingly, PINCH Q38A protein is mislocalized away from the cell membrane during dorsal closure. Conversely PINCH D303V transgenes, in which the RSU-1 interaction is specifically disrupted, only partially rescue the lethality of PINCH null mutants. This is in contrast to the RSU-1 null mutants which are viable, suggesting that the association of PINCH and RSU-1 is critically important. Both Q38A and D303V rescued flies demonstrate a high degree of wing blisters. This observation is indicative of a disruption of integrin mediated adhesion in the wing epidermis and is a mild phenotype of PINCH loss of function. Finally, PINCH ΔLIM5 transgenes were unable to rescue the lethality of PINCH null mutants, suggesting that LIM5 has functions beyond its interaction with RSU-1. Taken together, our preliminary analysis of the molecular scaffold PINCH indicates that its modular structure integrates multiple signals in order to execute developmental programs and maintain adult structures.

257B Function of singed during Drosophila development: a new role in blood cell migration. Jennifer Zanet1, Brian Stramer2, Tom Millard2, Paul Martin2, François Payre1, Serge Plaza1. 1) Centre de Biologie du Dévelopement, Toulouse, France; 2) Department of Physiology, University of Bristol, Bristol, UK. During development, cell migration remains a fundamental process used by cells to take up their final location within the organisms. Although the basic cellular mechanisms involved in cell migration have been intensively studied in cultured cells, in vivo systems recently emerged for a better understanding of the mechanisms governing cell migration in vivo. Hemocytes are the Drosophila blood cells playing a role in the innate immune system. During embryogenesis, these cells originate from the head and disperse throughout the embryo along invariant and developmentally programmed pathways. However, the molecular mechanisms controlling hemocytes migration are not well understood. As a first step to identify genes involved in this process, we noticed that hemocytes highly express the singed (sn) gene at the onset of migration. Sn encodes the Drosophila Fascin, an actin-bundling protein recently implicated in cell motility in vertebrate cells. However, in Drosophila, sn is known to be required for actin bundle formation during oogenesis and bristle extension. We find here that in absence of sn, hemocytes migration is impaired. Moreover, live imaging highlights a decrease of the lamellipodia dynamic and an altered cell polarity. The function of sn can be widespread since sn is also involved for hemocytes migration in response to injury. It was recently proposed that Fascin activity for cell migration is tightly regulated by phosphorylation on a conserved serine residue. We genetically addressed this question in vivo and our results show that phosphorylation of this residue regulates actin-bundling activity of sn during bristles extension but has no role for hemocyte migration. In summary, our data identify an evolutionary conserved role of sn during cell migration with an unanticipated function of sn in cell polarity. These results suggest a distinct post-translational regulation of sn in cell morphogenesis and in cell membrane dynamic of migrating hemocytes. 176 POSTERS: Cytoskeleton and Cellular Biology

258C Requirements for epidermal wound repair: Lessons from Drosophila genetics. Michelle Juarez, Efren Sandoval, William McGinnis. Dept Cell & Developmental Biol, Univ California, San Diego, La Jolla, CA. The epidermis is the largest organ of the body of most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a rapid but poorly understood genetic response that results in repair of the wound, increasing the likelihood of the animal’s survival. In Drosophila, this includes transcriptional activation of genes involved in crosslinking epidermal cuticle, e.g. genes encoding the enzymes Dopa-decarboxylase and Tyrosine hydroxylase. The transcription factor Grainy head plays a key role in the activation of immediate wound response genes that help repair epidermal breaks. We have screened deficiency collections to identify genes in pathways acting upstream of and parallel to Grainy head to induce epidermal wound repair genes. Three non-overlapping deficiencies have been identified that alter activation of epidermal wound reporter genes. Based on the initial results, we are currently investigating genes that: (1) act in serine protease cascades to provide signals for epidermal repair, (2) act in flotillin-mediated signal transduction and cell adhesion, and (3) act along with AMP kinase to sense cellular stress conditions. We expect that, as in previous studies in Drosophila, this genetic screen will reveal evolutionarily conserved pathways that mediate epidermal break repair in most animals, including humans.

259A Analysis of mutants in Strn-mlckand CG1776, two Drosophila myosin light chain kinase (MLCK) genes. Andrew Kreuz1, Ivan Tesic2, Deyra Rodriguez2, Rafael Acosta1, Amanda Simcox2. 1) Dept Biol, Villa Julie Col, Baltimore, MD; 2) Dept. Molecular Genetics, The Ohio State University, Columbus, OH. Phosphorylation of myosin light chain-2 (MLC2) at conserved myosin light chain kinase (MLCK) target serines is critical for normal functioning of the indirect flight muscle (IFM). Substitution of alanines for the target serines results in an increase in non-phosphorylated MLC2 in the IFM and causes a flightless phenotype. A single mlck gene, Stretchin-mlck(Strn-mlck) has been characterized and it encodes several MLCK isoforms expressed in the IFM that share homology with vertebrate smooth muscle MLCKs. We isolated a mutant (Strn-mlck1) that deletes the 3’ end of Strn-mlck, including the entire kinase domain. Complementation tests show that the gene is allelic to the classical mutant curved and has a similar held-out wing position. The wing phenotype precluded flight behavior testing but we found that MLC2 phosphorylation is unchanged in the mutant. This suggests either that MLC2 is phosphorylated in the IFM by another MLCK or that the genes function redundantly. We characterized a second mlck-like gene, CG1776, and show that the predicted protein shares about 50% homology to STRN-MLCK and vertebrate smooth and skeletal muscle MLCKs. Unlike STRN-MLCK and other MLCKs, CG1776does not contain a Ca2+/CaM regulatory domain. We show here the results of our analysis of the CG1776 gene, including the characterization of an insertion mutation and the effects of RNAi silencing.

260B Drosophila messy mitochondria, and fragmented mitochondria are required for mitochondrial morphology and imaginal disc development. Dennis LaJeunesse. Dept Biol, Univ North Carolina, Greensboro, NC. In F3 genetic screen, we have identified two genes messy mitochondria(mess), and fragmented mitochondria (frag) that are required for the proper mitochondrial morphology in the visceral muscles of the larval midgut. messy mitochondria (mess) maps to 96B10 on the right arm of the third chromosome and appears to be allelic to Oxysterol Binding Protein/CG6708; frag maps to the right arm of the third chromosome to the cytological region 88. mess and frag are essential genes and larvae homozygous for a null mutation in both genes die at the late third instar larvae/early pupal stage. In addition to the disruption of mitochondrial organization and distribution phenotype, both mess and frag mutant larvae also express a small imaginal disc phenotype with a loss of epithelial apical/basal polarity as well as disruption in larval growth. We are currently cloning and characterizing both genes and will present information regarding the relationship between mitochondrial morphology and cellular organization and function. POSTERS: Cytoskeleton and Cellular Biology 177

261C Atlastin promotes homotypic fusion of ER membranes. Genny Orso1, Diana Pendin2,3, Jessica Tosetto2,3, Andrea Daga2,4. 1) Scientific Institute E. Medea, Conegliano, Italy; 2) Dulbecco Telethon Institute at the E. Medea Scientific Institute, Conegliano, 31015 Italy; 3) Department of Pharmacology, University of Padova, Padova, 35131 Italy; 4) Department of Neurology, The David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA. The endoplasmic reticulum is a subcellular organelle comprised of anastomosing membrane networks. Formation and maintenance of the intricate ER architecture is essential for implementation of the multiple functions served by this organelle. Homotypic membrane fusion underlies both the biogenesis and maintenance of the ER and depends categorically on GTP hydrolysis but does not require cytosolic components, suggesting that a membrane bound GTPase may be responsible for this activity. Using in vivo analysis in Drosophila we demonstrate that D-atlastin, the fly homologue of the dynamin superfamily member atlastin-1 whose mutation causes Hereditary Spastic Paraplegia (HSP), specifically localizes on ER membranes, that loss of D-atlastin causes fragmentation of the ER and its overexpression induces the formation of aggregates of fused ER membranes in a strictly GTP-dependent manner because the GTPase deficient K51A mutant does not produce membrane aggregation. Since D-atlastin is capable of homo- oligomerization, we deduce that it connects ER membranes via a homophilic interaction and therefore is the key GTPase promoting homotypic fusion of ER membranes. We also propose that secretory pathway traffic impairment secondary to disruption of ER homeostasis caused by loss or gain of atlastin function is likely to be responsible for HSP pathology in humans.

262A Developmentally controlled sequestration of Importin α2 to lipid droplets. Naina Phadnis, Michael Welte. Department of Biology, Univeristy of Rochester, Rochester, NY 14627. Lipid droplets (LDs) are organelles that store neutral lipids. They play key roles in lipid homeostasis, membrane biogenesis and cell signaling. Previous proteomic studies had suggested that purified LDs from early embryos carry the karyopherin Importin α2. This result was surprising since Importin α2 is a nuclear import factor that assists in the delivery of proteins from the cytoplasm into the nuclear compartment and has no known function on LDs. To test if droplet association occurs in vivo, we determined the distribution of HA-tagged Importin α2 by immunostaining. In cycle 14 embryos, Importin α2-HA was not present in the nucleus or cytoplasm, but localized to distinct spherical structures reminiscent of LDs. To determine the nature of these structures, we centrifuged embryos; under these conditions, LDs accumulate in a distinct layer. Both HA-tagged and endogenous importin α2 were highly enriched in this layer. Remarkably, droplet association is developmentally regulated as importin α2 was generally cytoplasmic before cycle 14. Recently it has been proposed that droplets may act as generalized sites to sequester proteins; yet neither the mechanisms nor the biological functions of such sequestration are known. Importin α2 may provide an inroad into both problems. On developmental Westerns, Importin α2 undergoes a mobility shift just around the time when sequestration occurs. Previous literature suggests that those mobility changes are due to changes in phosphorylation. Using phosphorylation mutants, we are now testing whether phosphorylation controls droplet association. If so, it will provide tools to address the biological role of sequestration. Because overall importin α2 levels drop subsequent to droplet sequestration, droplet localization may be a precursor to turnover.

263B Mononuclear muscle cells of the ovarian epithelial sheath. Akemi J. Tanaka, Andrew M. Hudson, Lisa N. Petrella, Lynn Cooley. Department of Genetics, Yale University School of Medicine, New Haven, CT. Despite the huge amount of research focused on oogenesis, the muscles of the Drosophila female reproductive system have received little attention. There are three types of muscle in Drosophila ovaries: the epithelial sheath, peritoneal sheath, and oviduct muscles. In our previous work, we characterized the morphology, and sarcomeric organization of these muscles using GFP protein trap lines. Protein traps in the Fasciclin 3 gene revealed Fas3::GFP localization to dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles, and provided a reliable marker for epithelial cell boundaries and novel cell-cell junctions. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. We used a protein trap with uniform muscle expression as a marker in experiments to produce genetic mosaics via the Flp/FRT system in these cells, and have generated marked mitotic clones at high frequency. We are presently working on epithelial sheath cell lineage tracing based on this technique, and investigating whether there is evidence of proliferation in adults. The results of these experiments will be presented. 178 POSTERS: Cytoskeleton and Cellular Biology

264C Water channels, oviduct osmolarity and egg activation in Drosophila. Ido Apel1, Menachem Moshelion2, Yael Heifetz1. 1) Dept. of Entomology; 2) Institute of Plant Sciences and Genetics, The Hebrew University, Rehovot, Israel. Unlike most organisms, egg activation in Drosophila is initiated independently of sperm, occurring during egg passage through the female reproductive tract. As the egg moves from the ovary into the oviduct, it is subjected to mechanical pressure and to a change in environmental conditions. An important trigger of egg activation in Drosophila is hydration of the oocyte. Microarray analysis of mating-responsive genes expressed in the female oviduct identified several Drosophila putative aquaporin genes (DrmAQPs), ion channels (e.g. chloride channels: CG7589, CG5284) and transporters (e.g. Na+/K+/Cl- symporter: CG10413) which are predicted to be involve in osmoregulation of the oviduct. Interestingly, the expression level of one of these genes, Drosophila Integral Protein (drip), significantly increased post-mating. We hypothesize that DrmAQPs play an essential role in regulating water potential homeostasis of the oviduct and thus the egg hydration rate. To test this hypothesis, we first confirmed that the identified genes are indeed expressed in the oviduct. We found that DrmAQPs are express in the oviduct, ovary, and oocyte. Drip was found to be expressed in the epithelia of the female reproductive system. Mating enhanced its expression level significantly in the epithelia of the lateral and common oviduct. To examine DRIP water transport efficiency, the protein was expressed in Xenopus lavies oocytes and in Arabidopsis thaliana protoplasts. We found that expression of DRIP in both systems significantly increased the cells’ water permeability coefficient, suggesting DRIP may have a role as a water channel in the Drosophila oviduct. We are currently silencing genes suggested to be involved in regulating water potential in the oviduct to examine their effect on female fertility. These results, in conjunction with localization studies, may allow us for the first time to better understand the osmoregulation mechanism in the female reproductive tract, and thus understand the mechanism of egg activation.

265A The transcriptional control of secretion in the Drosophila embryonic salivary gland. Rebecca M. Fox, Monique Marshall, Deborah J. Andrew. Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore, MD. Many specialized organs require high-level secretory activity to perform their normal functions. In the Drosophila embryo, the salivary gland (SG) is the largest secretory organ, making it an excellent system to study the mechanisms of secretion. Two SG expressed transcription factors, CrebA and Xbp1, are important for secretory function. We have previously shown that 34 secretory pathway component genes (SPCGs) require CrebA for high-level expression. Moreover, 32 of the SPCGs contain a putative CREB consensus site within 2 kb of the translational start site. Electrophoretic mobility shift assays reveal that purified CrebA protein is able to bind to this consensus site in vitro. This interaction is specific, as mutating this site abolishes CrebA binding. The relevance of these sites to in vivo regulation by CrebA will be tested using lacZ reporter constructs containing wild-type and mutant versions of the CREB consensus site(s). Since CrebA regulates SPCG expression, additional experiments are in progress to determine if the loss and/or gain of CrebA function is able to directly influence the size and integrity of the secretory machinery. In yeast and mammals, Xbp1 functions to upregulate the secretory machinery in response to ER stress. Although Drosophila xbp1 is highly expressed in the SG, its role in secretory function has not been explored. Using in situ hybridization, we are testing all known SG expressed genes to identify the full complement of CrebA and Xbp1 target genes. These studies will determine if CrebA and Xbp1 work in the same or in parallel pathways to mediate secretory activity, and whether they serve distinct functions in regulating secretion. Taken together, these results should delineate the genetic networks required for the high-level secretory capacity of the SG.

266B Characterization of sec61α during embryogenesis. Xiaochen Wang, Elspeth Pearce, Robert Ward. Dept Molecular Biosciences, Univ Kansas, Lawrence, KS. The elongation and eversion of leg imaginal discs during metamorphosis is an ideal system for studying hormone-regulated morphogenesis. In a screen for dominant modifiers of the malformed leg phenotype associated with br1, a key ecdysone-induced early gene involved in adult tissue differentiation, we identified a mutation in sec61α, which encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. Sequence analysis revealed a nonsense mutation that would truncate the protein in the fifth of ten transmembrane domains, suggesting that this is a strong loss of function mutation in sec61α. Consistent with this idea, homozygous mutant sec61αE(br)165 or sec61αE(br)165/Df(2L)BSC6 animals show completely penetrant embryonic lethality with defects in dorsal closure and cuticle deposition. We have rescued this embryonic lethality by expression of a sec61α genomic construct. Indirect immunofluorescence experiments using an antibody generated against the Dog Sec61α indicates that Sec61α is expressed ubiquitously in embryos and larvae but is enriched in some secretory tissues. Because the phenotypes of sec61αE(br)165 are very similar to those of Halloween mutants, which are genes that encode P450 enzymes required for ecdysone biosynthesis, we examined the expression of ecdysone-induced genes by Northern blot and in situ analyses. Preliminary results, however, suggest that ecdysone is not reduced in sec61α mutants. Alternatively, sec61α may be required for the secretion of specific transmembrane or secreted proteins during dorsal closure. Careful examination of dorsal closure in sec61α mutants suggest that the leading edge is specified and elongates initially, whereas the lateral epidermis is compromised in its ability to elongate, and thus dorsal closure never goes to completion. This observation raises the possibility that specific reductions in the Dpp signaling in a sec61α mutant background underlie the dorsal closure defect, a hypothesis that we are currently testing. POSTERS: Genome and Chromosome Structure 179

267C Chromosome segregation and meiosis I progression in Drosophila oocyte. Ounissa Aït-Ahmed, Régis Meyer, Michèle Delaage, Roland Rosset, Michèle Capri. Institut de Génétique Humaine UPR 1142, CNRS, Montpellier, France. Chromosome segregation is achieved as a result of two essential processes: mitosis and meiosis. Defects on the mechanisms on which chromosome segregation relies may result in aneuploidy, causing cancer if they occur in mitotic cells and birth defects if occurring during meiosis. Meiosis is characterized by two nuclear divisions (meiosis I and II) following a single round of DNA replication. As a result haploid gametes are formed. Chromosome segregation during reductional meiosis I requires three specific events: homologous recombination that results in chiasma formation, monopolar orientation of sister kinetochores and a stepwise degradation of cohesion at anaphase I. Indeed sister kinetochores must remain attached until metaphase II while cohesion is lost on chromosome arms before anaphase I proceeds. Meiosis I specific events must be supported by specific proteins. It was our reasoning for undertaking the screen that resulted in the identification of the Drosophila yemanuclein-alpha (yem-alpha), a DNA binding protein specific for the oocyte nucleus (Aït Ahmed et al, 1992). Its localization to the chromosomes is restricted to meiosis I. It is a new component of the synaptonemal complex (SC). After SC disappears yem-alpha remains on chromosome arms and centromeres. Its function is critical for meiosis I progression and homologue segregation; in a recombination defective background a yem-alpha mutant oocyte undergoes a single division. Such a phenotype has never been reported in Drosophila meiosis. Yem-alpha function is genuinely new and will be discussed in the light of these data and other unpublished data.

268A Evidence for Common Fragile Sites in Drosophila. Matthew C. LaFave1, Lewis J. Overton2, Jeff Sekelsky1,2. 1) Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC; 2) Department of Biology, University of North Carolina, Chapel Hill, NC. Common fragile sites (CFSs) are regions of DNA prone to damage and breakage. Instability at these sites may contribute to cancer, but the causes of this fragility remain poorly understood. We have developed a system to detect and characterize CFSs in Drosophila melanogaster based on spontaneous DNA damage. To do this, we are using a mutant of mus309, the ortholog of human BLM helicase. This allows us to locate CFSs by visualizing sites of spontaneous damage as mitotic crossovers, and then using standard mapping techniques to locate regions with more damage than expected. We are also carrying out complementary approaches, including mapping non-homologous DNA insertion sites and genetically reducing levels of Pol α, which should both have exacerbated effects at CFSs. We are initially focusing on the left arm of chromosome 2. Results to date indicate that there is a non-uniform rate of damage across this region, suggesting that our approach will facilitate the detection of CFSs. Future directions will take advantage of genome re-sequencing, high-throughput SNP detection, and other emerging technologies to increase the resolution of CFSs to a level higher than that attained in mammalian studies.

269B Nucleoskeletal proteins EAST and CP60 modulate activity of the gypsy insulator in Drosophila melanogaster. Pavel Georgiev, Anton Golovnin, Larisa Melnikova, Ivan Krivega, Margarita Kostuchenko, Ilya Volkov. Inst Gene Biology RAS, Moscow, Russia. The best-characterized Drosophila insulator found in the gypsy retrotransposon contains 12 binding sites for the Su(Hw) protein. Along with Su(Hw), enhancer/silencer blocking requires Mod(mdg4)-67.2, a BTB/POZ domain protein. In particular, inactivation of Mod(mdg4)-67.2 in mutants leads to direct repression of the yellow gene promoter by the gypsy insulator. We have found in this study that such repression is regulated by CP60 and EAST, protein components of the nuclear matrix. CP60 is physically associated with the CP190 protein and binds to the Su(Hw) insulators in vivo. Inactivation of CP60 in mutants enhances the repression mediated by the mod(mdg4) mutations, suggesting that CP60 cooperates with Mod(mdg4)-67.2 in preventing conversion of the gypsy insulator into a silencer. EAST is a regulator of the nuclear endoskeleton, and its effect on the activity of gypsy is opposite to that of CP60. These results are the first evidence for the functional role of the nuclear matrix in regulating insulator activity. 180 POSTERS: Genome and Chromosome Structure

270C The Genomics Education Partnership: The dot chromosome, a comparative genomics investigation by undergraduates. Sarah Elgin1, Anya Goodman2, Christopher Jones3, Gary Kuleck4, Gerald McNeil5, Ken Saville6, Joyce Stamm7, David Lopatto8. 1) Washington U, St Louis MO; 2) Cal Poly St U, San Luis Obispo CA; 3) Moravian C, Bethleham PA; 4) Loyola Marymount U, Los Angeles CA; 5) York C/CUNY, New York NY; 6) Albion C, Albion MI; 7) U Evansville, Evansville IN; 8) Grinnell C, Grinnell IA. The advent of genome sequencing technology has opened up new opportunities to understand how genomes are organized, a critical parameter in the regulation of gene expression, and has created new opportunities for student-scientist research partnerships. With appropriate training, undergraduate students can provide the needed human input to improve raw sequence data and carry out careful annotation. Over 30 primarily undergraduate institutions are working with the Biology Dept and Genome Sequencing Center of Washington University to create a partnership that provides students with an opportunity to work on a genomics research problem. Currently we are finishing and annotating the gene-rich region of the dot chromosomes of several Drosophila species. In D. melanogaster, this unique domain has properties of both euchromatin (including normal gene density) and heterochromatin (including a high density of repetitious DNA- remnants of transposable elements and retrotransposons). Student members of the GEP work in a lab course during the academic year as a research team, each student contributing to finishing, annotating and analyzing a fosmid. Systematic checks show that the student results are usually identical to those of professionals. A comparison of the D. melanogaster and D. virilis dot chromosomes is now being prepared for publication, and work to analyze the dot chromosomes of D. erecta and D. mojavensis is underway. Students participating in the GEP report intellectual and personal gains normally associated with summer research experiences, particularly in developing problem-solving skills; they are very enthusiastic about the project. We are looking for additional schools to join us, and additional partners from the scientific community to pose meaningful annotation problems for students to tackle. Funded by grants from HHMI, NIH.

271A A search for locus producing RNAi through a high-throughput analysis of heterochromatin transcription. Danielle Nouaud1, Clémentine Vitte1, Dominique Anxolabéhère1, Hadi Quesneville2. 1) Dynamique du Génome et Evolution, Institut Jacques Monod - CNRS -Universités Paris6 et Paris7, Paris, France; 2) URGI, INRA unit 1164, Evry, France. Transposable elements (TEs) are major components of heterochromatin and may play an important functional role. To improve the quality of TE annotations, we have developed a combined evidence TE annotation pipeline analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods (Quesneville et al. 2005). This approach differs from standard efforts to annotate TEs in genome sequences that rely on the results of only a single computational method. Our TE annotation pipeline produces annotations of a quality far beyond what is available today and gives us the first detailed annotation of heterochromatic sequences in any organism. This will allow us to understand the relationships between TEs, other repeats and heterochromatin. Hence we perform a functional annotation of TE expression in order to identify TEs that could generate transcripts involved in the RNAi process, which is known to repress gene expression but also to induce heterochromatin formation. Available sequences such as ESTs, cDNAs and small RNAs are mapped onto the genomic sequence. This annotation gives us the opportunity to explore heterochromatin transcriptional activity from a global perspective. Using RT-PCR amplifications on candidate regions that were identified from an in silico search, we show that regions harbouring TEs in opposite orientation are able to produce both sens and anti-sens RNAs. Small RNAs are detected in these regions by northern blot hybridizations, suggesting that these candidate regions are able to trigger heterochromatinization in cis. In order to profile transcription in Drosophila heterochromatin with a more global approach, we have designed a custom microarray. The first results will be presented.

272B Heterochromatin variations in evolution of malaria mosquitoes. Igor Sharakhov1, Maria Sharakhova1, Irina Brusentsova2. 1) Department of Entomology, Virginia Tech, Blacksburg, VA, USA; 2) Institute of Cytology and Genetics, Novosibirsk, Russia. Heterochromatin is an essential part of any eukaryotic genome playing a significant role in many biologically important processes such as cell division, meiotic pairing, regulation of DNA replication and gene expression. Anopheline mosquito polytene chromosomes is an ideal model for studying regulation and evolution of heterochromatic genes. The A. gambiae centromeric regions consist of mostly diffuse heterochromatin. The types of centric heterochromatin vary among chromosomal arms in A. stephensi, the centric regions of chromosome 3 consist only of compact heterochromatin. In this study an antibody against the Drosophila heterochromatin protein 1 (HP1) have been used to localize the regions of intercalary and pericentric heterochromatin on the mosquito chromosomes. HP1 was found localized in the mesh-like type of heterochromatin and in some telomeric and euchromatic regions. However, the HP1 localization have not been detected in compact centric heterochromatin of chromosome 3 of A. stephensi. These results suggest that the molecular structure of centric heterochromatin differs between A. gambiae and A. stephensi. Analysis of the physical maps and polytene chromosomes has revealed extensive variations in morphology and location of heterochromatin among A. stephensi, A. funestus, and A. gambiae. We found that not only heterochromatin morphology but also its location have been changing in mosquito evolution. Heterochromatic regions of polytene chromosomes are known to be associated with the nuclear periphery, the major protein of which is lamin. To test whether HP1 and lamin co-localize, immunostaining with the Drosophila antibodies against lamin Dm0 was performed on A. gambiae and A. stephensi chromosomes. The analysis has revealed that HP1 and lamin share the major heterochromatic sites. Thus, our findings suggest that A. stephensi and A. gambiae differ in terms of three-dimensional organization of their cell nuclei. POSTERS: Genome and Chromosome Structure 181

273C Arginine kinase isoforms from Drosophila melanogaster. Sohini Ghosh, Glen Collier. Dept Biological Sci, Univ Tulsa, Tulsa, OK. Arginine kinase (AK), the phosphagen kinase of invertebrates including the fruitfly Drosophila melanogaster, has a tissue distribution similar to that of vertebrate creatine kinase. It is most abundant in muscle, particularly indirect flight muscle, with less activity present in the central nervous system and very little activity in imaginal discs and elements of the reproductive tract. However, unlike vertebrate creatine kinases(CK) that are encoded by separate genes, Drosophila arginine kinase is encoded by a single locus (Argk). Also, while knock-outs of mouse CKs are not lethal, a null mutant of Drosophila Argk is an embryonic lethal. Six putative alternative transcripts of the single locus for Argk have been annotated at the Drosophila Genome database Flybase (http:// www.flybase.org). We have identified the tissue and stage specificity of expression of these isoforms, identified the proteins produced using isoform-specific antibodies, and demonstrated the mitochondrial specificity of at least one isoform. Lethal rescue experiments utilizing isoform-specific cDNA constructs will test the functional role of phosphagens and their kinases.

274A Analysis of chromatid segregation in Drosophila. Amber Hohl1, Ruth Griffin1,2, Jack Bateman1, C.-ting Wu1. 1) Genetics, Harvard Medical School, Boston, MA; 2) Centre National de la Researche Scientifique, UMR 5092, Biochimie et Biophysique des Systemes Integres, Grenoble. Proper chromosome segregation is crucial to ensure the accurate transfer of genetic material from parent to daughter cells. When recombination occurs in G2 of the mitotic cell cycle, two main types of segregation events are possible. Recombinant chromatids can segregate to different daughter cells, known as X segregation (G2-X), or to the same daughter cell, known as Z segregation (G2-Z) [2, 6]. Recombination can also occur during G1, in which case both daughter cells carry two recombinant chromatids. It is generally believed that the ratio of G2-X to G2-Z segregation is 1:1; that is, the two sister chromatids of one dyad are thought to segregate randomly with respect to the two sister chromatids of the homologous dyad. Evidence from Drosophila and mice, however, has suggested that chromatid segregation after recombination in G2 may be non-random [2, 4, 5]. In particular, early work in Drosophila showed that G2-X segregation was favored over other types of segregation and, more recently, a predominance of G2- X segregation has been observed in mouse cells [2, 5]. Interestingly, the segregation pattern in mouse (G2-X, G2-Z, or G1) appears to vary depending on cell type [1, 4]. My research focuses on sister chromatid segregation in Drosophila using a newly developed method for mosaic analysis [3]. In particular, I am a) asking whether non-random chromatid segregation is cell type specific and b) establishing an experimental system for identifying the genes that control sister chromatid segregation. This work is supported by a graduate fellowship from the National Science Foundation. References: 1) Armakolas, A., & A. Klar. 2006. Science. 311(5764): 1146. 2) Beumer, K.J., Pimpinelli, S., & K.G. Golic. 1998. Genetics. 150: 173. 3) Griffin, R., Bakal, C., Wu, C.-t. & N. Perrimon, unpublished. 4) Liu, P., Jenkins, N.A., & N.G. Copeland. 2002. Nature Genetics. 30(1): 66. 5) Pimpinelli, S., & P. Ripoll. 1986. PNAS. 83: 3900. 6) Stern, C. 1936. Genetics. 21(6): 625.

275B Condensin II complex inhibits pairing in polyploid nurse cells and female meiotic chromosomes. Helen F Smith1, Justin Blumenstiel2, Tom Hartl1, Scott Hawley2, Giovanni Bosco1. 1) MCB, University of Arizona, Tucson, AZ; 2) Stowers Institute, Kansas City, MO. During oogenesis chromosome pairing is developmentally regulated in germline nurse cells and the meiotic oocyte chromosomes. We found that a mutation in a condensin II complex subunit, CapH2, gives pairing defects in both these cell types. The germ-line derived nurse cells of egg chambers are polytene early in development but normally lose this structure later in development. In mutants, 79% of stage 7 nurse cells exhibit persistent pairing of both sister chromatids and homologs compared to 0% in heterozygote controls as assayed by FISH. In meiosis, pairing of chromosomes is an important step for proper segregation of homologs and we find that homolog pairing is stabilized in the CapH2 mutant. In wildtype flies, euchromatic regions of the homologs begin to come apart by stage 6 to 8 of the developing egg chamber. In CapH2/Df flies, however, by FISH we find that during stage 6 to 8 the histone cluster remains paired. Furthermore, we have observed that synaptonemal complex component c(3)G is also stabilized in a threadlike formation in contrast to wildtype, where is becomes diffuse by stage 6 to 8. We are currently investigating (1) whether the increased pairing in the condensin mutant is c(3)G dependent, (2) whether pairing at different euchromatic and heterochromatic regions are equally affected and (3) initiation of pairing in embryogenesis is CapH2 mediated. These and other data (see abstract from T Hartl and G Bosco) suggest that CapH2 and other condensin subunits cooperate to modulate developmentally regulated chromosome pairing events, such as nurse cell polyteny and meiotic chromosome interactions. 182 POSTERS: Genome and Chromosome Structure

276C Single-gene duplications and transpositions in the Drosophila genome. Edwin C Stephenson, Ajinkya C. Inamdar. Department of Biological Sciences, University of Alabama, Tuscaloosa, AL. New genes and novel genetic functions may arise by gene duplication followed by the functional divergence of one of the two copies. Duplicated and transposed genes may be subject to altered cis regulation resulting in new patterns of gene expression, a process that may promote the acquisition of novel genetic functions. Classes of gene duplication events include: (1) Tandem duplication, in which the two gene copies are adjacent in the genome, (2) Duplication of mRNA sequences only, (3) Duplication of whole genes, including non-mRNA sequences, to non-tandem sites in the genome, (4) Transposition of whole genes to novel sites in the genome. The generation of tandem gene duplications is thought to occur through replication slippage at direct repeat sequences, and mRNA duplication by an uncharacterized reverse transcriptase-mediated process. We have examined the origin and fate of members of the third and fourth classes, which are even more poorly characterized, in the Drosophila genome. Relatively recent duplication and transposition events within the melanogaster species group were identified by pairwise genome synteny comparisons. The evolutionary history of the duplicated genes and the timing of the duplication events can be inferred in many cases. We have determined the extent of the duplicated regions, and have attempted to identify remnants of the duplication / transposition process that may be helpful in understanding the mechanisms by which these events occur.

277A A new example of trans-inactivation phenomenon in Drosophila melanogaster. Mikhail V. Kibanov, Sergey A. Lavrov, Yuri A. Abramov, Vladimir A. Gvozdev. Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Kurchatov Sq. 2, 123182, Russia. The expression of genes is regulated at many levels. Accumulating evidences point to the nuclear architecture and the spatial organization of genome as the major factors in regulation of single genes expression. One of the best known examples of gene inactivation as a result of trans-interactions between extensive regions of genome is mutation brownDominant, which is caused by an insertion of a large block (1.5 Mb) of heterochromatin AAGAG satellite sequence into the coding region of brown gene. In bwD/bw+ heterozygotes this insertion acts in trans to trans-inactivate wild type (bw+) allele of brown. Another genetic system has been recently described in our lab. This system is based on the inversion called A4 in the left arm of chromosome 2. Generally, similar inversions lead to cis-inactivation of the genes adjacent to the inversion breakpoints through their abnormal juxtaposition with heterochromatin - so called heterochromatic position-effect variegation. But A4 chromosome also causes trans-inactivation of the reporter genes located on the normal homologous chromosome, if these genes lie near the region surrounded by blocks of heterochromatin in A4 chromosome. We suspect that it is due to somatic conjugation of the homologous chromosomes and, as a result, relocation of the reporter genes into heterochromatic nucleus compartment. Using methods of 3D confocal microscopy, immunostaining and in situ hybridization in Drosophila tissues, we are planning to verify a correlation between reporter genes inactivation and their localization in heterochromatic nucleus compartment, to determine the topology of the region of homologous chromosomes conjugation. We are also going to examine the role of known components of heterochromatin and RNA-interference machinery and influence of mutations in these proteins on organization of the region of homologous chromosomes conjugation and reporter genes silencing.

278B Not Too Much of a Good Thing: Under-replicated Regions in Polytene Salivary Gland Cells. Noa Sher1, Helena Kashevsky1, Thomas Eng1, David MacAlpine2, Terry Orr-Weaver1. 1) Whitehead Institute, Cambridge, MA 02142; 2) Duke University Medical Center, Durham, NC 27710. Polyploid and polytene cells, cells that contain more than the normal diploid genomic content, are widespread in the plant and animal kingdoms. Polyploid and polytene cells result from a modified cell cycle in which DNA replication oscillates with a gap phase, but mitosis does not occur. In addition to the absence of mitosis, differential DNA replication can occur within the endo cycle: specific genomic regions may be blocked from replicating or undergo excessive replication to amplify gene copy number. We used comparative genomic hybridization to tiled microarrays to test for differential replication of the euchromatin of salivary glands from third instar Drosophila larvae. We did not identify domains amplified over the normal polytene copy number but found numerous under-replicated (UR) intervals. In addition to heterochromatin, which was known to be under-replicated, we found several euchromatic regions to be under-replicated up to 10 fold compared to the fully endoreduplicated genome. Strikingly, 11 of these regions are composed of single copy sequences and do not have transposons or repeats predicted to provide heterochromatic character. We used quantitative real time PCR to validate the results of the tiling array for these 11 UR regions. These regions previously were shown to be weak points in the polytene chromosomes of the salivary gland, to bind Polycomb group and the Su(UR) proteins, and to replicate late during the endo cycle S phase (Zhimulev et al. 2003. Chromosoma 111, 377-398). Interestingly, most of the UR regions analyzed show synteny in other Drosophila species, hinting at possible evolutionary conservation of these regions. The 11 UR regions harbor 43 genes, many of which are involved in cell adhesion and apoptosis. Genomic under-replication in polytene tissues may be a mechanism whereby cells limit the gene copy number as a way of downregulating transcription from genes with critical functions. POSTERS: Genome and Chromosome Structure 183

279C Effect of 2L TAS deficiencies on telomere-telomere associations. Radmila Capkova Frydrychova1, Trevor Archer2, James Mason1. 1) LMG, NIEHS, RTP, NC; 2) LMC, NIEHS, RTP, NC. Drosophila telomeres consist of two DNA domains: a terminal array of retrotransposons, HTT, and a subterminal repetitive telomere associated sequence, TAS, a source of telomere position effect, TPE. Deletion of the 2L TAS leads to a suppression of TPE on telomeric white transgenes located at homologous and nonhomologous telomeres. Flies with a TAS deficiency show an increase in telomeric w transcript during development, especially in pupae. Moreover, a read-through w transcript initiated from a HeT-A promoter also increased with the presence of a 2L TAS deficiency. One explanation for this genetic interaction is physical associations between telomeres. We, therefore ask whether absence of 2L TAS leads to changes in nuclear chromosome organization, telomere- telomere associations, or associations of telomeres with nuclear lamina. A second explanation, which we are also testing, is TAS may regulate HeT-A transcription through an RNAi mechanism.

280A Unprotected Drosophila telomeres activate the spindle assembly checkpoint. Giovanni Cenci1, Mariarosaria Musaró1,2, Laura Ciapponi2, Barbara Fasulo3, Maurizio Gatti2. 1) DISTeBA, Univ Lecce-Ecotekne, Leece, Italy; 2) Dept. of Genetics and Molecular Biology, Univ. “La Sapienza” (Rome); 3) Molecular Cell and Developmental Biology Dept., Univ. of California, Santa Cruz (Santa Cruz). We have previously shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. We have found that HOAP-depleted telomeres activate the spindle assembly checkpoint (SAC). The cav -induced SAC is partially overridden not only by mutations in the SAC genes zw10 and bubR1 but also by mutations in mei-41 (ATR), mus304 (ATRIP), rad50, grp (CHK1) and tefu (ATM). As expected, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulate at the kinetochores of cav mutant cells. Surprisingly, BubR1 also accumulatees at the cav mutant telomeres in a mei-41, mus304, rad50, grp and tefu -dependent manner. Collectively, our results suggest that recruitment of BubR1 by dysfunctional telomeres inhibits the Cdc20/APC function, preventing the metaphase-to-anaphase transition.

281B Mapping of the Telomere elongation gene. James Mason, Radmila Capkova Frydrychova. Lab Molec Genetics, D3-01, NIH/ NIEHS, Res Triangle Pk, NC. The Telomere elongation (Tel ) mutation increases telomere length by increasing the rate of addition of the telomere-specific retrotransposons to chromosome ends. After a few years the longer chromosome ends can be identified easily on polytene chromosomes. Previous data suggested that Tel maps in the middle of chromosome 3R at about 91-93. The E(tc) mutation has the same phenotype and maps to the same place. We used P element-mediated male recombination to locate Tel between 92A2 and 92A11, a region of about 320 kb and 27 annotated genes. We sequenced genes whose annotation suggested a function in DNA or protein metabolism, or had no annotated function. Two genes, CG5316 (damaged DNA binding, single-strand break repair) and CG15025 (unknown function), showed sequence differences from the available genome sequence and from our Oregon R control in both the Tel and E(t)c mutant lines. We are currently sequencing these two gene regions in other strains, e. g. Tel recombinant lines, to ask which of these sites corresponds to the Tel gene. A P element inserted between these two genes will also be used to generate recombinants in males to distinguish between these two genes as candidates for Tel. 184 POSTERS: Regulation of Gene Expression

282C MEF-2 and CF-2 collaborate in activation of the myogenic program in Drosophila. Anton Bryantsev, Kathleen Kelly-Tanaka, Thai Lee, Richard Cripps. Department of Biology, University of New Mexico, Albuquerque, NM. The process of myogenesis requires the coordinated activation of many structural genes whose products are required for myofibril assembly, function and regulation. While numerous reports have documented the importance of the myogenic regulator Myocyte Enhancer Factor-2 (MEF2) in muscle differentiation, it is also known that the interaction of MEF2 with co-factors is critical to the realization of muscle fate. Here, we identify a promoter region required for full MEF2-mediated activation of muscle-specific structural Actin57B gene in the Drosophila embryo, and we identify the zinc finger transcriptional regulator Chorion Factor-2 (CF2) as an additional factor binding to the Act57B promoter. We map two functional CF2-binding sites along the promoter sequence, in the close proximity to the single MEF2-binding site. We demonstrate that both MEF2 and CF2 can be independent activators of the Act57B promoter and, furthermore, they synergistically collaborate in activating the Actin57B target gene in vitro and in vivo. More globally, MEF2 and CF2 synergistically activate the enhancers of other tested structural muscle genes: Mhc and TnI. In vivo, loss of CF2 results in reduction of expression levels of Act57B, Mhc, and TnI. These findings validate a general importance of CF2 alongside MEF2 as a critical regulator of the myogenic program, identify a new transcriptional regulator capable of functioning with MEF2 to control cell fate, and provide further insight into the network of regulatory events that function to shape the developing musculature.

283A Weckle is required for the transcriptional activities of Dorsal in Drosophila. Dechen Fu, Mike Levine. Molecular and Cell Biology, U. C. Berkeley, Berkeley, CA. 94720. Weckle, a Zinc finger domain protein, is identified to be essential for dorsal-ventral pattern formation in early embryos of Drosophila by F1 Genetic Screen. Mutations of weckle result in dorsalized embryos, which is similar to the dorsal loss function mutation. Recently, Weckle is found to be associated with Toll pathway, which initiates the gradient of Dorsal along dorsal-ventral axis, and thus establishes the embryonic dorsal-ventral polarity. However, the role of Weckle protein in regulating the transcriptional activity of Dorsal protein has not been characterized. Here, we found that Weckle protein, which present in both cytoplasma and nucleus in early embryos, can also work as a transcriptional co-factor by binding specific DNA sequences via its Zinc-finger domain in vitro. Mutations of those Weckle binding sites in the enhancers of downstream genes of dorsal, including brinker (brk), ventral neuroblasts defective (vnd) and Short gastrulation (sog), result in either loss or reduced dorsal-dependent expression of these genes in early embryos. Interestingly, our Chromatin-immunoprecipitation (CHIP) results show that weckle site mutation in brk enhancer leads to disassociation of both Weckle and Dorsal protein from this enhancer in early embryos, and the recruitments of RNA polymerase II in inhibited. Thus, in addition to affect the nuclear translocation of Dorsal protein via Toll pathway, Weckle protein may also affect the DNA binding ability, and thus the transcriptional activity, of Dorsal protein in early developmental processes of Drosophila.

284B Atonal and Senseless regulate rhomboid expression during embryonic chordotonal organ development. David Li-Kroeger, Lorraine Witt, Brian Gebelein. Division of Developmental Biology, Cincinnati Childrens’ Hospital, Cincinnati, OH. The reiterative use of signaling pathways during development necessitates their precise temporal and spatial regulation. The epidermal growth factor (EGF) signaling pathway is controlled by the cell-specific expression of Rhomboid (Rho), a protease that cleaves the EGF ligand Spitz (Spi). As spi is ubiquitously expressed but sequestered in the Golgi unless processed, EGF signaling occurs when and where Rho is present. A subset of sensory organ precursor (SOP) cells transiently up-regulate rho to secrete Spi during development, inducing the recruitment of additional SOP cells and oenocytes. Our goal is to understand the transcriptional inputs regulating rho in these chordotonal (ch) SOP cells. We have identified a cis-regulatory element of rho, Rho654, that is specifically expressed in abdominal ch SOP’s. Co-staining with the proneural factor Atonal (Ato) demonstrates that Rho654-LacZ is expressed in ch SOP cells shortly after specification. Furthermore, the location of β-gal in the most anterior neuron of mature lateral ch organs (lch5) show that Rho654-LacZ is active predominantly in the SOP cell responsible for inducing oenocytes. Using the Gal4/ UAS system, we show that Rho654-LacZGal4>rho is sufficient to induce oenocytes in an otherwise rho mutant background. We then chose as candidates the genes ato and the conserved zinc-finger gene senseless (sens), since ch organ SOP cells require Ato and Sens for development. Furthermore, Sens participates in a positive feedback loop to cell-autonomously stimulate ato expression in SOP’s. Using a combination of genetics and expression analysis, we find that ato is necessary for Rho654-LacZ activation. Moreover, Sens plays two distinct roles in Rho654-LacZ regulation: 1) Sens indirectly activates Rho654-LacZ through the stimulation of ato, and 2) Sens directly binds to a conserved site in Rho654-LacZ to repress its expression in SOP cells potentially limiting the duration of Rho654-LacZ expression in these cells. These data support a model where the transient expression of ato and accumulation of Sens controls the timing of EGF signaling. POSTERS: Regulation of Gene Expression 185

285C Analysis of the DNA binding activity of the Drosophila Zic family member, odd-paired (opa). Aditya Sen, Heuijung Lee, Brian Stultz, Deborah Hursh. Division of Cell and Gene Therapies, CBER/FDA, Bethesda, MD. The Drosophila pair-rule gene odd-paired (opa) is a homolog of the Zic (Zinc finger protein in the cerebellum) family of mammalian transcription factors. Post-embryonic loss of function of opa causes defects in ventral head formation. These defects are indistinguishable from a specific class of cis-regulatory mutations, called dpps-hc, in decapentaplegic (dpp), the Drosophila homolog of BMP 2/4. opa is necessary for the activation of peripodial-specific dpp transcription in the eye/antennal disc, associated with dpps- hc mutations. The role of opa in adult Drosophila head development suggests conservation of genetic pathways across animal development, as mutations in vertebrate Zic genes are associated head malformations, such as holoprosencephaly. We are analyzing whether the role of Opa in the activation of Dpp/BMP expression in fly head development is direct, or if other factors contribute. We have taken a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) approach to determine potential targets for Opa.

286A Identification of Nuclear Localization Signals in the Drosophila OVO Protein. Akram Abou-Zied1, Mark Garfinkel2, Anthony Mahowald3. 1) Suez Canal University, Department of Zoology, Faculty of Science, Ismailia, Egypt; 2) University of Alabama at Birmingham, Department of Environmental Health Sciences, Birmingham, AL, USA; 3) Stanford University School of Medicine, Department of Developmental Biology, Stanford, CA, USA. In flies, ovo is critical for female germ cell viability, as judged by its most severe mutant phenotypes. Many of these phenotypes show female-specific defects that include cell death, aberrant cell proliferation, partial transformation of sex identity and abnormal egg chamber differentiation. Several lines of evidence support the proposal that OVO proteins act as transcription factors. The C- terminal sequences of all known OVO proteins contain four zinc-fingers of the C2H2. The C2H2 zinc-finger is composed of 22-26 amino acids that form a two-strand ?-sheet region and an ?-helical region that binds DNA with strong affinity. In this study we obtained germline-transformants of Drosophila that ectopically express GFP-OVO zinc-finger fusion protein in the third instar salivary glands, and this protein is nuclear-localized. We also found that the signal required for localizing OVO protein into the nucleus in the carboxy-terminus domain of OVO protein. No nuclear localization signal was found in the N-terminus or in the other domains. A stretch of 25 amino acid residues (YLCTFCGKGFNDTFDLKRHTRTHTG) within the second zinc-finger of this transcription factor is required for its nuclear localization in Drosophila S2 culture cells and this signal is constitutively active. This suggests a potential interaction between the OVO zinc finger and a signal for nuclear localization. Identification of nuclear localization signals of OVO will be a key advance in understanding the structural basis of how this subfamily of C2H2 zinc finger proteins recognizes and regulates the transcription of developmentally target genes.

287B Transcription elongation controls expression of Hox genes during Drosophila development. Vivek S. Chopra, Michael Levine. Center for Integrative Genomics, University Of California, Berkeley, Berkeley, CA. The Hox genes control the anterior-posterior patterning of most metazoan embryos. During Drosophila development, the Hox genes are initially regulated by the segmentation cascade, but these patterns are maintained by the Polycomb and trithorax complexes during the remainder of the life cycle. The precise spatio-temporal limits of Hox gene expression require rapid transcription responses. We provide genetic as well as molecular evidence that the Hox genes are in a “poised state”. An active form of Pol II is bound to the promoter regions of the Hox genes prior to their activation. Upon induction, Pol II is released from the pause site via regulation of elongation factors such as Elongin A. 186 POSTERS: Regulation of Gene Expression

288C A screen for transcription factors that coordinate cell fate determination and patterning of photoreceptors in the Drosophila eye. Hui-Yi Hsiao, Robert Johnston, David Jukam, Claud Desplan. Biology, New York University, NY. In the Drosophila eye, each ommatidium contains eight photoreceptors (PRs) arranged in a trapezoid shape. The different PRs are distinguished by several characteristics including position and Rhodopsin (Rh) expression. The inner PRs (R7 and R8) lie in the center of the trapezoid shape which is formed by the outer PRs (R1-R6). After initial PR fate specification, outer PRs rotate relative to the dorsal-ventral midline resulting in a mirror-image symmetric pattern in the retina. The mechanism underlying PR rotation is regulated by the Notch and Frizzled pathways. In addition to position, another distinction between outer and inner PRs is Rh expression. Outer PRs express Rh1 whereas inner PRs express Rhs involved in color vision (Rh3, Rh4, Rh5, and Rh6). Several transcription factors are known to regulate inner and outer PR fate. For instance, spalt prevents inner PRs from acquiring the outer PR fate, and sevenup prevents some outer PRs from acquiring the inner PR fate. Though some genetic programs controlling ommatidial rotation and outer/inner fate determination are well characterized, many regulatory steps still need further study. We are performing a UAS-RNAi screen knocking down all known transcription factors to understand the regulatory mechanisms underlying the determination and position of outer PRs. We use Rh1-GFP as readout to visualize polarity and cell fate phenotypes. Preliminary results of screen will be presented.

289A Regulation of toy, a Pax6 gene in Drosophila. Linn Jacobsson1, Jesper Kronhamn2, Åsa Rasmuson-Lestander1. 1) Molecular Biology, Umea, Sweden; 2) UCMP, Umea University, Umea, Sweden. The Pax-6 gene was first identified in mammals and encodes a transcription factor containing two DNA-binding motifs, a paired domain, and a paired-type homeodomain. Heterozygous mutations of the human Pax-6 gene cause various forms of congenital eye abnormalities; aniridia and Peters’ anomaly. The discovery that the eyeless (ey) gene is the pax-6 homologue in Drosophila, revealed an important function for Pax-6 also in the developing insect eye, which have a different organization and structure. Another Pax-6 homologue was identified, which has more features in common with Pax-6 proteins of other phyla than with ey, namely twin-of- eyeless (toy). The two Drosophila Pax-6 genes are expressed in the developing central nervous system and visual systems. During embryogenesis, transcripts of the toy gene are first detected at the cellular blastoderm stage in the posterior procephalic region. During subsequent development, toy is expressed in the dorsolateral head ectoderm that gives rise to the brain and the visual system, including the optic lobe and the imaginal eye discs. Since It has been shown that Toy regulates Ey transcription by binding to an eye-specific enhancer within the second intron of the ey gene or main focus lays on the regulation of the toy gene. To be able to dissect the regulation of the toy gene, transgenic flies with various overlapping parts of the presumed toy regulatory region driving a reporter gene (GAL4) have been made. The use of the yeast GAL4-UAS system enables us to use different reporters (UAS-LacZ, UAS-GFP) to analyse the expression patterns. The analyses from these constructs show that an 800bp region, just upstream of and including the toy transcription start is sufficient to obtain reporter gene expression in the embryonic eye-antennal primordium and brain as well as in larval eye discs. Further analysis of presumptive toy enhancer regions will be done focusing on the introns. These regions will be analysed more thoroughly for binding sites and consensus sequences for candidate regulatory proteins.

290B Two leg-arista-wing complex mutations disrupt different coding regions of TBP related factor 2 in Drosophila melanogaster. Simonova B. Olga1,2, Burdina Natalia.1, Vorontsova Yulia2, Cherezov Roman1, Modestova Elena1, Kulikova Dina1,2, Mertsalov Ilya1,2. 1) Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russian Federation; 2) Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russian Federation. The viable P-element induced leg-arista-wing complex (lawc) mutation has pleiotropic phenotype effect: homeotic transformation of arista into tarsus, morphological leg defects, abnormal wing formation, ectopic bristles etc. Early it was shown that all insertional viable and lethal lawc alleles occupy 5’-UTR of TBP related factor 2 gene (trf2) and the lawc mutants’ viability was restored in rescue experiments with transgenic constructs that express the trf2 gene domain homologous to TATA-box binding domain of TBP. All lethal mutations, except EMS-induced lawc520, partially complement to viable lawcp1. Heterozygotes lawc520/lawcp1 demonstrate strong lawc phenotype: transformed aristae, low viability, disturbed oocyte-specific protein transport and disorganized embryo polarization. To clear out whether lawcp1 and lawc520 changes trf2 coding region we analyze genomic sequences, containing trf2 ORF by PCR-method. We found correspondently the deletion of 54 bp in lawcp1 3‘-coding region and the deletion of 9 bp in lawc520 5‘-coding region. So, we propose the different significance of concrete TRF2 domains because the deletion of 3 aa in C-terminal region unlike the deletion of 18 aa in N-terminal region causes embryonic lethality. POSTERS: Regulation of Gene Expression 187

291C The Role of Paused Polymerase in the Regulation of Drosophila Developmental Genes. Jessica Piel, Joung-woo Hong, Michael Levine. Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA. Traditional views of transcription claim that the rate-limiting step of gene activation is recruitment of the polymerase to the promoter. This step is facilitated by transcription factors that recruit RNA polymerase to bind, and that once polymerase is bound, gene activation will inevitably follow. While this model suggests that all genes are activated by polymerase recruitment, certain genes, in contrast, are regulated at the point of elongation. These genes, such as hsp70, have PolII primed at their promoters prior to activation. Once stimulated by heat shock, the polymerase is released and allowed to continue transcription through the entire gene. It was previously thought that this type of regulation was limited to a small set of genes. However, recent PolII ChIP-Chip data indicate several developmental genes that also exhibit paused PolII at their promoters when inactive. It is possible that polymerase pausing may be a key form of regulation that allows quick activation of important genes in the rapidly-developing Drosophila embryo. It is our goal to identify all of the paused developmental genes and the role of pausing in embryonic gene regulation.

292A Characterization of tfiia-s-2, a germline-specific homolog of the small subunit of the General Transcription Factor, TFIIA. Margaret Wood, Cynthia Maddox, Haley Adams, Leah Cohen, Rebekah Turk, Mark Hiller. Biology, Goucher College, Baltimore, MD. Eukaryotic General Transcription Factors are necessary to position RNA polymerase at promoters and initiate transcription. Isoforms of several General Transcription Factor subunits are known to be important for tissue-specific gene expression. The General Transcription Factor TFIID is comprised of TBP (TATA-binding protein) and up to fourteen TAFs (TBP-associated factors). In Drosophila, testis-specific homologs of several TFIID subunits (testis-TAFs) are necessary for the transcription of some genes in the testis. Mutations in the testis-TAF encoding genes leads to defects in transcription of many genes normally expressed from testis-specific promoters and result in an accumulation of primary spermatocytes and a block in spermatid differentiation. Another General Transcription Factor, TFIIA, physically associates with TFIID at promoters. TFIIA consists of three protein subunits. A single gene, tfiia-l, encodes a 48 kD polypeptide which is protolytically cleaved to form two proteins, a 30 kD and a 20 kD species. A separate gene, tfiia-s, encodes the small subunit, a 14 kD protein. Here we show that the gene tfiia-s-2 (CG11639) encodes a germline- specific homolog of the 14kD subunit. in situ hybridization indicates that tfiia-s-2 is expressed in gonial cells and primary spermatocytes of the testis. This is in contrast to the TFIID subunit homologs where expression is restricted to primary spermatocytes. Alternative splicing of a single tfiia-s-2 exon, encoding twenty seven amino acids, may lead to the expression of two different 14 kD-like proteins. In primary spermatocytes, a testis-specific TFIIA-like complex may interact with the testis-specific TFIID-like complex to regulate gene expression.

293B Characterizing the transcriptional regulation of the Drosophila E75 gene through analysis of upstream non-coding DNA. Travis Bernardo1, Habiba Jannat1, Bill Maughan1, Veronica Dubrovskaya1, Edward Berger2, Edward Dubrovsky1. 1) Biology, Fordham University, Bronx, NY; 2) Biology, Dartmouth College, Hanover, NH. Drosophila development is regulated by two hormones, 20-hydroxyecdysone (ecdysone) and juvenile hormone (JH). Ecdysone binds to the EcR/Usp heterodimer, which then binds to an ecdysone response element and increases gene expression. While ecdysone action is well-characterized, both the receptor and the mode of action of JH are currently unknown. We previously found that expression of the E75 gene is induced by both ecdysone and JH. The E75 gene occupies 100 kb of genomic DNA; it has 4 alternative promoters, producing isoforms E75A, B, C, and D, as well as two polyadenylation sites. Our current efforts are focused on identifying DNA elements involved in the hormonal regulation of the E75A and E75D isoforms. To that end, we are employing several experimental approaches. First, since conserved non-coding elements may be of functional importance, we are identifying sequences that are conserved among distantly related drosophila species and among functionally distinct genes which share a common hormonal inducibility. Second, we are using chromatin immunoprecipitation (ChIP) followed by real-time PCR to identify non-coding regions with elevated levels of two known markers for enhancer regions, monomethylated histone 3 at lysine 4 and nucleosomal depletion of histone 3. Finally, we are testing the functionality of putative regulatory elements identified by both the sequence conservation and ChIP analysis using transient transfection assays. We have performed ChIP on Drosophila Schneider Line 2 (S2) cells using antibodies to a region common to all forms of histone 3, as well as antibodies specific to histone 3 monomethylated at lysine 4. We have found several non-coding regions upstream of the E75A and E75D isoforms that display peaks of H3K4 monomethylation concomitant with depletion of histone 3. The results suggest the presence of enhancers at some of these regions which may be involved in the transcriptional regulation of E75 possibly by ecdysone or JH. 188 POSTERS: Regulation of Gene Expression

294C The TCF Helper site, a new cis-regulatory element in the Wingless signaling pathway. Mikyung Chang, Jinhee Chang, Anu Gangopadhyay, Andrew Shearer, Kenneth Cadigan. MCDB, University of Michigan, Ann Arbor, MI. The Wingless (Wg) signaling pathway plays important roles during development through transcriptional regulation. The major transcription factor in the pathway is the sequence-specific DNA-binding proteins, TCF/pangolin. Due to considerable degeneracy, the consensus TCF binding sites can be found at high frequency throughout the genome. However, we found that only some TCF site clusters can respond to Wg signaling in vivo. This suggests that there is sequence information in Wg response elements (WREs) in addition to TCF binding sites. We have identified another motif that is required for activation of two WREs in the naked cuticle (nkd) gene by Wg signaling. The new motif was termed the TCF Helper site (Helper site). Similar to TCF binding sites, the Helper sites in the WREs of nkd are functionally important in numerous fly tissues as well as in cultured cells. We found that WREs in other Wg target genes such notum and sloppy-paired 1 also contain functional Helper sites. These results clearly show that the function of Helper sites is required in several WREs in a variety of fly tissues. Helper sites have no activity on their own, but greatly enhance the ability of TCF sites to respond to the pathway. However, they do not alter activation of UAS reporters by several Gal4 fusion proteins. It shows specificity of Helper sites towards TCF sites. Binding of TCF to a nkd WRE was reduced when Helper sites were mutated. Our results support a general role for the Helper site in WRE activation and suggest that they help recruit TCF to WREs. We will present our efforts to identify the protein(s) that bind to the Helper motif.

295A Characterization of Enhancer of split regulatory region sequences conserved in multiple Drosophila species. Deborah Eastman, Morgan Maeder. Biology, Connecticut Col, New London, CT. The Enhancer of split-Complex (E(spl)-C) genes are targets of the Notch signaling pathway. During Drosophila neurogenesis Su(H) dependent Notch activation up-regulates transcription of the (E(spl) genes, which function to down-regulate proneural gene activity. Although these genes have overlapping expression patterns during embryonic development due to common Su(H), proneural and bHLH reperessor binding sites, their expression patterns are distinct during later embryonic stages and in larval imaginal discs. This suggests the presence of different binding sites upstream of the various E(spl) genes. Our phylogenetic footprinting analysis of the E(spl) promoters from 12 different Drosophila species has identified conserved sequences that may provide insights into these differential regulators. We have identified conserved known transcription factor binding sites and novel sites upstream of the E(spl) genes and are now characterizing the possible roles of corresponding transcription factors in the regulation of E(spl) mgamma.

296B A cellular resolution atlas of gene expression in Drosophila pseudoobscura reveals interspecies variation in embryonic patterning. Michael Eisen2, Cris Luengo2, Charless Fowlkes4, Soile Keränen2, Clara Henriquez2, Lisa Simirenko2, Gunther Weber2, Oliver Rübel5, Min-Yu Huang5, Jitendra Malik3, David Knowles2, Mark Biggin2, Angela DePace1. 1) Department of Systems Biology, Harvard Medical School, Boston MA; 2) Genome Sciences Department, Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA; 3) Department of Computer Science, UC Berkeley, CA; 4) Department of Computer Science, UC Irvine, CA; 5) Computer and Data Visualization Department, UC Davis, CA. Understanding how transcriptional regulatory sequences evolve requires us to link changes in sequence with changes in function. We have applied high-resolution microscopy and image processing methods to blastoderm embryos of D. melanogaster and D. pseudoobscura to determine the expression patterns of key transcriptional regulators and a subset of their targets in their native context at cellular resolution in 3D over the hour prior to gastrulation. These techniques allow multiple types of statistically rigorous inter-species comparisons to be made, both between individual embryos and between composite multi-gene models, revealing widespread quantitative changes in expression patterns. We measure multiple types of variation, including changes in spatial position and number of cells comprising a pattern. These changes are gene specific; there is no overall trend in how expression patterns vary. Moreover, we see that portions of a gene expression pattern driven by distinct regulatory elements can change independently. Together these results argue that there are functional consequences of the regulatory sequence variation between D. melanogaster and D. pseudoobscura. Interpreting these functional differences in the context of specific sequence changes will shed light on cis-regulatory architecture, and the functional constraints under which cis-regulatory elements evolve. POSTERS: Regulation of Gene Expression 189

297C Hox and Senseless competition forms a molecular switch to regulate gene expression in the PNS. Brian Gebelein, David Li- Kroeger, Lorraine Witt. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Hox transcription factors regulate the patterning of many tissue types in metazoan organisms by regulating downstream target genes. How broadly expressed Hox proteins control gene expression in a cell-specific manner has remained unclear. Here, we show that the Drosophila Abdominal-A (Abd-A) Hox factor directly stimulates rhomboid (rho) expression in specific sensory organ precursor (SOP) cells by antagonizing repression by the Senseless (Sens) zinc finger transcription factor. Previous studies revealed that rho activation in SOP cells results in the secretion of Spitz (Spi), an epidermal growth factor (EGF) ligand, to induce hepatocyte-like cells (oenocytes) and secondary SOP cells in the abdomen. We identified a conserved rho cis-regulatory element that integrates both Hox and Sens inputs resulting in abdominal-specific rho expression. In the thorax, Sens directly represses rho, while in the abdomen, Abd-A, together with the Hox co-factor proteins Extradenticle (Exd) and Homothorax (Hth), antagonizes Sens through direct DNA binding competition. Thus, Hox-Sens competition provides a simple molecular switch for the regulation of rho and thereby EGF signaling. As sens encodes a conserved transcription factor essential for many aspects of development and the core Sens and Hox binding sites share significant overlap, we propose that Hox-Sens antagonism will be a general mechanism of gene regulation.

298A Molecular dissection of the IAB5 cis-regulatory module in Drosophila. Sara E. Goetz, John M. Allen, Robert A. Drewell. Biology Department, Harvey Mudd College, Claremont, CA. The three homeotic genes that comprise the bithorax complex (BX-C) control segment identification in the posterior thorax and abdominal regions during embryonic development. Cis-regulatory modules (CRMs) are non-coding regions of DNA in the BX-C that regulate the expression of the three homeotic genes: Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). IAB5 is a transcriptional enhancer that drives the expression of Abd-B. Using a combination of bioinformatics, cell culture and transgenic experimental approaches we are conducting a molecular dissection of the IAB5 region in order to determine which sequences are necessary for the functional activity of the enhancer.

299B Characterization of Ultrabithorax-responsive regulatory sequences. Bradley Hersh, Jamie Wood. Dept Biological Sciences, Clemson University, Clemson, SC. Though regulation of development by Hox proteins is important in the evolution of animal morphology, relatively few direct in vivo- regulated target genes have been identified and the organizational features of regulatory sequences directly bound by Hox proteins remain poorly characterized. We identified candidate direct targets of Ultrabithorax (UBX) by microarray analysis and analyzed regulatory regions of several candidates. We isolated a cis-regulatory element (CRE) upstream of the CG13222 gene that is activated in the haltere imaginal disc in response to UBX and is directly bound in vitro by UBX homeodomain. Mutation of a single UBX binding site in the CRE abolishes in vitro binding and leads to loss of in vivo activity-the first demonstration of a directly activated target CRE by UBX in the haltere. This critical UBX binding site and sequence surrounding the site are broadly conserved within Drosophila. We have undertaken three approaches to identify additional sequence requirements beyond the single UBX binding site. First, we have generated a minimal regulatory element to determine the extent of additional sequence involved. Next, we have introduced the homologous CRE from four Drosophila species into D. melanogaster, thereby defining conserved sequence regions that may contribute to its activity. Finally, we have mutated conserved sequence flanking the critical UBX binding site and demonstrated its contribution to the activation of this CRE. We also identified a CRE upstream of the anachronism gene that drives expression in four small clusters of cells in the wing, but drives no expression in the haltere, indicating repression by UBX. By identifying direct target regulatory sequences of UBX in the haltere, we will better understand the primary sequence requirements and organizational features that mediate regulation by Hox proteins. Based on these features, we can explore the evolution of Hox-regulated target gene networks in other insects and relate these network changes to morphological differences. 190 POSTERS: Regulation of Gene Expression

300C Functional activity of rapidly evolving cis regulatory modules in the Drosophila bithorax complex. Margaret C.W. Ho1, Holly Johnsen1, Esther Bae1, Ben Schiller1, Jason W.H. Chan1, William Fisher2, Susan E. Celniker2, Robert A. Drewell1. 1) Biology, Harvey Mudd College, Claremont, CA; 2) Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, CA. The homeotic genes regulate segment identity during development of the embryo and are important in the evolution of animal morphology. The bithorax complex (BX-C) in Drosophila contains three homeotic genes, which are regulated in the embryo by cis- regulatory modules (CRMs), including enhancers and insulators. How the CRMs functionally evolve is not clear. The recent publication of the genomic sequences of a number of different Drosophila species has allowed us to examine the underlying evolutionary conservation of the CRMs. We applied bioinformatic and computational approaches to perform cross-species analysis of the CRMs. The CRMs demonstrate a striking lack of underlying evolutionary conservation, suggesting that they are evolving rapidly. We are currently testing the functional activity of the CRMs in embryonic transgenic assays.

301A The vnd/nk-2 homeobox gene is activated in different neuroblasts by different combinatorial sets of enhancers in the 5’- upstream enhancer region. Andrey Ivanov, Siuk Yoo, Marshall Nirenberg. Lab Biochemical Genetics, NIH/NHLBI, Bethesda, MD. We have continued to study how a pattern of neuroblasts is generated in the central nervous system (CNS). Nucleotide sequences within the vnd/NK-2 5’-upstream enhancer (regions A and B) that have been conserved during evolution were identified by comparing genomic nucleotide sequences from seven Drosophila species, which diverged between 3 and 40 million years ago. Twelve short nucleotide sequences between 20 and 41 bp in length were found that are highly conserved within the 2.5 kb late enhancer (regions A and B) of the vnd/NK-2 gene. We prepared 12 DNA constructs, each with a different deletion of a highly-conserved sequence within region A or region B within the vnd/NK-2 gene, 7 DNA constructs, each with two deletions of highly conserved sequences, and 4 DNA constructs with large deletions. The 23 DNA constructs were cloned into pChs-Gal4 or pHStinger GFP vectors and at least 4 transgenic fly lines of each kind were obtained. Transgenic fly lines containing Gal4 constructs were crossed with UAS-lacZ and with wingless-lacZ; UAS-GFP flies. Embryos were incubated to allow development to embryonic stages 11-12 and β-galactosidase and GFP/RFP were visualized by immunohistochemistry staining or fluorescence. The results show that different combinations of enhancers activate the vnd/NK-2 gene in the 7 NBs examined. The DNA sites required for activation of the vnd/NK-2 gene in NB 1- 2, 3-1, 4-1, and 7-1 are cooperative. For example, deletion of any one of the 4 sites required for expression of the vnd/NK-2 gene in NB 7-1 in the -4.0 to -2.8 kb DNA region resulted in no expression of the vnd/NK-2 gene in NB 7-1. Our hypothesis is that the combinatorial set of DNA binding sites for transcription factors for each NB may function as a molecular mechanism for selection of NB cell type, position, and time of appearance of the NB.

302B Analysis of cis-regulatory elements controlling repo transcription in Drosophila. Robert W. Johnson, Jamie L. Wood, William C. Colley, Bradley W. Jones. Biology, University of Mississippi, Oxford, MS. The glial cells missing (gcm ) gene was identified as a “master regulator” of glial cell fate in the fruit fly Drosophila . However, gcm is also expressed in and required for the development of larval macrophages and tendon cells, and lamina neurons in the adult CNS. Thus, Gcm protein activates the transcription of different sets of genes in different developmental contexts. How Gcm regulates these different outcomes is not known. Our goal is to identify collaborators that act with Gcm to promote the transcriptional activation of Gcm target genes specifically in glial cells, or prevent their activation in the other tissues in which Gcm is expressed. We have focused on the transcriptional regulation of a well-characterized glial-specific Gcm target gene, the transcription factor reversed polarity (repo); we aim to understand how the transcription of the glial-specific Gcm target gene repo is regulated by Gcm and other factors. We have defined a minimal cis-regulatory element that recapitulates the endogenous repo expression dependent on a single Gcm binding site. We have also located three different cis-regulatory elements that drive cell-specific expression independent of Gcm binding sites: 1) A distal element that promotes expression in dorsolateral epidermis; 2) A repressor element that suppresses expression in the epidermis; 3) A proximal element that promotes expression in a subset of cell body glia. Currently we are characterizing these elements to define minimal sequences required for expression or repression with the goal of identifying interacting factors using genetic, biochemical and bioinformatic approaches. POSTERS: Regulation of Gene Expression 191

303C Systems biology analysis of the cis-regulatory control of the expression of Drosophila even-skipped stripes 2 and 3. Ah- Ram Kim1, Shuling Hou2, David Sharp3, John Reinitz1. 1) Department of Applied Mathematics and Statistics,and Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-3600, U.S.A; 2) Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; 3) Chief Science Office, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. The classical picture of the regulatory regions of metazoan genes is that complete promoters are collections of independent enhancers, and each enhancer is composed of a cluster of binding sites. The foundations of the above description, as well as clear evidence of its incompleteness are seen in the biology of the DNA that regulates the expression of stripes 2 and 3 of the Drosophila pair-rule gene even-skipped(eve). Here we present a quantitative and predictive model of the the transcriptional readout of two enhancer fusions of the -3.8 to -3.3 kb (stripe 3 enhancer) and the -1.6 to -1.1 kb (stripe 2 enhancer) regions of the gene eve in either orientation without spacer sequence between them. The model is based on a set of transcription factor binding sites and quantitative, time-resolved expression data at celluar resolution. We utilized the Cre-mediated site-specific transgenesis system to obtain quantitative gene expression maps of different reporter genes. Site-specific integration allows for direct comparison of gene expression data in the reporter genes. The model produces the correct gene expression pattern and shows that interactions between enhancers are important for generating novel patterns of gene expression during development.

304A Functional Analysis of the nerfin-1 Neuroblast Promoter. Mukta Kundu, Alexander Kuzin, Thomas Brody, Ward Odenwald. Neural Cell Fate Determinants, NINDS, Bethesda, MD. nerfin-1 belongs to a conserved subfamily of Zn-finger transcription factors present in all metazoans including man. Our characterization of loss- and gain-of-function mutants reveals that nerfin-1 is required for interneuron axon guidance (Kuzin et al., Dev. Biol. 277: 347-65, 2005). During embryonic nervous system development, nerfin-1 mRNA is dynamically expressed in both neural precursor cells and nascent neurons. For example, nerfin-1 expression is detected in all early delaminating neuroblasts (NBs). Use of the phylogenetic footprinting tool EvoPrinter (Odenwald et al., Proc. Natl. Acad. Sci. 102: 14700-5, 2005) identifies multiple putative enhancer regions both upstream and downstream of the transcribed region. In vivo transgene analysis of each of these regions reveals the modularity of cis-regulatory enhancer elements that regulate nerfin-1 expression in early NBs and subsets of nascent neurons in both the embryo and developing adult CNS and PNS. EvoPrint analysis reveals that the NB enhancer consists of multiple conserved sequence blocks (CSBs) that are nearly genus invariant. Based on this analysis, transgenic reporter lines were created that systematically analyzed NB enhancer CSB function; this analysis shows that CSBs perform identifiably distinct roles. We have developed an alignment tool, cisDecoder, that scans CSBs, generating libraries of cisDecoder tags (cDTs) that represent shared regulatory elements within functionally related enhancers (Brody et al., Genome Biol. 8: R75, 2007). cisDecoder analysis reveals the sub-structure of CSBs, consisting of overlapping sequence elements that are found in related or divergent enhancers. We have initiated a systematic in vivo analysis of these elements. This study has provided new insights into the internal structure and function of enhancers.

305B Transition from Notch-dependent to Notch-independent regulation of high-level Suppressor of Hairless expression in the socket cell. Feng Liu, James W. Posakony. Division of Biological Sciences/CDB, University of California San Diego, La Jolla, CA. In Drosophila, the transcription factor Suppressor of Hairless [Su(H)] is the primary mediator of the activity of the Notch cell-cell signaling pathway. For this function, it is expressed at low-to-moderate levels broadly or ubiquitously. In addition, its expression is subject to high-level autoactivation in response to Notch signaling, but only in the socket cell of external mechanosensory organs. Only the initiation of this autoregulatory activity of the Su(H) gene is dependent on Notch signaling; maintenance is independent of Notch function and requires Su(H) itself in cooperation with other, as yet unidentified, factor(s). Previously our laboratory identified an autoregulatory socket enhancer (ASE5) that directs the socket-specific up-regulation of Su(H) expression. We have now further dissected the functional elements within this enhancer in an attempt to understand the mechanism that mediates the transition from Notch-dependent to Notch-independent control of high-level Su(H) expression in the socket cell. So far, we have identified several cis-regulatory elements that are required for the socket-specific activity of the enhancer during different developmental stages. One of these cis-elements is required for ASE5 activity in pupal stages, but is dispensable in adults. On the other hand, ASE5 activity in adults requires the cooperation of at least two separate cis-regulatory motifs. This study will provide the basis for identification of the unknown factors that control the temporal program of Su(H) expression regulation in the differentiating socket cell. 192 POSTERS: Regulation of Gene Expression

306C Transcription factors bind thousands of active and inactive elements in the Drosophila blastoderm. Stewart MacArthur1, Xiaoyong Li1, Michael Eisen1,2, Mark Biggin1. 1) Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA; 2) Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Identifying the DNA regions bound by sequence specific transcription factors is central both to deciphering the complex genomic cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here we map sequences bound in Drosophila melanogaster embryos by transcription factors that control early developmental patterning. We show that these transcription factors bind with quantitatively different specificities to an overlapping set of several thousand genomic regions in blastoderm embryos. The more highly-bound regions include all of the well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the miRNAs transcribed in the blastoderm. These poorly-bound regions are, on average, far more distant from genes transcribed in the blastoderm than highly-bound regions, and are preferentially found in protein-coding sequences, suggesting that many are not involved in early- embryonic transcriptional regulation and may be non-functional. Specific high and moderate affinity in vitro recognition sequences for each factor are enriched in their bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies depending on which other factors bind to the same genomic regions in vivo, uncovering higher-order rules that must govern targeting of transcription factors. Comparative analysis demonstrates that predicted binding sites in the most strongly-bound regions are preferentially conserved compared to those in weakly bound regions, further supporting the idea that strong and weakly bound regions differ in function.

307A A Cell Non-Autonomous Feedback Loop Regulates neuralized Expression and Function Downstream of the Proneural Proteins During Sensory Organ Precursor Selection. Steven W. Miller, James W. Posakony. Division of Biological Sciences/CDB, UC San Diego, La Jolla, CA 92093-0349 USA. The Drosophila peripheral nervous system comprises a stereotypic array of mechanosensory bristles. The cells that make up each bristle are descendants of an individual sensory organ precursor cell (SOP), which derives from a region of the epithelium— termed a proneural cluster (PNC)—made competent by expression of the proneural bHLH proteins, Achaete and Scute. The number of PNC cells that adopt an SOP fate is regulated through the process of lateral inhibition, mediated by the Notch signaling pathway. Genes residing in the Enhancer of split [E(spl)-C] and Bearded (Brd-C) complexes are directly activated in non-SOP cells by the proneural proteins and the Notch pathway to inhibit the SOP cell fate. The E3 ubiquitin ligase Neuralized (Neur) is known to be expressed at high levels in the SOP cell and participates in Notch pathway function through the ubiquitylation and endocytosis of the ligand Delta. We have identified a cis-regulatory module from neur that orchestrates expression in SOPs; it is directly activated by the proneural proteins and directly repressed by E(spl)-C bHLH repressor proteins. We also provide evidence of both enhancer and endogenous neur expression in the PNC prior to SOP identification, suggesting that restriction of neur expression to the SOP is an important function of, and not just a consequence of, lateral inhibition. Consistent with this interpretation, defects in lateral inhibition can result either from mis-expression of neur in non-SOPs or inhibition of Neur function in the SOP. Together, our results support a model of SOP specification in which proneural protein function activates neur expression, which in turn is able to negatively regulate both its own expression and Neur protein function through activation of bHLH repressor and Brd family genes, respectively, in neighboring PNC cells.

308B The Homeotic selector genes and the NK homeodomain transcription factor Tinman togther control cardiac svp expression in the Drosophila dorsal vessel. Kathryn M. Ryan, Richard M. Cripps. Dept Biol, Univ New Mexico, Albuquerque, NM. A complex regulatory cascade is required for normal cardiac development, and many aspects of this network are conserved from Drosophila to mammals. In Drosophila, the seven-up (svp) gene, an ortholog of the vertebrate chick ovalbumin upstream promotor transcription factors (COUP-TFI and II), is initially activated in the cardiac mesoderm and is subsequently restricted to cells forming the cardiac inflow tracts. Here, we investigate svp regulation in the developing cardiac tube. The Hox segmental patterning genes and Tin are required and sufficient for cardiac svp expression, making these proteins likely regulators of svp. Using bioinformatics, we have identified a 1007-bp enhancer of svp which recapitulates its entire expression in the embryonic heart and other mesodermal derivatives, and we show that this enhancer is initially activated by the NK homeodomain factor Tinman (Tin) via two conserved Tin binding sites. Mutation of the Tin binding sites significantly reduces enhancer activity both during normal development and in response to ectopic Tin. Through deletion analysis a minimal cardiac svp enhancer of 282bp has been identified. This is the first identification of an enhancer for the complex svp gene, demonstrating the effectiveness of bioinformatics tools in assisting in unraveling transcriptional regulatory networks. svp expression is mediated axially by the Hox gene products and cardiac specific factors such as Tin; thus characterizing svp cardiac regulation will help the general understanding of how diverse signals are integrated to generate organ specific gene expression. Our studies define a critical component of the svp regulatory cascade and place gene regulatory events in direct apposition to the formation of critical cardiac structures. POSTERS: Regulation of Gene Expression 193

309C Functional analysis of promoter-enhancer interactions at the Drosophila bithorax complex. Ben Schiller, Margaret Ho, Omar S. Akbari, Esther Bae, Robert A. Drewell. Biology Department, Harvey Mudd College, Claremont, CA, USA 91711. There are many examples within gene complexes of shared transcriptional enhancers interacting only with a subset of target promoters. Promoter competition and insulators are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C) over 300 kb of intergenic DNA sequence is responsible for directing expression of the 3 homeotic genes during embryonic development. We have been examining the mechanisms which regulate promoter-enhancer interactions at the BX-C. In the BX-C, the IAB5 enhancer is located 55 kb 3’ of the Abdominal-B promoter and 48 kb 5’ of the abdominal-A promoter. Although roughly equidistant from the two promoters, IAB5 specifically interacts only with the Abdominal-B promoter, even though the enhancer and promoter are separated by at least two insulators. Our experiments demonstrate that a novel 254bp cis-regulatory element located 5’ of the Abd- B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. Furthermore, the promoter tethering element (PTE) sequence is sufficient to direct IAB5 to an ectopic site in competition assays. We are currently deleting the PTE from its endogenous locus by imprecise excision of a P element insertion to investigate the in vivo function of this element.

310A Structure, function, and evolution of a Notch- and EGFR-regulated eye enhancer. Christina I Swanson, Scott Barolo. Cell and Developmental Biology, University of Michigan, Ann Arbor, MI. Expression of the Drosophila Pax2 gene in cone cells of the developing eye is required for proper cone cell fate specification (Fu and Noll, 1997). Cone cell expression of Pax2 is controlled by Notch signaling, EGFR signaling, and the Runx-family protein Lozenge (Lz), all of which directly regulate the sparkling (spa) enhancer of the Pax2 gene (Flores et al, 2000). The spa enhancer contains 12 binding response elements for Notch, EGFR, and Lz; these sites are necessary for proper gene patterning. However, we have found that simply combining these 12 sites is insufficient to drive gene expression in vivo. We have identified multiple novel sequences within spa that are essential for enhancer function, suggesting that a surprising number of additional regulatory factors may bind within spa. We have also discovered that the spatial organization of spa is remarkably flexible, unlike the structure of those enhancers known as “enhanceosomes.” Intriguingly, reorganization of spa can affect the pattern of gene expression. Our data suggest that the regulation and organization of spa are unexpectedly complex. Identification of novel regulators of spa and elucidation of this enhancer’s spatial organization will provide insights into enhancer function as well as the mechanisms of cone cell fate specification.

311B In situ detection of Hox protein interactions with DNA regulatory elements in single nuclei. Elvira Tour, Adam Parè, William McGinnis. Department of Cell & Developmental Biology, University of California, San Diego, La Jolla, CA. We are developing assays to simultaneously visualize nascent mRNA transcripts, DNA cis-regulatory elements, and transcription factors at single genetic loci in fixed embryonic cells. As a way of increasing the “resolution” of the transcription factor/DNA regulatory element interactions, the degree of protein/DNA colocalization on a transgene carrying a wild type regulatory element is compared to the degree of colocalization on another transgene with a regulatory element with mutant binding sites, all in the same nucleus. We are currently assessing the degree of co-localization of the Hox proteins Dfd, Ubx, and their presumed cofactors, with endogenous DNA regulatory elements that are known targets of these transcription factors. This method of assaying transcription factor occupancy on cis-regulatory DNA can be thought of as a single cell “ChIP on Chip” assay, with the advantage that an embryonic field of nuclei contains many “control” nuclei with different combinations (and concentrations) of transcription factors. The assay should serve as a powerful tool to measure the relationships between the intranuclear concentration of transcription factors, their binding occupancy at specific regulatory elements, and the transcriptional activation (or repression) of specific genes in each nucleus of an embryonic developmental field. 194 POSTERS: Regulation of Gene Expression

312C Study of the transcriptional regulation of unpaired expression in Drosophila eye development. Chuan-Ju Wang, Ya-Hsin Liu, Y. Henry Sun. Academia Sinica, Inst Molecular Biology, Taipei, Taiwan. unpaired (upd) encodes a secreted ligand for the Jak/STAT pathway. It is expressed at the central point of the posterior margin in the eye disc, the initiation site of the morphogenetic furrow (MF). The expression of upd at this site is important for MF initiation and for the long-range control of cell proliferation in the eye disc. It had been shown that several signaling pathways (N, Dpp, Hh and Wg) are involved in positive and negative regulation of MF initiation. A prediction is that the combinatory action of these signaling defined the site of upd expression and hence the site of MF initiation. To understand the regulatory network controlling upd expression, we checked whether upd is regulated by these signaling pathways. Testing of gain-of-function and loss-of-function effects of these pathways on the expression of upd-lacZ suggested that Hh and Dpp pathways cooperate with N signaling to activate upd expression at the posterior center of the eye disc. Furthermore, we found that Wg signaling negatively regulate upd expression. We have also analyzed the eye enhancer region of the upd locus, and identified potential binding sites of several transcriptional factors. Further analyses are in progress.

313A Temporal switching of regulation and function on eye gone (eyg) in Drosophila eye development. Lan-hsin Wang1,2, Y. Henry Sun1,2. 1) Instittute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China; 2) Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. eye gone (eyg) encodes a Pax transcription factor and is important for Drosophila eye development. eyg expression begins in the embryonic eye-antennal primordia (EAP) and continues to the larval eye disc. The dorsoventral midline expression of eyg in the eye disc was shown to be regulated by N signaling which becomes activated at the DV midline at second instar (L2) stage. However, the regulation of eyg expression before and after this stage is still unknown. To understand the dynamic changes in the transcriptional regulation of eyg during eye development, we dissected the eyg-toe locus to identify its eye-specific cis-regulatory elements. We identified two cis-regulatory elements, B8 and E2, which represent distinctly different enhancer activities. Both elements drove GFP expression ubiquitously in EAP and first instar (L1) eye-antenna disc. E2 expression paused at L2 and resumed from early L3 on in an anterior stripe in the eye disc. In contrast, B8 continued its expression in eye disc of L2 and L3 larvae. Further dissections showed that the regulation of eyg/toe expression during eye development can be defined by three time windows: EAP- L1, L2, and L3. We further showed that the transcription of eyg at these different stages is regulated through distinct enhancers. We then examined the regulation by Notch signaling and found that N signaling regulates eyg only at L2 and the regulation is through the B8, but not the E2 enhancer. Loss- and gain-of-function analyses of eyg suggested that eyg has two temporally distinct functions: at EAP-L1 or L3 for head and antennal development, and at L2 for eye development. These results suggested that the eyg Pax gene is temporally regulated by separate enhancers to achieve distinct roles on eye development, and implies a temporal switch of the regulatory mechanism.

314B Regulation of the pro-neural gene atonal by selector factors and signaling pathways during eye development. Tianyi Zhang, Swati Ranade, Qingxiang Zhou, Francesca Pignoni. Dept Ophthalmology, Harvard Medical Sch/MEEI, Boston, MA. During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of ‘eye identity’ at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. To better understand the molecular mechanisms driving this transition, we have dissected the regulation of early atonal expression in the visual system. Our data show that neurogenesis in the Drosophila eye is directly regulated by RD factors and signaling pathways. POSTERS: Regulation of Gene Expression 195

315C Combinatorial regulation of Drosophila muscle enhancers - a systems-level approach. Robert P. Zinzen, Julien Gagneur, Charles Girardot, Eileen E. Furlong. Dev Biology Unit, EMBL, Heidelberg, Germany. The ordered recruitment of transcription factors (TFs) to cis-regulatory modules (CRMs) governs the spatio-temporal patterns of gene expression leading to differentiation of developing tissues. However, the transcriptional networks deployed and how they achieve temporally and spatially distinct expression patterns remain largely elusive. Several studies have focused on the binding of single TFs across genomes, but CRMs identified tend to direct wide varieties of expression patterns that cannot be explained by binding of single TFs. We focus on the global regulatory networks leading to the progressive subdivision of the Drosophila embryonic mesoderm into muscle primordia. To this end, we consider the genome-wide binding of 5 myogenic TFs over time. Our goal is to determine in how far differential co-regulation by these 5 TFs over time yields expression in mesodermal subsets, thus allowing insights in how mesoderal subdivision is achieved. We combine high-resolution ChIP-on-chip data with analysis of published and novel CRM activity. Furthermore, we test predictions made by our ChIP-on-chip analyses in vivo using novel vectors designed for detailed and even quantitative comparisons of CRM activity.

316A Oligomeric state of Drosophila CtBP plays a role in transcriptional regulation of Wingless signaling targets. Chandan Bhambhani, Jinhee Chang, Mikyung Chang, Kenneth Cadigan. Molecular, Cellular & Developmental Biology, Univeristy of Michigan, Ann Arbor, MI. CtBP acts in concert with several transcriptional regulators to control gene transcription. Although the role of CtBP as a corepressor has been well established, it also plays a positive role in directly regulating some Wingless (Wg) targets. The basis of our investigation is to explore the mechanism underlying this differential regulation by CtBP. Here we present evidence to support a model where monomeric form of CtBP is a positive regulator of Wg signaling while CtBP oligomers play a negative role in Wg target gene transcription. Structural analysis of the Human CtBP1 (hCtBP1) suggests that it is a dimer, and oligomerization is required for CtBP to function as a co-repressor. Based on the high homology between the hCtBP1 and Drosophila CtBP (dCtBP), a site directed mutagenesis approach was used to create a mutant which looses the ability to dimerize. This monomeric form of CtBP is as potent as the wildtype in enhancing the activation of a Armadillo-dependent reporter assayed in cultured cells. In wing imaginal discs, misexpression of the wildtype or monomeric CtBP leads to expansion of a Wg target Dll-lacZ, but the monomeric form is defective for repression of another Wg target nkd-lacZ. When expressed at similar levels, wildtype CtBP antagonizes Wg signaling leading to formation of wing notches and suppresses an Armadillo mediated small eye phenotype while monomeric form enhances the small eye phenotype with no effect on wing development. Currently the mutant and the wild type forms are being tested for their oligomeric state in vitro. However in vivo analysis suggests that out of the two differential activities shown by wildtype CtBP, the forced monomer retains only the positive transcriptional role. Hence to test if CtBP dimers are repressors, different approaches are being used to create a forced dimer predicted to retain only the negative role of the wildtype form.

317B Dicing Sex. Jamila Horabin, Ursula Olcese. Dept Biomedical Sciences, Florida State Univ, Tallahassee, FL. Sex-lethal (Sxl), the master switch of Drosophila sex determination, controls all aspects of sexual development. Its activity is regulated in response to the ratio of X chromosomes to autosome sets (X:A ratio); females with an X:A ratio of 1 turn the gene on, while it remains off in males where the ratio is 0.5. The RNA-induced silencing complex (RISC) influences chromatin silencing, translation repression and mRNA stability in plants, fungi and animals. RISC utilizes small RNAs, microRNAs (miRNA) for translation repression and/or mRNA cleavage, processed long double stranded RNAs (siRNA) for RNA interference. The two Drosophila dicers, required for either miRNA or siRNA maturation, strongly impact the female sex determination process. This effect appears to be from elevating the effective value of A in the X:A ratio, altering the timing as well as the strength of expression of the specialized X:A ratio sensitive promoter of Sxl. 196 POSTERS: Regulation of Gene Expression

318C Genomic imprints, chromatin proteins and Wolbachia. Daisey Y Kent. Mount Allison University, Biology, 63B York St, Sackville, Canada. Genomic imprinting is a process that marks chromosomes to record their parental origin. Imprinting can cause differential gene expression or chromosome loss. Imprinting is comprised of four stages; establishment, transduction, maintenance and erasure. The imprint is established in the germ line of parents and then is transduced into the differential chromatin structures that are maintained in all of the somatic cells of the progeny. We are investigating this transduction stage by monitoring the timing of the embryonic transition in DNA packaging proteins from HMG-D to histone H1. We’ve found that histone H1 associates with chromatin at the 9th cleavage division in Su(var)205 and Su(var)3-9 mutant embryos in which the paternal imprint is lost, three cell divisions earlier than in wild-type. Thus, histone H1 recruitment may respond to the parental origin of the DNA and be responsible for transducing the initial imprint into parent-specific chromatin conformations. We have also found that Wolbachia, bind histone H1 in Drosophila embryos. This suggests that Wolbachia may alter their host’s chromosome transmission by disrupting genomic imprinting.

319A Quantitative 3D Analysis of Expression Patterns Driven by cis-Regulatory Modules. Soile V.E. Keränen1, Barret D. Pfeiffer1, Bill Fisher1, Ann Hammonds1, Cris L. Luengo Hendriks1, Charless Fowlkes2, Clara N. Henriquez1, David W. Knowles1, Jitendra Malik3, Michael Eisen1, Mark D. Biggin1, Susan Celniker1. 1) Lawrence Berkeley Natl Lab, Berkeley, CA; 2) University of California Irvine, Irvine, CA; 3) University of California Berkeley, Berkeley, CA. The Berkeley Drosophila Transcription Network Project (http://bdtnp.lbl.gov/) has recently developed methods for collecting cellular resolution quantitative expression data in whole blastoderm embryos in a computationally analyzable format. Baseline data has been collected on the mRNA and/or protein expression patterns of over 100 genes as part of a larger effort to predict transcriptional output from genomic DNA sequences. However, the complete pattern of each gene’s expression is built up by the combined action of multiple cis-Regulatory Modules (CRMs). To investigate how complete patterns are established, we are quantitating the expression driven by individual CRMs. This is critical, as previous transgenic promoter experiments have been limited by lack of quantitative resolution and computational analysis. We find that a number of CRMs drive patterns of transcription that differ from those of the corresponding pattern elements of the intact gene in native context, e.g., by having less distinct borders at expression boundaries. Thus our quantitated CRM expression patterns will provide a more precise basis for modeling transcriptional output from specific DNA sequences and allow analysis of how sequences outside of individual CRMs help refine the output of the CRM. In addition, we are comparing transcript levels in nuclei vs cytoplasm to characterize the expression dynamics of different CRMs.

320B Post-translational modifications during formation of Da/Sc/E(spl) complexes. Marianthi Kiparaki1,2, Christos Delidakis1,2. 1) Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece; 2) Department of Biology, University of Crete, Heraklion, Greece. During the development of Drosophila peripheral nervous system, two groups of bHLH transcription factors have determinative role in neural fate assignment. Neural fate is promoted by proneural proteins (e.g. Scute), which activate neural target genes via heterodimerization with Daughterless. On the other hand, bHLH E(spl) proteins act as transcriptional repressors and inhibit neural fate by binding onto DNA, either directly or through interaction with the activation domain of Scute. Using tissue culture and Drosophila tissues we studied the post-translational modifications that occur during these protein interactions and their role in protein stability and function. POSTERS: Regulation of Gene Expression 197

321C Regulation of Vasa stability by Gustavus and Wallenstein. Jan-M. Kugler1, Jae-Sung Woo2, Byung-Ha Oh2, Paul Lasko1. 1) DBRI, Dept. of Biology, McGill University, Montreal, Canada; 2) Dept. of Life Science, Pohang University, Pohang, Korea. Accumulation of the germ line RNA helicase Vas in the perinuclear nuage and in the posterior pole plasm during oogenesis is important for the maternal effect function of Vas in embryonic germ cell formation and posterior somatic patterning. The mechanisms that mediate asymmetric Vas distribution are largely unknown. The paralogous proteins Gustavus (Gus) and Wallenstein (Wals) have highly similar SPRY-domains. This domain mediates physical interaction of Gus with Vas. The presence of an F-box in Wals and of a SOCS-Box in Gus suggests that each protein could act as substrate recognition subunit of distinct Cullin RING E3 ligase complexes involved in Vas ubiquitinylation. Vas co-purifies with transgenic Gus and Wals from ovarian extracts. Wals binding is sensitive to competition with a Vas peptide containing the established Gus binding site while a peptide containing a single amino acid substitution within the binding site does not interfere with co-purification. This indicates that both Gus and Wals can bind to a common site in Vas. Vas levels are significantly reduced in ovarian extracts from females bearing a piggyBac insertion in gus, indicating that Gus is involved in Vas stabilization. Interestingly, this phenotype can be strongly enhanced by concurrently over-expressing Wals, suggesting that Wals promotes Vas degradation. Consistent with lower Vas levels in gus mutant or Wals over-expressing ovaries the number of germ line cells in offspring embryos is significantly reduced. Both Wals and Gus are enriched perinuclearly in nurse cells. Interestingly, Gus is more prominent than Wals in cytoplasmic particles within the nurse cells and accumulates in the stage 10 pole plasm in a vas-dependent manner. This is consistent with our working model that local Vas stability is regulated differentially by distinct ubiquitin E3 ligase complexes recruited by Gus or Wals.

322A Computational model of eggshell patterning by the EGFR and Dpp pathways. Jessica Lembong, Nir Yakoby, Stanislav Y. Shvartsman. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ. The roof of dorsal eggshell appendages is formed by the follicle cells expressing a Zn-finger transcription factor broad (br). Genetic approaches identified the EGFR and Dpp pathways as key regulators of br expression, but the dynamics of br expression remained poorly understood. Based on our recent experiments, we propose a dynamic model for br expression. First, br expression is induced by an incoherent feedforward loop downstream of EGFR signaling. Second, br induces expression of the Dpp receptor Thickveins (Tkv). This activates Dpp signaling, which then represses br expression in a negative feedback loop. This module limits the duration of br expression. Third, Brinker (Brk), activated by EGFR and repressed by Dpp, provides a gating mechanism, whereby high levels of Brk block the repression of br by Dpp. Finally, Tkv expression is regulated in a positive feedback loop, where Dpp signaling activates Tkv expression. At this level of complexity, a modeling approach is essential for exploring the patterning capability of this mechanism. We will present a computational model of br regulation and use this model to describe and predict ~10 mutant backgrounds. The model is based on the formalism of piecewise linear dynamical systems and is ideally suited for exploring the dynamics of networks with a large number of uncertain parameters. We will show how a computational approach can test the feasibility of the proposed patterning mechanism and analyze its robustness and evolvability.

323B Targeted Deletions of the slowpoke transcriptional control region through Homologous Recombination. Xiaolei Li, Nigel Atkinson. Section of Neurobiology, The university of Texas at Austin, Austin, TX. Our lab use Drosophila as a model to study anesthetic drug induced tolerance. In flies, the slowpoke (slo) gene encodes the big conductance Ca2+ and voltage activated K+ channel that is broadly expressed all over the neural system and plays an important role in regulating the animal’s neural activity. This channel is found to be essential for a fly to acquire anesthetic drug induced tolerance. Moreover, the expression of the channel is up-regulated after the exposure to anesthetic drugs, and the increased amount of channels in return enables the fly to acquire tolerance to drug induced sedation. The expression of BK channel is modulated by a 7 KB promoter region, which includes many DNA elements that could be recognized by transcription factors such as CREB, AP-1 and HSF. In current project we utilize the endogenous machinery of DNA repair and homologous recombination to generate deletion mutants of the transcription factor binding sites within slo promoter. More specifically a P element containing the flanking sequences of the target site is introduced into the animal. And by introducing a transgenic recombinase and a transgenic endonuclease, the donor DNA is released from the chromosome and linearized in order to aim at the target chromosomal loci through homologous recombination. This gene targeting technique is an efficient and rapid way to generate gene specific mutants in fly. We plan to use a collection of these deletion mutants to identify DNA binding elements within the slo promoter that are important for drug tolerance. 198 POSTERS: Regulation of Gene Expression

324C Medea SUMOylation restricts the signalling range of the Dpp morphogen in the Drosophila embryo. Wayne Miles1, Shugaku Takeda1, Ellis Jaffray2, Laura Bayston1, Sanjay Basu1, Laurel Raftery3, Ron Hay2, Hilary Ashe1. 1) Faculty Life Sci, Univ Manchester, Manchester, United Kingdom; 2) School of Life Sciences, University of Dundee, Dundee, UK; 3) Cutaneous Biology Research Centre, Massachusetts General Hospital and Harvard Medical School, Charlestown, USA. Morphogens are secreted signalling molecules which form concentration gradients and control cell fate in developing tissues. One of the best characterised morphogens is Drosophila Decapentaplegic (Dpp), a conserved TGF-β signalling molecule which patterns the dorsal ectoderm of the embryo by activating the Smad transcription factors, Mad and Medea. We have investigated the role of the SUMO post-translational modification in regulating Dpp signalling. We demonstrate that embryos carrying a mutation affecting an essential enzyme in the SUMO conjugation pathway have increased expression patterns of Dpp target genes. We identify Medea as a SUMOylation target in the embryo, and locate three sites in the protein at which SUMOylation occurs. Moreover, our results demonstrate that failure to SUMOylate Medea leads to the increased Dpp signalling phenotype observed in the SUMO pathway mutant, suggesting that Medea is the primary SUMOylation target in the Dpp pathway. Our results identify an unusual strategy for regulating morphogen range which, rather than impacting on the morphogen itself, targets an intracellular transducer. Current work is focused on identifying additional SUMO targets which may also impact on the Dpp pathway. Overall, we anticipate that the SUMO post-translational modifier will be used in many contexts to modulate signalling outputs during development.

325A Identification of functional domains and target genes of the Hindsight zinc-finger protein. Liang Ming1,2, Ronit Wilk2, Amanda Pickup2, Howard Lipshitz1,2. 1) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 2) Program of Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.

The hindsight (hnt) gene encodes a C2H2-type zinc-finger transcription factor that regulates epithelial morphogenesis of specific tissues during Drosophila development. At the cellular level, HNT maintains epithelial integrity and regulates the actin cytoskeleton, Jun kinase and Hedgehog signaling. Two HNT-binding sites and their corresponding DNA-binding domains in the HNT protein have been identified. The sites resemble those bound by the human HNT homolog, RREB1/FINB, which functions as a transcriptional modulator, either potentiating or down-regulating target gene expression. HNT is expressed in late-third-instar salivary glands, enabling identification of endogenous binding sites by immunostaining of polytene chromosomes. Two particularly strong sites map to 4C and 60C. The HNT-binding site at 4C is associated with the hnt locus itself. Data obtained using a hnt reporter transgene as well as tissue-specific RNAi-mediated knockdown, suggest that HNT down-regulates expression of its own gene. The HNT-binding site at 60C has been mapped to nervy (nvy). Preliminary data suggest that nvy expression, too, is modulated by HNT.

326B Determining the in vitro DNA-binding specificities of transcription factors regulating early embryogenesis. Nobuo Ogawa, Stuart Davidson, Xiao-Yong Li, Lucy Zeng, Hou-Cheng Chu, Michael B. Eisen, Mark D. Biggin. Genome Sci Dept, Lawrence Berkeley Natl Lab, Berkeley, CA. Sequence specific DNA binding proteins bind many tens of recognition sequence variants over a wide range of affinities. Rarely, if ever, has this complexity been captured for a single protein, let alone for the many factors acting in a network, presenting a challenge for efforts to interpret the transcriptional cis regulatory information encoded in animal genomes. As part of the Berkeley Drosophila Transcription Network Project, we are developing methods to obtain extensive in vitro DNA binding specificity data and are applying them to the 37 factors that convey the bulk of spatial patterning information in the pregastrula embryo. First, we have developed an affinity resin based SELEX method that accumulates over 1,000 individual sequences of selected oligonucleotides per round. We have obtained such data for 12 early factors and have calculated Position Weight Matrices (PWMs) for these proteins using an algorithm that determines the relative affinity for each nucleotide at each site position. Second, to confirm the biochemical accuracy of the PWMs and to determine if affinities at one nucleotide position are independent of those at the other positions, we have also developed a multiplex method that can measure binding to 13 different sequences at once. This analysis shows that Gt protein’s specificity is accurately described by a PWM that assumes position independence, but that Bcd’s is not. Third, we have developed an additional assay in which sheared genomic DNA is bound to a transcription factor in vitro and then the bound DNA is analyzed with an Affymetrix tiling array. The resulting in vitro/chip data should allow us to determine the role of homomeric cooperativity and binding site competition in establishing occupancies to the complex clusters of different affinity sites present throughout the genome. The results of all three assays have been compared to the patterns of binding of the same proteins in the embryo as measured by ChIP/chip assays. POSTERS: Regulation of Gene Expression 199

327C E23 is an ABC transporter that regulates the hormone 20-hydroxyecdysone in a cell autonomous manner. Elana A Paladino1, Lauren Besquillo1, Dan Garza2, Andrew J Andres1. 1) School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV; 2) Novartis Institutes for Biomedical Research, Cambridge, MA. Systemic pulses of the steroid hormone 20-hydroxyecdysone (20E) result in the initiation of novel gene expression cascades. We use the salivary gland as a model system for understanding how these global 20E pulses result in tissue specific genetic programs. In the salivary gland, three major physiological changes are brought about by 20E: production of a polypeptide glue mixture in the third instar, secretion of this glue protein late third instar, and autophagy of the larval gland at the end of the prepupal period. Although much is known about the physiological responses of the salivary gland to 20E, the genetic pathways that direct these changes have not been completely deciphered. E23 is a direct gene target of 20E signaling that has similarity to ABC transporters. Through the hydrolysis of ATP, ABC transporters actively pump substrates across membranes. We tested the hypothesis that E23 regulates intracellular levels of 20E by overexpressing E23 before the critical pulses of 20E that initiate glue synthesis, glue secretion, and salivary gland cell death. We found that ectopic E23 displays a phenotype similar to those of loss-of-function receptor mutations. In addition, we used FLP/FRT technology to create cells in the salivary gland that express different levels of E23 protein, and showed that the E23-induced block could be overcome with increased concentration of hormone. These observations lead to a model where 20E induces E23 to regulate the intracellular exposure of a cell to hormone. This model is being tested using RNAi technology to silence E23.

328A Identification of Broad-regulated genes during leg development in Drosophila. Elspeth Pearse1, Xiaochen Wang1, Xue-Wen Chen2, Robert Ward1. 1) Department of Molecular Biosciences, University of Kansas, Lawrence, KS; 2) Department of Electrical Engineering & Computer Science, University of Kansas, Lawrence, KS. The elongation and eversion of leg imaginal discs in Drosophilais an ideal system for studying hormone-regulated morphogenesis. A pulse of the steroid hormone ecdysone at the onset of morphogenesis triggers the transformation of the imaginal disc into an immature adult leg through coordinated changes in cell shape. The transcription factor broad (br) is a key ecdysone-inducible early gene involved in adult tissue differentiation. To identify br-dependent ecdysone-inducible genes during imaginal leg disc morphogenesis, we probed Affymetrix whole Drosophila genome microarray chips with RNA isolated from leg discs of white prepupae from amorphic br5mutant and wild type animals. Using a statistical approach we have identified 42 induced and 71 repressed genes whose expression differs significantly between wild type and br5mutant leg discs (P<0.005). Through this analysis we identified ImpE3 as a gene induced by broad, consistent with our prior studies showing that ImpE3 is induced by ecdysone and broad at the onset of metamorphosis in leg imaginal discs. Other broad-induced genes include three chitin binding proteins, three putative adhesion molecules and four components of the Notch signaling pathway. We have validated ~ 20 of these genes using quantitative RT-PCR or northern blot analysis. We plan to conduct functional analysis on several candidate genes using the Vienna RNAi stock collection.

329B rasiRNA pathway components participate in posttranscriptional silencing of telomeric retrotransposon HeT-A. Sergey G. Shpiz, Yuri A. Abramov, Alla I. Kalmykova. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russian Federation. In most eukaryotes, telomeric DNA is maintained by the telomerase activity. By contrast, Drosophila telomeres consist of mixed tandem arrays of specialized telomeric retrotransposons HeT-A, TART and TAHRE. HeT-A, TART and TAHRE transcripts were shown earlier to accumulate in ovaries of the rasiRNA (repeat-associated short interfering RNA) pathway mutants, but their abundance is not affected by mutations in miRNA or siRNA pathway components. In order to understand the mechanism of RNA silencing of retrotransposons, we used the lines with the HeT-A attachments to the truncated X chromosome with a break in the yellow locus. In case the attachment occurred after degradation of a yellow promoter, the yellow gene expresses from the HeT-A promoter located at its 3’ region. Fused HeT-A/yellow transcript contains 30 or 100 nucleotides of HeT-A 3’ region as a result of the presence of two transcription initiation sites. The abundance of spliced HeT-A/yellow transcripts is under the control of rasiRNA silencing pathway components Spn-E, Aub, and Piwi and a product of vasa locus containing vig and vasa genes. No increase in the amount of non- spliced HeT-A/yellow transcripts, which abundance reflects the level of transcription, was detected in spn-E, aub, and vasa locus mutants. These data suggest that rasiRNA pathway components spn-E and aub and product/products of vasa locus are involved in the posttranscriptional silencing of HeT-A expression and that 30-100 nucleotides of HeT-A 3’ region are enough to support rasiRNA- mediated silencing of a reporter gene. 200 POSTERS: Regulation of Gene Expression

330C Post-transcriptional Regulation of nanos mRNA During Early Drosophila Development. Danielle R. Snowflack, Elizabeth R. Gavis. Molecular Biology, Princeton University, Princeton, NJ. Precise spatial and temporal regulation of gene expression is essential during early Drosophila development for proper patterning of the anterior-posterior (A-P) axis. Establishment of the A-P axis requires both selective repression of the maternally contributed mRNA nanos (nos) and its localization to the posterior where the mRNA is actively translated. Although the mechanism by which nos mRNA is translationally repressed is not well understood, repression in the bulk cytoplasm is mediated by the translational control element (TCE), a structural motif in the nos3’ untranslated region consisting of two major stem loops (II and III) that are bound by the repressor Smaug (Smg) in the embryo and Glorund (Glo) in the ovary, respectively. Previous results from our lab have shown that there are at least two modes of TCE-mediated translational repression, one operating at the initiation step and one that acts at a more downstream event during elongation or termination. I will further characterize the dependence of this latter mode of repression on Smg, the embryonic repressor, as well as on other factors that interact with Smg to repress translation of nos mRNA during early embryogenesis. In addition, I will use affinity purification methods to identify proteins that may contribute to this mode of repression. These studies will shed light on a poorly understood mechanism of translational control.

331A The Drosophila MRP gene functions as an inducible pesticide transport. Jolene Tarnay-Cogbill1, Flora Szeri2, Andras Varadi2, Steven Robinow3. 1) Cell & Molecular Biology, University of Hawaii, Honolulu, HI; 2) Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary; 3) Department of Zoology, University of Hawaii, Honolulu, HI. The Drosophila dMRP gene is an ortholog of the human MRP1, MRP2, and MRP3 genes. These genes encode multiple drug resistance associated proteins (MRP) that are members of the ATP-binding cassette (ABC) transporter superfamily and are involved in integral cellular processes such as ion transport, signal transduction and detoxification of endogenous and exogenous compounds. dMRP mutant animals, when compared to control animals, show no diference in response to fenitrothion and allethrin pesticide exposure but display increased sensitivities to DDT and aldicarb. Currently this pesticide sensitivity assay is being repeated using dMRP RNAi knockdown flies to attempt to phenocopy our previous results, and thereby confirm that loss of dMRP is responsible for the increased sensitivity seen in response to DDT and aldicarb exposure. Based on these pesticide sensitivity data we hypothesized that DMRP transports DDT and aldicarb, but not fenitrothion or allethrin. Preliminary studies indicate that DDT, as well as fenitrothion and allethrin, may be substrates for DMRP transport. Further studies are being conducted to clarify these data. Exposure to xenobiotics has also been found to cause transcriptional up-regulation of human MRP genes. Therefore, we tested the hypothesis that DDT and aldicarb, but not fenitrothion or allethrin, will induce transcriptional up-regualtion of dMRP. Our results indicate, however, that all four pesticides tested are capable of inducing dMRP expression.

332B Inhibition of RNA interference by cell death signaling. Weiwu Xie, James Birchler. Biological Sci Div, Univ Missouri-Columbia, Columbia, MO. Targeted gene silencing can be achieved in Drosophila melanogaster by RNA interference through a transgene expressing dsRNA homologous to the target gene. One such construct, driven by an eye-specific promoter GMR, encodes inverted repeat sequences of the white (w) gene exon 3 and has been shown to repress w expression to a pale yellow color. Several commonly used eye mutant markers partially restore the eye color whereas others cannot. Interestingly, the restored color in the Bar eye mutants surrounds the region where cell death occurs. By comparing the two groups of marker genes, those that restore color produce irregular cell death during eye development. Therefore, we hypothesized that the inhibition of RNAi is caused by cell death signaling. Excessive cell death can be induced by over-expression of the canonical apoptotic genes grim, hid and rpr and other genes strica and ttk. When combining these transgenes with the RNAi construct, we observed eye color restoration in all cases. When a cell death inhibitor gene is added and the eye shape restored, w gene silencing is also restored. The restored color pattern of the Bar eyes also disappeared when the cell death was suppressed by acetamine. We also tested other constructs used to silence either w or the EGFP gene. Although differences occur, we observed inhibition of RNAi of all transgenes when combined with at least some transgenes causing cell death. Further analysis indicates that the phenotype is proportional to RNAi and cell death in strength. Mutagenesis was performed seeking for alteration of the color restoration in the Bar eyes with w RNAi. A mutational analysis has recovered several isolates that reduce or increase the effect of the cell death signaling on RNAi. POSTERS: Regulation of Gene Expression 201

333C Disruption of CP190’s C-Terminal domain genetically reduces Gypsy insulator functionality. Omar Akbari, Chi-Yun Pai, Daniel Oliver. Dept Biol, Univ Nevada, Reno, Reno, NV. Chromatin insulators or boundary elements are important players for the organization of the chromatin structure in the cell nucleus. Insulator elements have been found in many organisms from yeast to mammals. However, the distribution of chromatin insulators in the genome is not well characterized and the molecular mechanisms remain unclear. The gypsy chromatin insulator of Drosophila melanogaster is one of the best understood chromatin insulators. It is comprised of a DNA sequence bound by a complex of at least four characterized proteins. We recently discovered that CP190, a centrosomal protein, is a component of the gypsy insulator complex. CP190 colocalizes with Su(Hw) and Mod22, two other gypsy insulator components on the polytene chromosome. In addition it is also present in complexes independent of Su(Hw) and Mod22. To further characterize CP190’s role at the gypsy insulator complex we generated a point mutation in CP190 disrupting it’s C-terminal domain. This point mutation genetically disrupts gypsy insulator functionality. Additionally, polytene staining reveals that this disruption of gypsy insulator functionality is due to reduced CP190 binding affinity to the gypsy insulator complex. These results suggest that the CP190 C-terminal domain is necessary for CP190 localization to the gypsy insulator complex.

334A Activity of Gt protein gradient differentially positioning pair-rule stripes. Luiz P Andrioli, Ligiane Oliveira, Thiago Caséé. Depto Genéética e Biologia Evolutiva, IB, USP, Sãão Paulo, Sãão Paulo, Brazil. Still one intriguing question in the segmentation studies is the understanding of how the aperiodic expression domains of maternal and gap genes give rise to expression of the seven striped pattern of each pair-rule gene. One possibility is that the activity of gap genes as morphogens is an important requirement for the transition of these expression patterns. Here, we propose to investigate whether minimal differences in the Gt protein concentrations is able to repress enhancers and differentially position two groups of overlapping stripes of the pair-rule genes even-skippedeven-skipped (eveeve), hairyhairy (hh), runtrunt (runrun) e fushi-tarazzu fushi-tarazzu (ftzftz). In gtgt null mutant embryos, the anterior border of eveeve 22, runrun 22, ftzftz 22 and hh 33 and the posterior border of hh 55, eveeve 55, runrun 55 and ftzftz 55 are expanded, consistent with the absence of the common repressor Gt which is normally expressed in an anterior domain ahead of these stripes 2 and in a posterior domain behind stripes 5. We next analyzed these stripes differential susceptibility for gt repression. To do that, we generated transgenic flies misexpressing a gtgt ectopic ventral domain intersecting the ventral portion of the striped pattern. Thus, we detected ventral repression of hh 33, eveeve 22, runrun 22 and ftzftz 22 and all stripes 5. According to the expected activity of a morphogen, stripes more distantly positioned to gt expression domains (hh 33 and hh 55) exhibited greater repression effects when compared to closer stripes (eveeve 22 and ftzftz 55). However, we were not able to distinguish differences between runrun 22 and ftzftz 22 or eveeve 55 and runrun 55 which overlap considerably. We are now investigating the striped pattern in gtgt- embryos also carrying the gt ventral misexpression construct, to check whether in the absence of the gt endogenous domains we could distinguish differential repressions between stripes. Finantial support: FAPESP.

335B Characterizing the Role of Self-Association in Transcriptional Repression by Yan. Thomas G.W. Graham1, Maureen P. Cetera1, Pavithra Vivekanand1,2, Ilaria Rebay1. 1) Ben May Department for Cancer Research, University of Chicago, Chicago IL; 2) Department of Biology, Franklin & Marshall College, Lancaster, PA. The receptor tyrosine kinase (RTK) pathway plays a role in numerous cell-fate determination processes during Drosophila development. Two key effectors of RTK signaling are the transcriptional repressor Yan and the transcriptional activator Pointed (Pnt), which bind to the same Ets-binding sites on DNA. Phosphorylation by MAPK induces nuclear export and degradation of Yan and activates the P2 isoform of Pnt (PntP2), thereby derepressing and activating target genes. We seek to understand how Yan functions as a repressor and how this repression is regulated by signaling. It was recently shown that the isolated SAM domain of Yan self- associates to form head-to-tail polymers in vitro. Data from our lab and others reveal that missense mutations in the SAM domain that abolish Yan-SAM self-association also impair Yan repressor activity, suggesting that self-association influences Yan function and regulation. We are taking an interdisciplinary approach to study Yan self-association and to investigate its in vivo relevance. As the self-association affinity has so far been measured only for the isolated SAM domain of Yan, we are attempting to express and purify full-length Yan protein for biochemical characterization. In order to study Yan self-association in cells, we are developing Yan- EGFP fusion proteins for use in fluorescence imaging. Using fluorescence recovery after photobleaching (FRAP) experiments, our goal is to detect dynamic changes in the size of Yan oligomers in response to signaling based on associated changes in the diffusion rates of these complexes. Using molecular genetic analysis of Yan monomeric mutants in vivo and in cultured cells, we are attempting to elucidate further the role of self-association in repression by Yan. Finally, through mathematical modeling, we are exploring under what conditions the “Yan module” gives rise to the switch-like behavior observed in vivo and whether Yan self-association may play a role in this ultrasensitivity. 202 POSTERS: Regulation of Gene Expression

336C The Polycomb group of genes control the repression states of genes that subdivide compartments in Drosophila. Luis Gutierrez, Jürg Müller. Gene Expression Unit, European Molecular Biology Laboratory, Heidelberg, Baden, Germany. The Polycomb group of genes (PcG) encode proteins that control the stable silencing of Hox genes during the development of Drosophila and vertebrates. Studies analzying genome-wide binding of PcG proteins in Drosophila, mice and humans identified many potential target genes of the PcG system. Here, we present evidence that PcG proteins indeed control expression of a wide variety of transcription factors with known roles in development. These include genes required for antero-posterior, dorso-ventral and proximo-distal patterning of limbs. In addition, the PcG system is required for repressing the caudal HOX genes caudal and even-skiped in anterior limbs. We conclude that PcG proteins directly repress expression of transcription factors that control the major regionalization events in Drosophila. Since these PcG target genes are highly conserved in mammals, it seems likely that the mammalian PcG system is required for regulating expression of the orthologous genes not only in ES cells but also during ontogeny.

337A Regulatory mechanism of temporal expression of Blimp-1, an important factor for determination of pupation timing. Akagi Kazutaka1, Hitoshi Ueda1,2. 1) The Grad. Sch. of Nat. Sci. and Tech., Okayama Univ., Okayama, Japan; 2) Dept of Biol, Fac of sci., Okayama Univ., Okayama, Japan. In insect, ecdysone works as a master hormone to control molting and metamorphosis during the development. It has been shown that ecdysone induces so-called early genes directly and some of the early genes encode transcription factors which play important roles during the process of ecdysone responses. On the other hand, the transcription factor βFTZ-F1 is induced only after decline of ecdysone and the temporally restricted expression is necessary for the progression of embryogenesis, molting and metamorphosis. We have recently identified Blimp-1 as a binding factor to the promoter region of the ftz-f1 gene. This factor is expressed during high ecdysone period and plays an important role in determining the precise timing of βFTZ-F1 expression and also pupation timing. To elucidate the regulation mechanism of the Blimp-1 gene, we measured Blimp-1 mRNA level in developing animals by RT-PCR. Expression pattern of Blimp-1 mRNA showed small difference compared to that of E75A mRNA, which is one of the well-known early gene product. Furthermore measurement of the transcripts in the cultured salivary glands in the presence of ecdysone reveal that the Blimp-1 gene is induced by ecdysone directly but profile of its induction is different from the early gene E75A. These results suggest that Blimp-1 gene is regulated by different mechanism compared to the early genes and this mechanism is important to determine the timing of βFTZ-F1 expression and pupation.

338B Windei is a binding partner of the histone methyltransferase Eggless and is required for efficient trimethylation of histone 3 lysine 9 during oogenesis. Carmen M. Koch1, Diane Egger-Adam2, Andreas Wodarz1. 1) Department of Stem Cell Biology, CMPB, Georg-August-University Goettingen; 2) Faculty of Biology, University Kostanz. The repression of gene expression plays a very important role in many stages of development and is often mediated by the methylation of histones. Trimethylation of histone 3 lysine 9 (H3K9) is known to play an important role in chromatin silencing and the formation of heterochromatin. The recruitment of heterochromatin protein 1 (HP1) to chromatin regions rich in trimethyl H3K9 subsequently leads to the formation of stably condensed chromatin. Recently it has been shown that Eggless (Egg), the ortholog of the human methyltransferase SETDB1 is involved in H3K9 trimethylation in Drosophila. Here we show that the protein Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF binds to Egg and precisely colocalizes with Egg in ovaries. We have generated null mutations in wde, which are semilethal and can be fully rescued by a transgene encoding a fusion protein of Wde with green fluorescent protein. Surviving homozygous mutant females are sterile and possess only rudimentary ovaries. Elimination of wde function in germ line clones leads to the arrest of oogenesis before stage 10 and subsequent degeneration of mutant egg chambers. Germ line cells mutant for wde show strongly reduced H3K9 trimethylation, similar to egg mutant cells. The distinct localization of both proteins in 1 to 3 focal spots within the oocyte nucleus close to HP1 silenced regions points to a function of the Wde/Egg complex in the transcriptional silencing of the oocyte. POSTERS: Regulation of Gene Expression 203

339C Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci. Eric Lai, David Tyler, Katsutomo Okamura, Wei-Jen Chung, Joshua Hagen. Dev Biol, Sloan Kettering Inst, New York, NY. Many microRNA (miRNA) loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. We report here that antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. Interestingly, iab-8 miRNAs are relevant to Hox gene regulation, since ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A and induce a dramatic homeotic transformation of halteres into wings. We generalize this principle by showing that other miRNA loci in both invertebrates and vertebrates are processed on their antisense strands into endogenous mature small RNAs. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes.

340A Translation of germ cell-less is regulated by Bruno in a BRE-independent manner. Jocelyn Moore, Paul Lasko. DBRI, Department of Biology, McGill University, Montreal, QC, Canada. During Drosophila oogenesis and embryogenesis, gene products are sequestered in discrete regions to ultimately achieve embryonic asymmetry. germ cell-less (gcl) RNA, like other germ plasm components, is present throughout the oocyte during oogenesis, then accumulates in the germ plasm, at the posterior pole of the embryo, after fertilization. gcl RNA accumulates at the posterior in advance of detectable Gcl expression in the germ plasm. Although gcl RNA is detected throughout the embryo during early embryogenesis, Gcl protein is detected only in the germ plasm. In addition, previous studies have shown that somatic expression of Gcl is developmentally detrimental. These lines of evidence suggest that translation of gcl RNA is repressed outside of the germ plasm. Examination of the gcl 3’UTR sequence led to the identification of a single Bruno Response Element (BRE) that is conserved among several Drosophila species. Reciprocal effects on Gcl expression are observed in an arrest heterozygote and when Bruno is overexpressed. These results are consistent with Bruno acting as a translational repressor of gcl mRNA outside of the pole plasm. In accordance with this hypothesis, reduction in the maternal dosage of Bruno also leads to ectopic Gcl expression in the embryo, in turn, causing repression of anterior hückebein RNA expression. Our results indicate that Bruno can bind directly to the gcl 3’UTR in vitro, but, surprisingly, this binding appears to be independent of the BRE. A short probe consisting of the gcl BRE region fails to compete with recombinant Bruno binding to the full length gcl 3’UTR, and recombinant Bruno binds efficiently to the gcl 3’UTR even when the BRE has been deleted. We conclude that the gcl-Bruno interaction may represent a novel mode of BRE-independent translational control by Bruno.

341B Pumilio-dependent repression of CyclinB mRNA in the prospective somatic cytoplasm of the embryo. Krystle J Nomie, Robin Wharton. Molecular Genetics and Microbiology, Cell Biology, Duke University, HHMI, Durham, NC. During early development, the embryo relies heavily on post-transcriptional gene regulation. Pumilio (Pum), a founding member of the PUF domain RNA-binding protein family, recognizes sequences within the 3’-untranslated regions (3’-UTRs) of target mRNAs, thereby inhibiting translation and/or promoting degradation. In the prospective somatic cytoplasm of the syncitial cleavage stage embryo, Pum represses hunchback (hb) mRNA to allow abdominal segmentation. In the primordial germ cells (PGCs), Pum represses CyclinB (CycB) mRNA to restrict proliferation. CycB protein accumulates throughout the embryo; thus, it was thought that Pum does not repress translation of the mRNA in the soma. However, Vardy and Orr-Weaver (2007) have shown that Pum indeed does repress CycB mRNA in the somatic cytoplasm and that action of the PanGu kinase is required to antagonize this repression. We have shown that Pum-dependent regulation of CycB mRNA in the somatic cytoplasm occurs without Nanos, the canonical Pum co-factor required for regulation of hb in the posterior and CycB in the PGCs. We are currently defining binding sites for Pum in the CycB 3’-UTR as a first step to elucidating the mechanism by which it regulates translation in a Nanos-independent manner. 204 POSTERS: Regulation of Gene Expression

342C Lamin interacts with the gypsy insulator complex. Daniel K Oliver. Biology, University of Nevada, Reno, Reno, NV. DNA organization in the eukaryotic cell nucleus is dynamically regulated. Regions of the chromosomes are compacted to various degrees according to their transcriptional activities. The mechanism through which this is accomplished is yet unknown. However, these higher order structures appear to be accomplished through a class of cis transcriptional regulators deemed “chromatin insulators” or “boundary elements,” as previous studies have suggested. These insulators compartmentalize transcriptionally active regions of the genome, separating active regions from inactive. However, accumulating evidence suggests that chromatin insulators are involved in the organization of higher order structures in chromatin. The gypsy insulator complex of Drosophila melanogaster is the best-understood insulator of all those identified to date. It consists of a DNA sequence containing twelve copies of a binding site for the DNA-binding protein Suppressor of Hairy Wing [Su(HW)], which recruits various other insulator proteins and organizes them into a functional complex. Mod(mdg4)2.2 has long been acknowledged as a contributor to the gypsy insulator complex, while Centrosomal Protein 190 (CP190) has recently been identified as a key component of the gypsy insulator. A recently discovered possible contributor to gypsy insulator activity is lamin. Lamin is a known participant in nuclear envelope functioning and has been verified as a component of the nuclear matrix. It also shows strong evidence of association with the gypsy insulator, particularly mod(mdg4)2.2. It has demonstrated notable enhancer properties of gypsy phenotypes, primarily that of yellow2. In this study, two lamin phenotypes were explored, lamin Ari3 and lamin 83, a weak and strong phenotype, respectively. They were assayed for interactions with mod(mdg4)2.2 and CP190, which both suggested an association of lamin to the gypsy insulator complex.

343A dBlimp-1, a developmental timer protein controls pupation timing through its stability. Moustafa Sarhan1, Hitoshi Ueda1,2. 1) Biol. Dept., Fac. of Sci. Okayama Univ., Okayama, JP; 2) The Grad. Sch. of Nat. Sci. and Tech., Okayama Univ.Okayama, JP. In living organisms, the timing of some developmental events is precisely controlled. In Drosophila, ecdysone plays a fundamental role in controlling the developmental timing. Recently, we identified dBlimp-1 as a binding factor to the promoter region of the ftz-f1 gene and demonstrated that dBlimp-1 is induced by ecdysone and can repress βFTZ-F1 expression when tether to the promoter region of the ftz-f1 gene. Furthermore, dBlimp-1 is instable and its rapid loss is required for the proper timing of βFTZ-F1 expression and pupation. To analyze the functional domains of dBlimp-1, we started with deleting the conserved regions and the proline-rich region and check the effects of these deletions in the context of βFTZ-F1 expression, pupation timing and stability of the expressed proteins. Results indicated that, dBlimp-1 posses two repression domains; one is within the proline-rich region and the other lies on a conserved region which is present in N terminal region. Instability of dBlimp-1 is also caused by middle part of its proline-rich region. A deletion of certain region in the proline-rich region renders dBlimp-1 to become stable and retain its repression function. Induction of this stable but functional dBlimp-1 in mid stage prepupae leads to far delayed in pupation timing as compared with that line carrying full length gene. Taken together, dBlimp-1 is a timer protein which can determine the precise timing of βFTZ-F1 expression and pupation timing by working as a repressor for the ftz-f1 gene.

344B Post-transcriptional control of cyclin B during Drosophila spermatogenesis. Alicia Shields, Byung Soo Gim, Margaret T. Fuller. Department of Genetics, Stanford University School of Medicine, Stanford, CA. Developmental control of the core cell cycle machinery is critical to ensure proper execution of cell type and stage specific cell cycles. One of the most important developmentally regulated cell cycles occurs during meiosis. Understanding how the developmental program controls meiotic divisions of the germ line will provide insight into cell cycle control in vivo during development and differentiation. Meiosis features a specialized cell cycle with an extended G2 phase known as meiotic prophase. In Drosophila males, the G2 period of meiosis I lasts 3.5 days and is characterized by the transcription of genes required for subsequent spermatid differentiation. Many of these transcripts, as well as those required for entry into meiotic division, are translationally repressed until their protein products are required. We have identified a pathway involving translational control of the cell cycle regulator cyclin B that may play a critical role during meiotic prophase to delay transition from the G2 phase to the first meiotic division. In this pathway, the cyclin B 3’ UTR acts in cis and the RNA binding protein Tsr acts in trans to delay expression of cyclin B protein until just prior to the G2/M transition. We are currently investigating cis-acting sequences in the cyclin B 3’ UTR that specify translational repression of cyclin B in immature spermatocytes, and the mechanism of action of the repressor Tsr and other trans-acting factors that impose translational repression via the cyclin B 3’ UTR. POSTERS: Regulation of Gene Expression 205

345C The role of oligomerization and histone deacetylation in Groucho-mediated silencing during wing development. Clint Winkler, Wiam Turki-Judeh, Alberto Ponce, Albert Courey. Deptartment of Chemistry & Biochemistry, University of California, Los Angeles. The Drosophila Groucho (Gro) protein is a prototype for a large family of co-repressors, including the human TLE proteins. As a global co-repressor, Gro is recruited to DNA by transcriptional repressors, and plays diverse roles in development and signal transduction. Upon recruitment to DNA, Gro mediates long-range silencing independent of its distance from the promoters and enhancers driving expression. This process is completely dependent on the ability of Gro to oligomerize through a putative coiled- coil motif and partially dependent on an interaction with the class I histone deacetylase Rpd3. Genetic and biochemical approaches are being used to study the relevance of oligomerization and histone deacetylation to Gro- mediated silencing. The expression domains of the Decapentaplegic (Dpp) target genes optomotor-blind (omb) and vestigal (vg), genes that regulates anterior/posterior patterning in early wing development, are regulated by Gro in the wing imaginal disc. Repression of these genes in the peripheral boundaries of the wing pouch is a result of Gro recruitment by the sequence specific repressor Brinker, which opposes Dpp signaling. Wing disc chromatin immunoprecipitation (ChIP) experiments are being used to understand the role of oligomerization in the repression of omb and vg. Additionally, ChIP experiments and HDAC inhibitor experiments seek to illuminate the relationship between repression and histone deacetylation.

346A 1,2 Mechanisms of metabolic suppression in Drosophila melanogaster surviving extremely low O2 environments. Dan Zhou , Jin Xue1,2, Orit Gavrialov1,2, Gabriel Haddad1,2. 1) Departments of Pediatrics and neuroscience,University of California, San Diego, La Jolla, CA 92093-0735, USA; 2) The Rady Children’s Hospital - San Diego, San Diego, California, USA. Metabolic suppression is an important survival strategy in many animal species when exposed to a severe hypoxic environment. Although a number of biological mechanisms have been proposed for the transition to a hypometabolic state in hypoxia, the molecular basis that mediates this suppression of metabolic activity is still largely unknown. Currently, we have generated a Drosophila melanogaster strain that survives extremely low, normally lethal, level of O2. In order to identify the molecular basis of metabolic suppression and its role in hypoxia-tolerance, we determined the differences in expression profiles between hypoxia-selected and control flies in larvae. We found a significant down-regulation in the genes encoding metabolic enzymes which include glycolytic enzymes, TCA cycle enzymes, lipid and protein metabolic enzymes. In order to investigate the mechanisms that regulate the coordinated suppression of these genes, a de novo analysis was performed to identify transcription factor binding elements in the cis-regulatory regions of the functionally related genes. We found that the binding element of hairy was highly enriched in the down- regulated, but not up-regulated or not changed, genes encoding TCA cycle enzymes. Furthermore, loss-of-function mutation of hairy significantly enhanced hypoxia-sensitivity in flies. These results demonstrated that the hairy transcription suppressor mediates, at least in part, the transcriptional suppression of metabolic genes and plays an important role in hypoxia-tolerance. 206 POSTERS: Signal Transduction

347B The Ca+2-dependent protease Calpain A modulates embryonic dorsal-ventral patterning. Helena Araujo1, Katia Carneiro1, Marcio Fontenele1, Rodrigo Agrellos1, Adriana Oliveira-Silva1, Ethan Bier2. 1) Dept Histology & Embriology, Fed Univ Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil; 2) Section on Cell and Developmental Biology, University of California at San Diego. During Drosophila embryogenesis, a nuclear gradient of the NFκB/c-rel protein Dorsal subdivides the dorsal-ventral (DV) axis in a threshold-dependent manner. The central process that induces formation of the Dorsal gradient is ventral activation of the Toll receptor pathway, which induces proteassomal degradation of the IκB homologue Cactus. Although signaling through Toll seems to be suficient to generate all Dorsal thresholds, other pathways may provide precision in the location of DV territories. Here we show that the Calcium-dependent protease encoded by Calpain A alters DV patterning. CalpA degrades Cactus as part of a Toll-independent pathway. Through double-stranded RNA interference and mRNA injections we show that CalpA degrades Cactus dependent on residues in the C-terminal PEST domain. Furthermore, CalpA seems to be inhibited by maternal Dpp signals, which contributes a dorsalizing activity contrary to the ventralizing Toll signal. The mechanisms for regulation of CalpA by dpp are under investigation.

348C Fine Dissection of the BMP-dependent Regulation of brinker. Enrica Charbonnier, George Pyrowolakis. Developmental Biology Unit, Institute for Biology I University of Freiburg, Freiburg, Germany. Signaling by the ligand Decapentaplegic (Dpp), a member of the TGFβ superfamily of cytokines and the fly ortholog of vertebrate BMP2/4, plays a crucial role in many developmental processes in Drosophila melanogaster. Recently, we reported and characterized a novel branch of the Dpp signaling pathway that results in the direct repression of key developmental target genes. Dpp-induced repression is mediated by short silencer elements (SEs) located in the regulatory region of target genes. A specific arrangment of Smad binding sites in the SEs instructs the signal-induced assembly of a repressor complex consisting of Smad proteins and the cofactor Schnurri. A primary role of the repressive branch of Dpp signaling consists in the transcriptional downregulation of brinker (brk) in many different tissues throughout development. This regulation is critical for the read-out of the Dpp-signaling pathway, as Brk has been shown to be a direct transcriptional repressor of Dpp-target genes in the absence of signaling. The significance of the SE-dependent downregulation of brk is particularly striking in the wing imaginal disc where Dpp acts as a long-range morphogen to control both growth and patterning mostly through the establishment of an inverse and instructive gradient of nuclear Brk protein. The importance of the Dpp-mediated repression of brk is probably reflected in the high number of potential SEs found in the genomic locus of the gene. A total of 10 evolutionary conserved SEs decorate the regulatory region of brk, much more than in any other gene in the Drosophila genome. Here we present initial efforts in understanding the impact of the SEs in brk regulation during both embryonic and larval development. Using reporter constructs and transgenic approaches, we are testing if any given SE is affiliated with an individual, tissue specific, enhancer of brk, or if, alternatively, SEs act in an additive manner to confer graded expression of brk in tissues such as the wing imaginal disc.

349A TGF-β signaling in the ring gland. Scott Gesualdi, Theodor Haerry. Dept Biol, Florida Atlantic Univ, Boca Raton, FL. Drosophila has adapted a developmental strategy to reach the sexually mature stage as fast as possible. However, like every animal, the limiting factor is often the availability of food. The prothoracic portion of the ring gland plays an important role in the regulation of development in response to available nutrients through the secretion of the steroid hormone 20-hydroxy-ecdysone (20E). While it has been found that Insulin-like growth factor signaling promotes synthesis of 20E, we find that activation of the TGF- β pathway through the Activin receptor BABO inhibits the release of 20E. Currently, we are analyzing the role TGF-β signaling and two potential downstream transcription factors play in ring gland development and the regulation of P450 cytochrome hydroxylases involved in 20E synthesis. Our results suggest that sexual maturation is controlled by an evolutionary conserved interaction between Insulin-like and TGF-β type growth factors to control the release of steroid hormones. POSTERS: Signal Transduction 207

350B Fused-Costal2 regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation. Yajuan Liu1, Xuesong Cao1, Jin Jiang2, Jianhang Jia1. 1) Sealy Center for Cancer Cell Biology and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555; 2) Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390. The seven-transmembrane protein Smoothened (Smo) acts as a signal transducer in the Hedgehog (Hh) pathway that mediates many key developmental processes. In Drosophila, Hh-induced phosphorylation promotes Smo cell-surface accumulation and signaling activity; however, the mechanisms controlling Smo phosphorylation and cell-surface accumulation are still unknown. The intracellular signaling complex containing Fused (Fu) and Costal2 (Cos2) is thought to transduce the Hh signal downstream of Smo. Here, we identify a novel feedback mechanism that regulates Smo through the Fu-Cos2 complex. We found that Hh-induced Smo accumulation is inhibited in fu mutant clones or by expressing a dominant-negative form of Fu, and such inhibition is alleviated by removal of Cos2. Conversely, overexpressing Cos2 blocks Smo accumulation, which is reversed by coexpressing Fu. Cos2 blocks Smo accumulation through its C-terminal Smo-interacting domain; and Fu antagonizes Cos2 by phosphorylating Cos2 at Ser572. Furthermore, we found that Ser572 phosphorylation attenuates Cos2-Smo interaction and promotes Cos2 instability. Finally, we provided evidence that Fu and Cos2 control Smo cell-surface accumulation by regulating Smo phosphorylation. Our data suggest that Cos2-Smo interaction blocks Hh-induced Smo phosphorylation, and that Fu promotes Smo phosphorylation by antagonizing Cos2.

351C Hedgehog-induced phosphorylation regulates Smoothened activity by promoting a conformational switch in its C-terminal cytoplasmic tail. Yun Zhao, Jin Jiang. Department of Developmental Biology, UT SW Medical Ctr, Dallas, TX. Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins is essential for metazoan development and its malfunction causes congenital disorders and cancer. The seven-transmembrane protein Smoothened (Smo) transduces the Hh signal across the plasma membrane by direct association with a cytoplasmic signaling complex containing the kinesin-related protein Costal2 (Cos2) and the Ser/Thr kinase Fused (Fu), but how Smo is activated remains poorly understood. In Drosophila, Hh induces phosphorylation at multiple Ser/Thr residues in the Smo C-terminal cytoplasmic tail (C-tail), leading to Smo cell surface accumulation and activation. Here we provide evidence that phosphorylation regulates Smo activity by inducing a conformational switch in its C- tail. Using fluorescence resonance energy transfer (FRET) assay, we find that Smo exists as a preformed dimer/oligomer, but in the absence of Hh, the Smo C-tails within the dimer/oligomer adopt an inhibitory conformation that prevents their association. Hh- induced phosphorylation promotes a conformational change that increases the proximity between Smo C-tails. Point mutations that impair Smo dimerization/oligomerization compromise Smo activity, which can be restored by forced oligomerization through a heterologous system. Furthermore, induced oligomerization of Smo C-tails suffices to trigger pathway activation independent of endogenous Smo. We find that Hh-induced phosphorylation of Smo facilitates recruitment of the cytoplasmic signaling complex containing Cos2 and Fu, leading to Fu dimerization and activation. We propose that Hh-induced phosphorylation regulates Smo activity by inducing an active conformation that promotes the recruitment and activation of the cytoplasmic signaling complex.

352A A structure-function analysis of the Drosophila STAT Stat92E. Laura Ekas, Timothy Cardozo, Foster Gonsalves, Erika Bach. Dept Pharmacology, New York Univ Sch Medicine, New York, NY. The JAK/STAT pathway is critical for many biological processes in both vertebrates and invertebrates. In contrast to mammals, Drosophila has a single JAK (Hopscotch) and a single STAT (Stat92E). A detailed structure-function analysis of the 761 amino acid Stat92E protein has not been reported. To address this issue, we made truncations and substitutions in Stat92E and tested these variants in in vivo and in vitro assays. A full-length Stat92E protein tagged at the N-terminus (3HA-Stat92EFL) rescues the small eye and the larval lethality in stat92E mutants. In addition, Stat92EFL strongly activates an in vitro Luciferase reporter. We also find that Stat92E with a Y711F substitution is non-functional in both assays, as predicted by the inability of Stat92EY711F to form dimers and bind DNA. Moreover, we find that Stat92E variants lacking either the first 134 (3HA-Stat92EΔN) or the last 35 residues (3HA-Stat92EΔC) are fully functional in both assays. Our results differ from a previous study, which demonstrated that Stat92E lacking residues 1-134 behaved as a negative regulator of JAK/STAT signaling (Henriksen et al., 2002 Genes Dev 16:2379). Furthermore, Stat92E lacking both the N- and C-terminal domains (3HA-Stat92EΔNΔC) activates the Luciferase reporter significantly more than wild type and is a gain-of-function allele in vivo. These findings are particularly interesting considering that both the N- and C-terminal domains of mammalian STATs have been shown to be critical for maximal transcriptional activation. Our study therefore reveals that there may be significant functional differences between Stat92E and mammalian STATs. Additionally, we have identified R442, which resides in the DNA binding domain and is conserved in mammalian STAT3 and STAT5a, as an amino acid critical for function. This residue is mutated to P in the stat92E85C9 loss-of-function allele. Non-conservative mutations to P or A result in a non-functional Stat92E, as does a conservative mutation to K. These results suggest that the guanidinium side chain group of R442 is critical for the Stat92E:DNA interaction. 208 POSTERS: Signal Transduction

353B The function of StIP in the JAK/STAT pathway. Linzhu Han, Douglas A. Harrison. Biol Dept, Univ Kentucky, Lexington, KY. The JAK/STAT pathway is a well conserved developmental pathway that is important in cell proliferation, differentiation, cell migration and apoptosis. It shares same mechanism in flies and vertebrates. Ligand-dependent activation of the JAKs results in phosphorylation of STATs. The phosphorylated STATs enter into the nucleus and regulate transcription of target genes. The fly JAK pathway controls many processes, including specification of follicle cells in oogenesis and regulation of hematopoiesis. In mammals, StIP is believed to be an adaptor facilitating JAK/STAT pathway activation. It can associate with both JAKs and unphosphorylated STATs, and may serve as a scaffold protein to regulate activation of STAT3 by JAK phosphorylation. In Drosophila, there is one StIP homolog, CG11887. Our lab has identified three mutations in Drosophila StIP. Stipc05390is a piggyback insertion in exon 2, and was generated in the Exelixis insertional mutagenesis screen. Two other alleles are deletions of StIP generated by the excision of a nearby P element. All of them are homozygous lethal. Two assays are being used to determine whether StIP mutations affect JAK pathway activity. One is interaction with hopTum-l, which causes upregulation of JAK/STAT signaling and hematopoietic neoplasia in flies. StIP is assumed to positively affect the JAK/STAT pathway and consistent with this, mutations in StIP dominantly suppress tumor formation. A second assay is follicle cell specification in oogenesis. High JAK activity is required for specification of the most terminal follicle fates, the border cells and posterior terminal cells. However, no obvious effect on follicle cell fate has been detected in StIP mutant clones. Furthermore, in situ hybridization indicates StIP has a distinct embryonic expression pattern from upd, the gene encoding the primary ligand for JAK activation. Results of these studies will be presented.

354C A Role for Ipk2 Kinase Activity in Regulating Cell Proliferation and Apoptosis of Imaginal Disc Tissue During Drosophila Melanogaster Development. Man-kin Marco Tsui, Andrew Seeds, John York. Howard Hughes Medical Institute, Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710 USA. Eukaryotic cells process an ensemble of highly phosphorylated inositol molecules, known as inositol phosphates (IPs,) such as IP4, IP5 and IP6. In recent years, the metabolic pathways of IPs have been studied in a number of organisms. In the yeast Saccharomyces cerevisiae and the fruit fly Drosophila melanogaster, inositol hexakisphosphate IP6 is generated from IP3 via the sequential actions of the kinases Ipk2 and Ipk1. Ipk2 is involved in the first step of IP synthesis and it is a 6-/3-kinase that phosphorylates I(1,4,5)P3 to I(1,3,4,5,6)P5. Here we report that loss of Drosophila melanogaster IPK2 results in developmental defects. Homozygous ipk2 nulls are pupal lethal, fail to eclose as adults and exhibit abnormalities of the eyes, wings and body. These defects can be rescued by the expression of wild-type fly Ipk2 but not a kinase inactive mutant, consistent with a role for the products of Ipk2, IP4 or IP5, in regulating development. Examination of ipk2 null larvae revealed that the imaginal tissues exhibit extensive spontaneous apoptosis. The apoptosis may partly be due to the activation of JNK signaling. In addition, the ipk2 null imaginal discs exhibit defects in the rate of DNA synthesis, partially through Ipk2-dependent modulation of JAK-STAT signaling pathways. Taken together, our results suggest Ipk2 plays important roles in proliferation by maintaining normal DNA replication rate in development, and loss of Ipk2 results in spontaneous apoptosis possibly due to activation of JNK signaling and other apoptotic pathways.

355A Molecular and genetic characterization of upd, upd3 and os. Liqun Wang, Douglas Harrison. Dept Biology, Univ Kentucky, Lexington, KY 40506. The JAK/STAT pathway is a well conserved signaling pathway from vertebrates to Drosophila. It responds to many ligands including cytokines and growth factors in vertebrates. In Drosophila, the secreted glycoprotein, Unpaired has been shown to activate the JAK/ STAT pathway. Unpaired has an essential role in many developmental processes such as embryonic patterning, sex determination, and more. Gene upd is located in polytene band 17A. Two predicted genes, upd2 and upd3, within 70kb of upd, have sequence similarity with upd and Upd2 can activate JAK signaling. Classical mutations, described as outstretched (os), have been defined as alleles of upd. Two upd alleles, updYM55 and updYC43, cause embryonic lethality. Three os alleles, oso, oss, os1, result in outstretched wings, small eyes, or both. Allelism of upd and os is based on the failure of the zygotic lethal upd alleles to complement the outstretched wing and small eye phenotypes of the os alleles. However, additional mutations in the upd region were identified that genetically separate the os and upd loci, suggesting that the molecular basis of the complementation is complicated. Because the upd-like genes are close to upd on the X chromosome and show sequence similarity with upd, it was possible that os phenotypes may result from mutations in upd-like genes. To test this, a mobilization screen was done to generate mutants of upd3 with a P element inserted in the last intron of upd3. The upd3 mutants show os phenotypes, small eyes and/or outstretched wings. Molecular characterization of upd3 mutants shows deletion of part or all of the last exon of upd3. Genetic characterization of upd3 mutants with upd and os shows that the upd3 mutants complement the upd mutants but fail to complement the os mutants. This suggests that the os may be caused by mutations in a common regulatory region of upd and upd3. Consistent with this, molecular characterization of os alleles has revealed lesions distal to upd3 in three independent alleles. Results of enhancer reporter assays for regions that are deleted in os alleles will be presented. POSTERS: Signal Transduction 209

356B Fucose modification is essential for Delta- but not Serrate-dependent activation of Notch signaling in Drosophila. Tomonori Ayukawa1, Hiroyuki O. Ishikawa2, Kenji Matsuno1,2. 1) Dept Biol Sci, Tokyo Univ of Science; 2) Genome and Drug Research Center, Tokyo University of Science. Notch (N) is a transmembrane receptor with homology to epidermal growth factor (EGF-like repeats) and mediates cell-cell interactions necessary for many cell-fate decisions. Some of these EGF-like repeats are O-fucosylated by O-fucosyltransferase 1(O-fut1) that is essential for N signaling in Drosophila and mouse. However, the specific roles of this O-fucose modification have been elusive, because O-fut1 has its enzymatic activity-independent roles, and the O-fucose can be a mere foothold for further modification by N-acetylglucosaminyltransferase Fringe (Fng), which is required for certain N signaling events. Therefore, in this study, we attempted to evaluate the specific requirements of the Notch O-fucose modification, using the mutants of two genes, GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer), which are essential for the GDP-L-fucose synthesis. Since, GDP-L-fucose is the universal donor for fucosyltransferases, these mutants lack all forms of fucose-modification and thus show defects in N signaling. We overexpressed two ligans for N, Delta (Dl) and Serrate (Ser), in the wing discs of Gmd mutant and found that Dl-N, but not Ser-N signaling preferentially requires fucosylaton of N. In agreement with these observations, overproduction of sensory organ precursor (SOP) cells was observed in the Gmd mutant disc, suggesting a failure in lateral inhibition, in which Delta but not Ser and fng have essential role. These results suggest that some class of fucose modification has a novel function in N signaling. However, in addition to O-fucose modification, fucose could be attached to N- glycans, proteoglycans, and glycolipids. Therefore, it is currently unknown what kind of fucose modification has a crucial role in N signaling. Experiments to determine the specific fucose modification, which is responsible for our observations, are under progress.

357C Mechanism of Bearded family protein activity during Notch-mediated lateral inhibition in Drosophila. Joseph R. Fontana, James W. Posakony. Division of Biological Sciences/CDB, University of California San Diego, La Jolla, CA 92093. The Notch (N) pathway is an evolutionarily conserved cell-cell signaling system that is required for the specification of a large number of different cell types in developing animals. In Drosophila, Notch signaling is involved in many post-embryonic developmental processes, including multiple cell fate decisions during mechanosensory organ development. Among the known Notch targets in Drosophila is the Bearded family of genes. These genes encode small proteins (8-26 kDa) that have important, but largely uncharacterized, roles in Notch-dependent processes such as lateral inhibition, where a single sensory organ precursor (SOP) cell is specified within a group of equipotent cells (proneural clusters), and binary cell fate decisions in the SOP lineage, which give rise to the differentiated cells of the mature mechanosensory organ. Misexpression of Bearded family genes leads to disruptions in both lateral inhibition and later cell fate decisions during mechanosensory organ development. We have carried out multiple genetic and structure/function studies of Bearded family proteins, seeking more detailed insights into the mechanism by which they participate in Notch-mediated lateral inhibition. Precise deletions of several Bearded family genes, created by homologous recombination, have been used to demonstrate their overlapping activities in lateral inhibition. Misexpression of numerous Bearded protein variants has revealed which protein domains contribute to their capacity to disrupt lateral inhibition. Cell culture assays have allowed us to correlate these results with Bearded protein localization, as well as with the ability of Bearded proteins to affect the localization of Neuralized, an E3 ubiquitin ligase and known binding partner of Bearded proteins. Our biochemical analyses have uncovered the domains required for interaction with Neuralized and the domain responsible for inhibiting the interaction between Neuralized and one of its targets, the Notch ligand Delta. The results of these studies will be presented.

358A The DEAD-box RNA helicase Belle differentially regulates Notch signaling during Drosophila development. John Poulton, Wu-Min Deng. Dept. of Biological Science, Florida State University, Tallahassee, FL. Identifying genes involved in a cell signaling pathway can help identify core components of the pathway, but can also help us understand how the pathway functions at the cellular level by uncovering the processes that modify and regulate various aspects of pathway activity. From a genetic screen for defects in Notch activity during oogenesis, we have identified Belle (Bel) as being needed for Notch activity in the follicle cells. At stage 6, Notch is activated in the follicle cells, resulting in differentiation and cessation of proliferation. Follicle cell clones of bel do not express markers indicative of differentiation, but instead show prolonged expression of immature fate markers and mitotic markers after stage 6. In addition, expression of the Notch activity reporter E(Spl):CD2 is reduced in bel clones. We also find that oocyte polarity is disrupted when the posterior follicle cells are mutant for bel. bel clones generated in imaginal discs cause severe defects in the adult eye and wing. Surprisingly, we find that Notch activity is upregulated in bel clones in both eye and wing discs, as indicated by increased levels of E(Spl):CD2 staining. We are currently working toward understanding the reason for the apparent inverse relationship between bel and Notch activity levels in imaginal discs compared to follicle cells. On this point, we have noticed that Notch protein accumulates in bel clones during oogenesis, suggesting possible defects in Notch trafficking. In discs, mutations affecting Notch trafficking have been shown to upregulate Notch activity, as we see in bel disc clones. We are therefore investigating the relationships between bel and Notch trafficking, and between Notch trafficking and Notch activity in oogenesis. Preliminary data suggest that mutations in some genes previously shown to disrupt Notch trafficking and upregulate Notch activity in discs, do have the opposite effect on Notch activity levels in the follicle cells, consistent with our findings in bel clones. 210 POSTERS: Signal Transduction

359B Role of Lqf in Notch signaling. Xuanhua Xie. Dept MCDB, Univ Texas at Austin, Austin, TX. Notch signaling pathway is involved in almost all kinds of tissue development in metazoans. In Drosophila, two different ligands, Delta and Serrate, are employed to send Notch signal. Interestingly, endocytosis of Delta is a necessity for signaling. Liquid facets (Lqf), a Drosophila epsin, plays a specific role for Delta endocytosis. Although several functional domains, like UIMs (ubiquitin interaction motifs), CBMs (clathrin binding motifs) and ENTH (epsin N-terminal homology), have been characterized, the specific roles are still unknown. We decide to prepare different genomic pieces of lqfs containing deletions of these motifs and tagged with GFP. Expression levels will be determined and rescue experiments will be done using lqf null or hypomorphic alleles. The subcellular localization of the mutant proteins will also be investigated. These results could be informative to elucidate why Delta endocytosis is important and the mechanism of Notch signaling.

360C Roles of Drosophila Deltex in Suppressor of Hairless-independent Notch signaling. Kenta Yamada, Kazuya Hori, Takashi J. Fuwa, Kenji Matsuno. Dept. Biol. Sci. / Tec., Tokyo Univ. Sci., Japan. Notch is a single-pass transmembrane receptor. Notch signaling controls many aspects of cell functions, such as cell-fate specification, morphogenesis, and programmed cell death. Deltex (Dx) family proteins, RING domain E3-ubiquitin ligases, are conserved modulator of Notch signaling. Dx regulates the endocytic trafficking of Notch in combination with other regulators of endocytosis, such as Nedd4 family proteins and β-Arrestin. We previously proposed that Dx plays a role in non-canonical and Suppressor of Hairless [Su(H)]-independent Notch signaling upstream of Notch, whereas canonical Notch signaling thoroughly depends on Su(H). In this study, we attempted to understand how Dx activates Su(H)-independent Notch signaling in Drosophila. We found that Mastermind (Mam) was also dispensable for Dx-mediated Notch signaling. However, Notch and Presenilin (Psn) were required for Dx-mediated Notch signaling, indicating that both Su(H)-dependent and Su(H)-independent Notch signaling requires the S3 site cleavage of Notch for its activation. Hrs and Vps28 are components of the ESCRT (endosomal sorting complex required for transport) machinery, which sorts ubiquitinated membrane proteins into intraluminal vesicles. We found that these two genes were not required for Dx-mediated Notch signaling. These results suggest that Dx-mediated Notch signaling occurs at endosomal membrane of early endosomes. We also found that loss of dx functions resulted in an accumulation of Notch in the subapical region of epithelial cells. Under this condition, Notch was reduced in early endosomes, but increased in late endosomes. These results suggest that Dx promotes the transportation of Notch from the plasmamembrane to early endosomes where Su(H)-independent Notch signaling is probably activated.

361A Regulation of Neuroblast Self-renewal in Drosophila by aPKC and Numb. Xiofei Chen, Krista Golden, Sahar Rahmani, Cheng- Yu Lee. Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109. Asymmetric cell division provides an efficient mechanism for precise regulation of stem cell identity and potential (self-renewal) and generation of cellular diversity (differentiation). Despite many seminal studies reporting a large number of genes that promote stem cell identity, regulation of stem cell self-renewal remain largely undefined. We have been using Drosophila neural stem cells (neuroblasts) as a model system to study regulation of self-renewal vs. differentiation in the context of development. Brain neuroblasts repeatedly divide asymmetrically for hundreds of times to self-renew themselves and to generate thousands of terminally differentiated neurons. Neuroblast self-renewal requires establishment and maintenance of proper apical/basal cortical polarity; the evolutionarily conserved cell polarity protein atypical Protein Kinase C activity at the cortex (aPKCcaax) is a potent activator of neuroblast self- renewal. However, how aPKC regulates neuroblast identity remains largely unknown. Here, we show that over-expression of aPKCcaax induces symmetric neuroblast-neuroblast cell division in a dosage-sensitive manner. A high level of aPKCcaax disrupts cortical cell polarity and triggers symmetric segregation of cell fate determinants including Numb (Nb), likely leading to decreased Nb function in both daughter cells. Consistent with this hypothesis, overexpression of fly Nb or mouse Nb rescues ectopic increase in neuroblasts induced by aPKCcaax indicating that Nb likely mediates aPKCcaax-induced neuroblast self-renewal. Domain analysis reveals that the conserved protein tyrosine binding (PTB) domain and the C-terminus of the Nb protein are both required for suppression of aPKCcaax- induced ectopic neuroblasts. Over-expression of a PTB-domain binding protein Numb associated kinase (Nak) enhances aPKCcaax- induced ectopic neuroblasts. Currently, we are focusing our effort on understanding the molecular mechanisms by which aPKC, Nb, and Nak regulate neuroblast identity. POSTERS: Signal Transduction 211

362B PDE1c and PDE11 and their role in Drosophila male fertility. Rachel A Clemens-Grisham, Anke Vermehren, David B Morton. Integrative Biosciences, Oregon Health and Science University, Portland, OR. Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides which make them an important regulator of the cAMP and cGMP signaling pathways. The Drosophila genome codes for six PDEs. PDE4 (Dunce) has been extensively studied for its role in a number of behavioral pathways including learning and memory and courtship. Other PDEs in Drosophila are PDE1c, 6, 8, 9, and 11. Both PDE1c and PDE11 are dual specific for cAMP and cGMP. Additionally, PDE1c is predicted to have two calcium/ calmodulin binding sites. Our evidence suggests that both PDE1c and PDE11 play a role in male fertility. A fly line with a PiggyBac PB insertion (PDE1cc04487) in the first intron of PDE1c is male sterile and PDE1cc04487/Df(2L)exel6030 is also male sterile. Precise excision of c04487 restores male fertility. Male PDE1cc04487 will copulate with w1118 females and their testes contain motile sperm. However, seminal recepticles removed from these females contain no visible sperm suggesting infertility is caused by an inability to transfer sperm to the female. We also generated a reporter construct (pPDE1c-GAL4) that drives UAS-dsRED expression in neurons in the CNS and peripheral sensory neurons and cells in the seminal vesicle, anterior ejaculatory duct and ejaculatory bulb of the male reproductive system. We have generated a fly line containing a transgene of PDE1c cDNA under heat shock control which has been crossed into the PDE1cc04487 mutant background and preliminary evidence suggests that ubiquitous expression of PDE1c after adult eclosion rescues the male infertility phenotype. We have also generated a deletion in the PDE11 gene (PDE11Δ4195-2198) that removes the translational start site of one of two predicted transcripts that is also male sterile. In addition, PDE11Δ4195-2198/Df(2l)M36F- S6 males are sterile. We are currently investigating the cause of infertility in this mutant using a PDE11 cDNA under heat shock control to rescue the infertility.

363C A novel ecdysone (20E) signaling pathway is used in the mid-third instar to turn on glue genes. Benjamin Costantino1, Daniel Bricker1, Kate Shen1, John Merriam2, J Callender3, Vincent Henrich3, Andrew Andres1. 1) University of Nevada-Las Vegas,School of Life Sciences, 4505 Maryland Parkway, Las Vegas, NV 89154-4004; 2) Department of Molecular and Cellular Biology, University of California-Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90024-1606; 3) Department of Biology, University of North Carolina- Greensboro, 312 Eberhart Ave, North Carolina 27412-5001. A major role of the larval salivary gland in Drosohpila melanogaster is to synthesize and secrete a polypeptide glue mixture, securing the developing pupa to a solid substrate. The steroid hormone 20-hydroxyecdysone (20E) is a key signaling molecule that modulates most of the salivary gland activity. At the end of the third instar, it has been shown that 20E activates a heterodimeric nuclear hormone receptor consisting of ecdysone receptor (EcR) and Ultraspiricle (USP) to directly induce the “early” polytene puff genes and cause stored glue granules to dump their contents into the lumen of the salivary gland. However, in mid-third instar larvae the glue genes are dramatically and synchronously expressed prompting many authors to speculate that this event is also regulated by a smaller pulse of 20E. Here we show that induction of the glue genes is 20E regulated, but the receptor responsible for these events is not a heterodimer consisting of EcR and USP. Using glue synthesis as an assay system, we are formulating a new model in which EcR heterodimerizes with another member of the nuclear-hormone receptor superfamily to induce glue synthesis. Presented here is evidence for a putative novel ecdysone signaling pathway.

364A Regulation of Drosophila Phototransduction by Ceramide Kinase. Ujjaini Dasgupta, Usha Acharya. Program in Gene Function and Expression, University of Massachusetts, Worcester, MA. Ceramide Kinase (CERK) is an enzyme that phosphorylates the sphingolipid ceramide to ceramide-1- phosphate thereby decreasing ceramide levels. We have identified Drosophila CERK (DCERK) as a key regulator of phototransduction. Drosophila phototransduction is a prototypic G protein coupled receptor (GPCR) signaling cascade that results in activation of transient receptor potential (TRP) and TRP-like channels downstream of Phospholipase C (Norp A) in response to light. Mutants in proteins of this cascade lead to retinal degeneration and have been used as models to understand human degenerative disorders. We have isolated mutants in DCERK and they show down-regulation of Rhodopsin levels accompanied by light induced retinal degeneration of photoreceptors. Genetic, molecular and biochemical analyses suggest that these mutants are defective in Phospholipase C mediated hydrolysis of phosphatidylinositol 4, 5, bis-phosphate to diacylglycerol and inositol trisphosphate. Our results not only reveal the role of DCERK in photoreceptor homeostasis, they also emphasize the crosstalk between sphingolipid and phospholipid mediated signaling in vivo. 212 POSTERS: Signal Transduction

365B Investigating Dscam Signaling. Maria-Luise Erfurth1,2, Dietmar Schmucker1,2. 1) Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA; 2) Department of Neurobiology, Harvard Medical School, Boston, MA. The transmembrane receptor Dscam (Down syndrome cell adhesion molecule), a novel member of the immunoglobulin superfamily of cell adhesion molecules, is required for axon guidance and establishment of dendritic connections in many parts of the nervous system of the fruit fly Drosophila melanogaster. Dscam is not only expressed in neuronal cells both of the central nervous system and peripheral nervous system but also in cells of the innate immune system of Drosophila . An extraordinary feature of Drosophila Dscam is that it potentially encodes 38,016 possible isoforms with 19,008 distinct extracellular domains by virtue of mutually exclusive splicing. This property renders Drosophila Dscam an extremely interesting molecule, but has simultaneously hampered the investigation of Dscam function and signaling, as only biochemical assays showing isoform-specific homophilic interactions have been reported. To date it is unclear whether and how selective homophilic interactions are utilized in vivo and whether any other presently unknown ligands of Dscam are also important in vivo. Genetic and biochemical studies suggest that the adaptor molecule Dock, the Drosophila orthologue of the human proto-oncogene Nck links the Dscam receptor to intracellular signaling via Pak kinase and thereby mediates cytoskeletal changes in response to extracellular guidance cues. Dock itself has been shown to interact with the tyrosine kinase Ack, the protein tyrosine phosphatase dPTP61F as well as the Wiskott-Aldrich syndrome protein (Wasp) and the sorting nexin DSH3PX which in turn can directly interact with Dscam. The underlying activation mechanisms and details of the proposed pathway however, remain unidentified. The aim of my studies is to better understand the regulatory mechanisms and signaling machinery of the Dscam pathway. To achieve this goal we are using Drosophila immune cells for biochemical assays. We have started to define activated and non-activated states of Dscam signaling and try to identify crucial signaling events that underly Dscam’s function in neuronal wiring or immune defense.

366C Examining the Activation of Slipper, a Drosophila JNKKK. Rebecca Gonda, Beth Stronach. Dept Biological Sci, Univ Pittsburgh, Pittsburgh, PA. The Jun N-terminal Kinase (JNK) pathway regulates many cellular processes. Thus, it is important to understand how this signal transduction pathway can elicit specific responses. In Drosophila, there are several JNKKKs through which the pathway can act. One of these, Slipper (Slpr), the Drosophila mixed lineage linase (MLK), is essential for tissue morphogenesis. Selective activation of Slpr among JNKKKs may contribute to specificity in signaling. To explore mechanisms of Slpr activation, we can gain insight from models of mammalian MLK activation, which requires several steps. First is relief of autoinhibition, followed by dimerization. Once dimerized, the protein autophosphorylates and can be phosphorylated by upstream kinases. Additionally, JNK phosphorylates MLK at a consensus site as a possible mechanism of positive feedback. We are interested in the mechanism of Slpr activation and observing the effect of activation in vivo. Our first approach to study Slpr activation is using in vitro translated Slpr labeled with S35- methionine. Labeled Slpr appears as a doublet and shifts in mobility to a single band on a denaturing gel upon treatment with λ- phosphatase, indicating that it is phosphorylated. Slpr derivatives expressed in MDCK cells also appear to be phosphorylated. Deletion of a JNK consensus site results in a single band, indicating that this may be the site of phosphorylation in the other constructs. Two forms of Slpr differentially localize in the MDCK cells. Full-length Slpr has cortical localization, whereas a truncated form is cytoplasmic, indicating a role of the C-terminus in Slpr localization. To address how active Slpr functions in vivo, I am currently creating transgenic animals with alanine mutations in putative phosphorylation residues. These mutants are being characterized in various assays to determine how blocking Slpr phosphorylation affects its function. Overall, we hope that these experiments elucidate the mechanisms by which Slpr is modified to elicit a specific response within the JNK pathway.

367A Receptor Mediated Endocytosis of rat-Neu in Drosophila: A model of Receptor Tyrosine Kinase down regulation. Noor Hossain, Nasrine Yacoub, Leena Patel, Roger Jacobs. Biology Department, McMaster University, Hamilton, Ontario, Canada. Neu/ErbB2, a vertebrate member of the Epidermal Growth Factor Receptor (EGFR) family, is a Receptor Tyrosine Kinase (RTK) that plays an important role in fundamental cellular processes, including cell metabolism, survival, proliferation and differentiation. Over activation of ErbB2 is correlated with 20-30% of human breast cancers with poor clinical prognosis. The failure of RTKs to be appropriately deactivated is regarded as an important factor contributing to enhanced receptor activity. The key mechanism of RTK signal termination involves Receptor Mediated Endocytosis (RME), but it is not known how activated RTKs trigger subsequent down regulation. Previous studies in vertebrate cell culture, and in transgenic Drosophila have demonstrated that one phosphotyrosine (pTyr), Y1028 suppresses Neu signaling and protein levels. In this study we provide genetic evidence suggesting pTyr Y1028 of rat- Neu (neuYA) is involved in endocytosis and attenuated Neu/RTK signaling in Drosophila. A genetic modifier screen of neuYA with amorphic alleles of RME pathway components, identified that Rab5, Rab11, Epsin, Alpha-Adaptin, Effete, Cbl and Syntaxin modify phenotypes generated by ectopic Neu expression in a YA dependent manner. We have isolated novel mutations in Drosophila genes required to modify neuYA signaling in a genome wide dominant modifier mutagenesis. A constitutively active form of neuYAE (pTyr residue 1028 and 1253) expressed under the control of Glass Multimer Reporter (GMR-neuYAE ) produced rough eye phenotypes. Approximately 61,000 mutagenized F1 flies were screened for dominant suppressors or enhancers of this rough eye phenotype. A total of 30 enhancers and 36 suppressors were isolated and mapped to 27 complementation groups on the 2nd and 3rd Chromosome. We will report the outcome of our mapping study that identifies novel attenuating components of Neu/RTK signaling in Drosophila or known components of identified RTK signaling. This approach may identify gene candidates for the development of future cancer therapeutics targeting RTK down regulation. Supported by NCIC and NSERC. POSTERS: Signal Transduction 213

368B Metal sensing mechanism of metal-responsive transcription factor 1 (MTF-1). Haiqing Hua1, Xiaohua Chen3, Kuppusamy Balamurugan2, Dominik Staiger1, Alisa Davis1, Viola Günther1, Oleg Georgive1, David Giedroc3, Walter Schaffner1. 1) Inst of Molecular Biology, Zurich, Switzerland; 2) Inst of Physiology, Zurich, Switzerland; 3) Department of Chemistry, Indiana University, Bloomington, IN. Metal-responsive transcription factor 1 (MTF-1) is a central regulator of metal homeostasis and metal detoxification in both insects and mammals. It specifically binds to DNA sequence elements called metal response elements (MREs) associated with a number of metal- and stress-responsive genes. We and others have shown that human MTF-1 contains a cysteine-rich cluster(632CQCQCAC638), found immediately C-terminal to the serine/threonine-rich transcriptional activation domain. MTF-1 proteins containing two Cys- >Ala substitutions (C632A/C634A) or a deletion in this region (Δ(632-644)) are very poorly induced by Zn(II) and Cd(II) but retain the ability to drive basal levels of transcription in an MRE-dependent manner. Here we have characterized a cysteine-rich cluster containing six cysteines in Drosophila MTF-1. Wild type Drosophila MTF-1 is able to induce the transcription of metallothioneins and of the copper importer Ctr1B, in response to copper load and copper starvation, respectively. Interestingly, Drosophila MTF-1 protein containing six Cys->Ala substitutions is not induced by copper load but is still functional during copper starvation. Our data suggests that the cysteine-rich domain in Drosophila MTF-1 is not simply part of an activation domain but rather a sensor for excess copper in the cell.

369C Warts pathway signalling requires merlin, but not expanded, for R8 photoreceptor specification. David Jukam, Desplan Claude. Dept Biol, 1009 Main Bldg, New York Univ, New York, NY. The decision to express one of four rhodopsin molecules establishes the basis for the retinal mosaic of photoreceptor subtypes in Drosophila color vision. The Drosophila eye consists of ~800 ommatidia which each contain 8 photoreceptor cells that respond to light, in addition to 11 accessory cells. The outermost six photoreceptors, R1-R6, express the broad spectrum rhodopsin 1 that is involved in dim light and motion detection23. The two innermost photoreceptors, R7 and R8, respond to UV and colored wavelengths, respectively, by expressing one of four rhodopsins (rh3, rh4, rh5, rh6)1,13. The R7 and R8 photoreceptor are paired within each ommatidium in two subtypes — ‘yellow’, whereby R7 expresses UV-rh4 and R8 expresses green-rh6, or ‘pale, where R7 expresses UV-rh3 and R8 expresses blue-rh5. The ‘yellow’ subtype is found in 70% of ommatidia and the ‘pale’ combination make up the remaining 30%1,13. The two subtypes are randomly distributed throughout the retina. Recently, our lab showed that a tumor suppressor, warts/D-lats, and a growth regulator, melted, control the post-mitotic specification of R8 subtype fate. warts and melted repress each other’s transcription to form a bistable feedback loop that directs expression of either blue Rhodopsin 5 (Rh5) or green Rhodopsin 6 (Rh6) in R8. But whether the Warts/Hpo/Sav complex specifies photoreceptor fate through a distinct signaling pathway or with its canonical signaling partners is unknown. The FERM domain protein merlin, can act upstream of warts in the tumor suppressor function. Here, we show that merlin is required for the Rh6 and yR8 subtype specification, but fat and expanded are not involved. In addition, we show that merlin is permissively required for warts function in R8. We also present our genetic analysis of yorkie and a model for how the warts pathway controls rhodopsin expression.

370A Role of the Eyes Absent retinal determination protein in phosphotyrosine signaling networks. Santiago A. Morillo1, Wenjun Xiong2, Andrea Roche3, Ilaria Rebay1,4. 1) Department of Molecular Genetics and Cell Biology; 2) Committee on Cancer Biology; 3) Committee on Developmental Biology; 4) Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL. Eyes absent (eya) is a key component of the conserved Retinal Determination Gene Network (RDGN), which controls eye specification and other developmental events in Drosophila and in vertebrates. The EYA protein is a transcriptional coactivator with an N-terminal transactivation region and the conserved C-terminal Eya domain (ED) that interacts with the DNA-binding protein Sine oculis (So). The ED has also been shown to possess catalytic activity as a protein tyrosine phosphatase, although its substrates are largely unknown. Data from our previous studies showed that both the transactivation and phosphatase activities of Eya are required for normal eye development. However, the relationship between Eya’s function as a transcriptional coactivator and its participation in phosphotyrosine signaling networks remains an intriguing and open question. We are considering two models as a framework to address this question. In one scenario, the primary or exclusive function of EYA’s phosphatase activity would be to modulate its function as a transactivator; thus, both the phosphatase and transactivation functions would participate in common nuclear signaling events. Alternatively, phosphatase and transcription functions could be largely independent, such that Eya would participate as a phosphatase in a separate set of signaling networks from those influenced by its transcriptional functions. To distinguish between these possibilities, we have performed a genetic screen designed to explore the signaling pathways with which Eya interacts. We are currently using a combination of genetic, cell biological and biochemical assays to study the phosphatase activity of EYA and how it relates to its role as a transcriptional coactivator in the RDGN and other signaling networks. 214 POSTERS: Signal Transduction

371B Identification of Ddok-interacting proteins involved in Dorsal Closure. Soumit Roy, Y.G Yeung, E. Richard Stanley. Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, NY. Dorsal closure (DC) during Drosophila embryogenesis involves bilateral dorsal movement of the epidermis leading to dorsal suturing and is widely used as a model system to study concerted epithelial sheet movement during development, morphogenesis and wound healing. Several signaling pathways have been shown to regulate different aspects of this morphogenetic event. Our lab has shown that the non-receptor Syk family tyrosine kinase, Shark, is required for DC, acting upstream of the Jun kinase pathway. Using a yeast-two-hybrid approach, we identified the adaptor protein, Ddok, as a Shark-interacting protein, showing that it is required upstream of Shark for DC. Ddok localizes Shark to the cell periphery and to the actomyosin cable in leading edge cells and is essential for maintenance of F-actin cytoskeleton organization in the epidermis during DC. We have screened for Ddok-interacting proteins using the yeast-two-hybrid system and have identified two proteins that interact with Ddok. One of these, Spaghetti squash (Sqh), is the regulatory light chain of non-muscle myosin II. The heavy chain of non- muscle myosin II, encoded by zipper, is required for DC and non-muscle myosin II is an essential component of the actomyosin cable in the dorsal-most epithelial cells. Since Sqh is required for the functioning of myosin II, its direct interaction with Ddok could explain the essential role of Ddok in the formation and/or maintenance of the actomyosin cable during DC. We have validated the interaction of Sqh with Ddok in S2 cells. Current studies are focused on formally demonstrating a direct interaction between Sqh and Ddok and characterizing the Sqh-Ddok protein complex in S2 cells.

372C Localization of the Toll-7 protein in Development of Drosophila melanogaster. Natalie A Sandoval, Elizabeth Eldon. Biological Sciences, California State University Long Beach, Long Beach, CA. The Toll-like receptors are transmembrane proteins that begin their expression during the embryonic phase in Drosophila melanogaster. Toll-7 is one of nine members of the Toll family of genes found in Drosophila, which are thought to be involved in innate immune responses and development. The genes encoding these proteins have already been identified, but their roles in development are still undefined. One important question is whether these genes play independent roles or have overlapping functions with other Toll family members. The Toll-7 receptor is a close homolog to another Toll-like receptor our lab is interested in called 18w (Toll-2). The sequences of 18w and Toll-7 suggest that these genes are the result of a duplication event. It is not known whether the two genes share overlapping functions. As a first step in addressing this question we are carrying out a detailed analysis of their protein expression domains. In this study, the TIR domain of the Toll-7 open reading frame was cloned into the expression plasmid pTYB11. Expression from the resulting fusion protein was induced into E. coli. The Toll-7 portion of this fusion protein (Tl7TIR) was purified and is currently been injected into guinea pigs to produce antibodies. The antibodies produced will be used for localization of Toll-7 activity in the fruit fly. The Toll-7 antibody will be used in colocalization studies with the 18w antibody using immunofluorescence and confocal microscopy. Our studies should begin to explain the observation that some Toll family receptors have dynamic expression patterns but mutant subtle phenotypes. This project is supported by the NIH-RISE #5R25GM071638.

373A Functional analysis of the proprotein convertase amon: Does amon regulate energy metabolism? Kate R Small, Michael Bender. Department of Genetics, University of Georgia, Athens, GA. Many peptide hormones are synthesized as much larger inactive precursor molecules that must be processed and activated by a class of enzymes known as the proprotein convertases (PCs). The Drosophila gene amontillado (amon) encodes a homolog of the mammalian PC2 protein that has been linked to human disorders in energy metabolism such as diabetes and obesity. To ask whether amon function is required to regulate energy metabolism in flies, we first examined lipid storage in the gut and fat body of amon null mutants. Oil Red O staining of lipids showed that while amon mutants exhibit normal lipid storage in their fat body, amon mutant gut tissues contain significantly less lipid than wild type controls. We also found that amon mutants have a significantly lower number of mouthhook contractions than controls suggesting that the decreased lipid storage seen in amon mutants could be due to decreased energy intake. We next performed several behavioral analyses and found that a majority of amon mutants do not wander towards a food source, but that this defect could be partially rescued by heat shock driven expression of an amon cDNA. These results seem to indicate that amon activity is involved in the ability of an animal to locate and move towards a food source. Our early data point towards a role for amon in energy metabolism by regulating the ability of animals to locate and move towards an energy source, and to take in and store the energy in the gut. POSTERS: Signal Transduction 215

374B Beta-arrestin Kurtz is an endocytic adaptor that functions in early embryonic patterning. Marla Tipping, Alexey Veraksa. Biology, University of Massachusetts Boston, Dorchester, MA. Mammalian beta-arrestins have been characterized as endocytic adaptors functioning in the regulation of GPCR and non-GPCR signaling pathways. The Drosophila genome encodes one non-visual beta-arrestin, Kurtz (Krz). krz mutant germline clones were generated to determine its role in Drosophila development. The embryos derived from the mutant germline clones show severe defects in both the ventral midline and anterior and posterior termini. This phenotypic data has led us to investigate the involvement of Krz in the regulation of signaling through the receptor tyrosine kinase Torso, which specifies terminal patterning, as well as through the Toll receptor that controls ventral midline development. We show that both pathways are regulated by Krz through investigation of downstream pathway components. We are now investigating the molecular mechanisms of Krz involvement in the regulation of embryonic patterning. We show that Krz associates with components of the AP-2 clathrin adaptor complex. This suggests that Krz acts as an endocytic adaptor regulating signaling activity by vesicular transport of pathway components.

375C Identification of substrates and regulators of the Eya phosphatase. Carolyn Wrobel, Justin Cassidy, Ilaria Rebay. Ben May Department for Cancer Research, University of Chicago, Chicago, IL. Eyes Absent (Eya) is a key regulator of proliferation and differentiation during development. Eya functions as a transcriptional coactivator and a tyrosine phosphatase, and perturbation of either activity causes developmental defects in flies and humans. While multiple Eya transcriptional targets have been identified, its phosphatase substrates remain unknown. In addition, the signals that regulate Eya activity are incompletely characterized. The developmental requirement for the Eya phosphatase suggests its substrates serve as critical downstream effectors. We will use optimized in vitro phosphatase assays to investigate Eya’s ability to act on candidate proteins phosphorylated by genetically interacting kinases. In addition, we are developing Eya substrate trap variants modeled on conventional cysteine phosphatase mutants to employ in identifying proteins whose phosphorylation is enhanced by their expression in S2 cells. Eya integrates input from multiple signaling pathways, and exploration of such interplay has shed light on upstream regulators of Eya and may likewise reveal Eya substrates. Genetic data suggest a potential interaction between Eya and JAK/STAT signaling; however, this crosstalk has not been explored. We are examining the effects of Eya and JAK/Stat proteins on each other’s transcriptional functions and determining the effects of modulating Eya and Stat gene dosage on their respective roles in promoting cell proliferation and survival during development. Finally, multiple signals that influence Eya function also alter its subcellular localization. Elevated expression of its transcriptional partner So increases the extent of Eya nuclear accumulation, while nuclear translocation of Eya is blocked by activation of Gαi proteins. To discover additional Eya regulators, we have generated a GFP-tagged Eya protein to employ in a genome-wide RNA interference screen for factors that alter its localization. Collectively, these studies should provide significant insight into Eya function and how its activity is coordinated with a diversity of developmental signals.

376A An in vivo UAS-RNAi based pilot screen for wound healing genes in Drosophila larvae. Yujane Wu, Yan Wang, Michael Galko. Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, Houston, TX. Wound healing is a highly conserved survival response whose function is to restore the structure and function of tissue that encounters physical trauma. We previously showed that epidermal specific expression of a Jun N-terminal kinase (JNK) dominant negative blocks wound closure in Drosophila larvae. However, the wound-induced signals that regulate and are regulated by JNK and its pathway components are still unidentified. To identify novel genes required for epidermal wound closure, we performed a pilot screen of eighty candidate wound healing genes focused largely on components of the canonical JNK pathway (the kinases misshapen, slipper, hemipterous, basket, and the transcription factors djun and dfos) and genes important for actin cytoskeleton dynamics. To rapidly score wound closure in live larvae, we developed a Gal4/UAS-based epidermal wound reporter line in which epidermal membranes and nuclei are fluorescently labeled. Reporter bearing flies were crossed to flies harboring UAS-RNAi inserts (from the Japanese NIG-fly consortium) to conditionally block target gene function in the larval epidermis. We generated wounds on progeny larvae using a pinch-wounding assay that creates an approximately 200-300 μM gap in the epidermal sheet. In normal larvae these wounds heal by 24 hours through cell rearrangement and migration. Using this conditional RNAi-based screening strategy, we identified nine genes that are required for normal epidermal wound closure in Drosophila larvae. This includes five of six canonical JNK pathway components as well as genes involved in actin remodeling (chickadee, Paxillin, G protein γ1). We have verified complete epidermal specific loss of the targeted protein in some cases. Our results suggest that Jun and Fos mediated transcription is likely to play an important role in the wound response. Further, the success of our pilot screen suggests that tissue- specific in vivo RNAi will be an efficient way to systematically identify genes required for epidermal wound closure. 216 POSTERS: Signal Transduction

377B A screen for dominant modifiers of PDZ-GEF/Dizzy, a Rap1-specific guanine nucleotide exchange factor. Zhongchun Zhang, Marc Therrien. Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada. The Ras/MAPK pathway is a key conduit regulating cell proliferation and differentiation. We previously conducted a screen for modifiers of a rough eye phenotype caused by the eye-specific expression of a dominant negative form of Connector eNhancer of KSR (CNK), which is a scaffolding protein of the Ras/MAPK pathway. In addition to isolating mutations in bona fide components of the pathway, the screen identified the small GTPase Rap1 and its upstream activator PDZ-GEF/Dizzy as dominant enhancers, thus suggesting that their activity is required in concert to Ras/MAPK signaling during eye development. It also opened the possibility that CNK regulates Rap1 signaling and may even link Ras and Rap1 signaling. While a functional connection between these two pathways has been suggested by other studies, the underlying molecular events are currently unknown. PDZ-GEF proteins play important roles during Drosophila, C. elegans and mouse development. However, among the numerous Rap1-specific GEFs that have been characterized to date, it is the only one for which no upstream activators have been identified. To investigate how Ras and Rap1 signaling may be connected and possibly elucidate how PDZ-GEF is regulated, we set out a genetic screen for dominant modifiers of a pdz-gef hypomorphic mutant background. Approximately 70,000 progeny derived from EMS-mutagenized parents have been screened so far and ~150 modifiers have been balanced. The pdz-gef mutant phenotype, the screen and a preliminary genetic characterization of the modifiers will be presented.

378C Spatial control of MAPK activation and signaling in the early embryo. Yoosik Kim1,2, Mathieu Coppey1,2, Gerardo Jiménez3, Stanislav Shvartsman1,2. 1) Dept of Chemical Engineering, Princeton University, Princeton, NJ; 2) Lewis-Sigler Institute for Integrative Genomics, Princeton University, NJ; 3) Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain. The terminal patterning of the embryo depends on the localized activation of the Torso receptor, which signals through the MAPK pathway. MAPK signaling leads to phosphorylation and nuclear exclusion of Capicua, a transcriptional repressor of the terminal gap genes tailless and huckebein. Using an image processing routine for quantifying the dynamics of MAPK and Capicua in the same embryo, we demonstrated that Capicua and MAPK are locally anti-correlated throughout the embryo length, as has been suggested by previous genetic and biochemical studies. At the same time, we established that the two gradients are globally correlated: both MAPK and Capicua concentrations are higher at the anterior than at the posterior of the embryo. Using a biophysical model which reproduces experimental data, we propose that this asymmetry can be explained by the presence of Bicoid, the anterior morphogen, which was shown to be a substrate of MAPK. We propose that a Bicoid-dependent substrate competition mechanism limits MAPK availability at the anterior pole of the embryo. We tested this mechanism in genetic and imaging experiments. Our work reveals a biochemical mechanism that couples the AP and terminal systems, well before they are integrated at the enhancers of target genes.

379A PLC-γ regulates differentiation and growth in Drosophila melanogaster. Juan Manuel Murillo-Maldonado1, Justin Thackeray2, Juan Riesgo-Escovar1. 1) Dept Developmental Neurobiol, Inst Neurobiologia, UNAM, Queretaro, Queretaro, Mexico; 2) Deparment of Biology, Clark University, 950 Main Street, Worcester MA 01610, USA. In vertebrates, phospholipase C-γ (PLC-γ) is involved in regulating cellular processes including differentiation, growth and proliferation. In Drosophila melanogaster, a single PLC-γ has been identified, encoded by small wing (sl). Homozygous mutant sl flies have extra- R7 photoreceptor cells in the eye, smaller wings, and ectopic wing veins1. We determined, by in situ hybridization, the spatial and temporal distribution of sl mRNA during Drosophila embryogenesis as well as in imaginal eye and wing discs. sl is expressed at all stages of embryogenesis2, suggesting participation of sl in embryonic development. In addition, sl is expressed in specific regions within the imaginal wing and eye discs. This can explain the subtle mutant phenotypes observed in the respective adult tissues. γ Genetic interaction experiments show that: a) PLC- participates, through the activation of the IP3 receptor (IP3R), and together with the insulin and EGF pathways, in the regulation of cellular growth in the wing; and b) PLC-γ regulates negatively, through the activation of the IP3R and with the insulin pathway, in the activity of EGF signaling regulating cellular differentiation in the wing. C- SH2 and N-SH2 domains are critical for PLC-γ function in differentiation whereas only the N-SH2 domain seems important in the regulation of cellular growth. 1 Thackeray J.R., Gaines P.C., Ebert P. and Carlson J.R. (1998). small wing encodes a phospholipase C-γ that acts as a negative regulator of R7 development in Drosophila. Development125, 5033-5042. 2 Emori Y., Sugaya R., Akimaru H., Higashijima S., Shishido E., Saigo K and Homma Y. Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages. J Biol Chem 269(30), 19474-19479. POSTERS: Signal Transduction 217

380B Genetic Dissection of Signaling from a Single Phospho-Tyrosine of an Oncogenic EGF Receptor in Drosophila. Leena Patel, J. Roger Jacobs. Biology Department, McMaster University, Hamilton, Ontario, Canada. Human Her2 (ErbB2) is overexpressed in 20-30% of breast cancers and is indicative of poor prognosis. In vitro and transgenic studies with a transforming allele of the rat orthologue, neu, are used to map second messengers to specific phospho-Tyrosines (pTyr). Neu receptor activation is coupled to the activation of the Ras/Raf/MAPK signal transduction pathway through the phosphorylation of specific carboxy-terminal tyrosine residues, causing transformation in mouse mammary epithelia. Signaling in a GRB-2-/SHC-dependent manner occurs strongly through the specific tyrosine residues Y1144 and Y1226/7 in rat fibroblasts. Signaling through tyrosine residue 1201 has also been linked to Ras, however, the adaptor proteins responsible for relaying signals are unknown. We are currently employing a genetic approach to dissect the pathways activated by each of the 5 tyrosine residues of Neu when mis-expressed in Drosophila melanogaster. We have previously shown that peptide sequences surrounding each pTyr of Neu are conserved in the Drosophila EGFR (DEGFR), and each Neu pTyr activates signals in Drosophila cells. Here we report the results for the specific tyrosine residue 1201 (Neu-YC). Study of Neu-YC in Drosophila was supported by the structural and functional similarities between Neu and DEGFR. In particular, the Neu-YC SH2-/PTB-binding domain motif (PEYL) is identical to the phi motif (Y1308) in DEGFR. Haplosufficiency modifier studies revealed mild suppression by mutation in Ras and Raf of phenotypes generated by expression of neu-YC in the wing margin and in the developing compound eye. We have also conducted an EMS mutagenesis screen to identify novel modifiers of Neu-YC signaling. We have screened over 15 000 mutagenized chromosomes and have isolated 15 mutations that function in either a canonical Ras-dependent manner or a Ras-independent manner. An analysis of these 2 screens will be presented. Supported by NCIC and NSERC.

381C Regulated secretion of the EGFR ligand Spitz via palmitoylation and proteolysis. Josefa Steinhauer, Jessica Treisman. Developmental Genetics, Skirball Institute, NYU Medical Center, New York, NY. Several signaling ligands have recently been found to carry post-translational lipid modifications. These modifications are thought to influence the secretion of these molecules and their distribution in the extracellular space. Our lab previously showed that the primary EGFR ligand Spitz carries a covalently linked palmitic acid. We propose that this modification functions to tether Spitz to the membrane of producing cells, thereby increasing its local concentration and allowing it to activate high threshold responses in neighboring cells. We have generated a chimeric Spitz molecule that is tethered to the membrane via an exogenous type II transmembrane moiety. This chimera activates the EGFR in vivo despite being unpalmitoylatable, suggesting that the palmitate does not serve another purpose aside from membrane-tethering. We find that both wild-type palmitoylated Spitz and our transmembrane chimera can signal to cells beyond their immediate neighbors. Expression of either construct in S2 cells results in release of the extracellular domain of Spitz into the culture medium. Sequencing of this secreted fragment reveals a strong start signal 16 amino acids downstream of the N-terminus. We postulate that release of soluble Spitz from producing cells requires a proteolytic cleavage within the N-terminus, which may enable long range signaling in vivo. We are testing the effects of protease inhibitors and mutation of the predicted cleavage site on Spitz signaling and secretion. Additionally, since active Spitz is produced from a type I transmembrane precursor that is functionally inactive, we have constructed two type I transmembrane Spitz chimeras of varying length to determine whether Spitz can signal in the type I configuration and whether the distance of the EGF domain from the membrane influences this capability. We hypothesize that palmitoylated Spitz differs from the transmembrane precursor in its orientation with respect to the cell membrane, which presents the EGF domain for receptor binding and exposes the extracellular cleavage site.

382A closca, a new element of the Torso tyrosine kinase receptor pathway. Gemma Ventura1, Rui Gonçalo Martinho2, Ruth Lehmann3, Jordi Casanova1, Marc Furriols1. 1) IRBB-IBMB-CSIC, Barcelona, Spain; 2) Instituto Gulbenkian de Ciencia, Oeiras, Portugal; 3) Skirball Institute, New York University Medical Center, NY, United States. In Drosophila melanogaster, the Torso tyrosine kinase receptor is distributed uniformly all over the surface of the plasmatic membrane of the embryo, but it is only activated at the poles, where it specifies the development of terminal structures. The responsible mechanism to locally activate Torso is the restricted processing of the trunk gene product at each pole of the embryo. In addition, Torso-like protein, which is expressed in a group of follicular cells located at the poles of the oocyte, is the key element to process the Trunk pro-ligand. Other elements are also necessary to activate the receptor, such as Nasrat and Polehole, which have a double function. On the one hand, they activate the pathway, allowing for the accumulation of Torso-like to the vitelline membrane, and on the other, they contribute to vitelline membrane integrity. To gain further knowledge about the mechanisms involved in the localized activation of the Torso receptor we aimed to identify new terminal system elements. Through an EMS female sterile mutation analysis (generated in Ruth Lehman’s laboratory), we identified a new element of the terminal system, which we named closca. Preliminary results suggest that Closca protein is required for Torso receptor activation, as well as the mainteinance of vitelline membrane integrity. Here we present the first results on the functional and molecular characterisation of closca as a new element of the Torso RTK pathway. 218 POSTERS: Signal Transduction

383B A genetic screen for G protein-coupled receptors involved in Rho1 signaling during leg imaginal disc morphogenesis. Nikette Benjamin, Rachel Morgan, Laurence von Kalm. Department of Biology, University of Central Florida, Orlando, FL. The Stubble type II transmembrane serine protease (TTSP) plays an integral role in leg and wing epithelial morphogenesis. Mis- expression of human TTSPs is associated with a variety of pathologies including many forms of cancer. Although clinically important, the mechanism of action of TTSPs is poorly understood. Previous work in our laboratory has shown that the Stubble TTSP acts upstream of the Rho1 (RhoA) signaling pathway in the control of actin cytoskeletal dynamics during imaginal disc morphogenesis. These findings raise the possibility that Stubble utilizes an “outside-in” mechanism in which extracellular proteolytic events affect Rho1 signaling within the cell. Members of the G protein-coupled receptor (GPCR) family are potential extracellular Stubble targets. This class of receptors has been shown to regulate RhoA signaling in vertebrates. In addition, three vertebrate TTSPs, Enteropeptidase, Matriptase/MT-SP1, and TMPRSS2, have been reported to activate GPCRs either directly, or indirectly through a proteolytic cascade. To investigate the possibility that Stubble regulates Rho1 signaling via activation of a GPCR, we utilized a second-site noncomplementation assay for genetic interactions between Stubble and GPCR mutations. Using a combination of specific mutations and deletions, we tested 96 of the 99 GPCRs in the Drosophila genome. Two deletions, collectively uncovering four GPCR loci, interact strongly with mutations in the Stubble locus potentially linking one or more of these GPCRs to Rho1 signaling during imaginal disc morphogenesis.

384C The proteolytic domain of the Stubble type II transmembrane serine protease is essential for proper regulation of apical cell shape change during epithelial morphogenesis. Rachel Morgan, Laurence von Kalm. Department of Biology, University of Central Florida, Orlando, FL. The type II transmembrane serine proteases (TTSPs) comprise a rapidly growing family whose members play critical roles in development and human health. The Stubble locus encodes a typical member of the TTSP family necessary for proper epithelial morphogenesis. Previous work in our lab has shown that the Stubble protease functions upstream of the intracellular Rho1 (RhoA) signaling pathway to regulate apical cell shape change in leg and wing imaginal discs. Stubble acts through an apparent “outside- in” signaling mechanism to control the apical actin-myosin contractile belt. Loss-of-function and gain-of function Stubble alleles are associated with malformed legs and wings in the adult animal. The Stubble protease has a modular structure and is composed of four domains: an N-terminal cytoplasmic domain, a transmembrane domain, and extracellular stem and serine protease domains. Within the stem region, a conserved disulfide-knotted motif specific to arthropods is present. One approach to understanding the mechanism of the interaction between Stubble and Rho signaling is to determine which of the domains in the Stubble protein are required for in vivo activity. We approached this question by sequencing 16 mutant alleles of Stubble, all of which have reduced in vivo function in the leg disc epithelium. Sequence data reveal only two classes of mutations, proteolytic domain and apparent regulatory, among the 16 alleles. Thus, we conclude that the extracellular proteolytic domain is essential for function, consistent with the proposed outside-in signaling role for this protease.

385A Investigating regulation of Armadillo protein stability in Drosophila. Kelly Alexandre, David Roberts, Daniel Schneider, Greg Rogers, Robert Duronio, Mark Peifer. Biology, University of North Carolina, Chapel Hill, NC. Inappropriate activation of the Wingless (Wg)/Wnt signaling pathway occurs in many human cancers. Wg signaling stabilizes the key effector, Armadillo (Arm) which otherwise is a substrate for ubiquitin-mediated proteasomal destruction. A major advance in understanding regulation of Arm stability was the discovery that inactivation of the Drosophila F-box protein Slimb (mammalian βTrCP) activates Wnt signaling and stabilizes Arm. This suggested that Arm is ubiquitinated by an SCF-class E3 ubiquitin ligase. SCF complexes consist of a Skp substrate adaptor, a Cullin scaffold, an F-box protein, and a RING-finger protein of the Roc1/Rbx1 family. Published data supports this model of SCF-Slimb-mediated ubiquitination of Arm, yet two sets of data suggest that Arm degradation is more complex. First, the major fly Roc1, Roc1a, is not essential for Arm degradation in wing imaginal discs, although it does mediate destruction of the Hedgehog effector Ci. This suggests that a different Roc protein may contribute to the E3 ligase, Roc proteins may function redundantly, or E3 ligases containing other RING-finger proteins may target Arm for degradation in some tissues. The RING-finger protein Sina/Siah is one candidate, as Siah mediates p53-dependent Arm/β-cat degradation. Second, published data suggest that loss of Slimb and Axin in larval tissues trigger high level accumulation of Arm, while our data suggest that loss of both APCs has a less dramatic effect on Arm levels, raising the possibility that multiple mechanisms of Arm regulation exist. We are exploring mechanisms of Arm regulation in Drosophila using three strategies. We are performing a small-scale RNAi screen of all Drosophila Skps, Cullins, Rocs, and F-box proteins in S2 cells to define the molecular components of the Arm E3 ligase. Second, we are evaluating potential redundancy and tissue-specific requirements of different RING-finger proteins. Finally, we are analyzing mitotic clones in the wing disc and brain to look at Arm accumulation after mutation of potential Arm E3 ligase components. POSTERS: Signal Transduction 219

386B Identification of novel mediators of the Wg signaling cascade. Tina Buechling, Thomas Horn, Michael Boutros. Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany. The Wnt/Wingless (Wg) signaling cascade, one of the most important evolutionary conserved signaling pathways, plays an imperative role in various developmental events and diseases, such as cancer. Even though the Wg signaling cascade has been studied intensively for decades, several steps in the signaling pathway are not yet understood. RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms and has opened new avenues to conduct systematic genetic screenings for cellular signaling pathways. In such an RNAi screen for Wg signaling components, we have previously identified Evi/Wls, a novel component of canonical Wnt signaling pathways. First-generation RNAi libraries used in many screens had a number of technical limitations, including off-target effects and an incomplete genome coverage. We have designed and generated a new RNAi library, removing off-target sequences and optimizing RNAi efficiencies with an updated genome coverage (R5.2). To dissect the Wg signaling cascade under less artificial conditions than previously reported, we have successfully established an endogenous Wg-responsive luciferase assay system based on the most downstream transcription factor pangolin (pan). In genome-wide screens in multiple cell lines, we identified known and putative regulators that affect Wg signaling in a positive or negative manner. Epistasis experiments positioned them in the pathway with respect to known pathway components. Specifically, we identified a gene that we named opossum (opo) among a set of positive regulators that have been validated in multiple cell lines. Epistatical analysis placed opo upstream of Wg, presumably acting in the secretory pathway. In vivo, opo knockdown causes severe wing margin defects, similar to known wg pathway phenotypes. A detailed functional characterization of opo is currently on-going.

387C The Wnt signaling pathway influences the activity of aPKC in polarity and adhesion. Pamela Colosimo, Nicholas Tolwinski. Developmental Biology Program, Sloan Kettering Inst, New York, NY. Various signaling pathways can affect the polarization of cells. The Wnt-PCP pathway, for example, determines organization within a plane. Several signaling events have been attributed to polarity establishment such as the asymmetric distribution and activation of Dsh and aPKC. This limits their activities to particular compartments of cells, which is essential for the development of polarized cells. Early studies suggested that aPKC can directly phosphorylate GSK-3B, and that this phosphorylation inhibits GSK-3B activity in polarizing cells. More recent work has challenged this finding, leaving no other mechanism that links the activity level of aPKC, GSK-3B, and Dsh. Here we present genetic and biochemical evidence that aPKC is a direct phosphorylation target of GSK-3B, and that GSK-3B phosphorylation of aPKC inhibits its activity. Further, GSK-3B regulation of aPKC plays an important role in the development of polarized epithelia in Drosophila. In the standard Wnt signaling model, Dsh directly inhibits the activity of GSK-3B. This suggests a mechanism where the specific localization of Dsh could lead to compartments with lower GSK-3B activity, resulting in localized activation of aPKC. We also investigated signaling events downstream of aPKC, and find that levels of aPKC activity affect levels of Armadillo (Arm) at adherens junctions. These results indicate that altering aPKC activity through Dsh and GSK-3B may be an important mechanism that cells could utilize to dynamically regulate cell-cell adhesion in many cell-polarity related processes, such as asymmetric cell division, directed cell migration, axon specification and formation of polarized tissues.

388A Regulation of Dishevelled in Fz/planar cell polarity and Wnt/β-Catenin signaling. Andreas Jenny1, Ekatherina Serysheva2, Hebist Berhane1, Michael Boutros3, Marek Mlodzik2. 1) Dept Mol and Dev Biol, Albert Einstein College of Medicine, Bronx, NY; 2) Dept. of Developmental and Regenerative Biology Mount Sinai School of Medicine, New York, NY; 3) Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany. The Wnt-Frizzled(Fz)/β-Catenin and Fz/planar cell polarity (PCP) pathways are related signaling pathways crucial for the induction and regulation of many developmental processes in vertebrates as well as in invertebrates. In addition to the Fz receptor, both pathways rely on the adaptor protein Dishevelled (Dsh) to transduce a signal to downstream effectors. A crucial question is how the specificity of the signal is achieved. Dsh phosphorylation correlates with PCP signaling, as well as canonical Wnt/β-Catenin signaling and is thought to be critical for the regulation of pathway activity. To gain a better understanding of the regulation and specificity of Dsh for PCP versus Wnt/β-Catenin signaling, we performed a systematic RNAi screen in order to identify kinases and phosphatases that affect the Dsh phosphorylation status in a Western blot based mobility shift assay. We have identified several new kinases and a phosphatase (PP2a) that affect Dsh phosphorylation in cell culture. We will present data addressing their function in vivo. The identification and characterization of Dsh kinases and phosphatases is a significant step forward in understanding the regulation of PCP signaling and in particular will shed light on the specificity of Dsh for Wnt/β-Catenin versus PCP signaling. 220 POSTERS: Signal Transduction

389B Negative regulation of Wingless signaling by the microRNA miR-8. Jennifer A. Kennell, Kenneth M. Cadigan. Dept of Molec, Cell & Dev Biol, Univ of Michigan, Ann Arbor, MI. Using a genetic screen to identify regulators of Wingless (Wg) signaling in Drosophila, we identified a microRNA, miR-8, that suppresses Wg signaling in vivo and in cell culture. Misexpression of miR-8 in the developing eye suppresses the reduction in eye size caused by Wg or Arm overexpression. In addition, misexpression of miR-8 in the developing wing inhibits expression of endogenous Wg target genes including senseless, distal-less, dFz3 and naked cuticle. In cell culture and in the developing wing and leg, we found that misexpression of miR-8 inhibits dTCF protein expression without affecting dTCF mRNA, suggesting that miR-8 may inhibit Wg signaling by downregulating this key transcription factor. However, dTCF does not appear to be a direct target of miR-8. This suggests that miR-8 may be downregulating a previously unknown factor required for dTCF stability. To test the biological relevance of miR-8 we generated mutants lacking the gene. Flies lacking miR-8 grow to be adults that are significantly smaller, display decreased abdominal cuticle pigmentation and have abnormally shaped legs. We are currently testing whether any aspect of this phenotype is due to elevated levels of dTCF or misregulation of Wg signaling.

390C The retromer complex is involved in Wingless/Wnt secretion by controlling retrograde transport of Wntless from endosomes to the trans Golgi network. Xinhua Lin1,2, Tatyana Y. Belenkaya1, Yihui Wu1, Xiaofang Tang1,2, Bo Zhou1,2, Longqiu Cheng1, Yagya V. Sharma3, Dong Yan1,2, Erica M. Selva3. 1) Division of developmental Biology, Cincinnati Children’s Hospital medical center, Cincinnati,OH 45229, USA; 2) The Graduate Program in Molecular and Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA; 3) Department of Biological Sciences, University of Delaware, Newark, DE 19716. Secreted Wingless (Wg)/Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wg/Wnt secretion. Recent studies have identified Wntless (Wls)(also known as Evenness Interrupted (Evi) or Sprinter (Srt)as an essential component for Wg/Wnt secretion in Wnt-producing cells. In addtion, retromer complex has been shown to be essential for Wnt signaling in Wnt producing cells. However, currently it is unclear whether retromer controls Wnt signaling by regulating Wnt secretion. To determine the molecular mechanism(s) by which retromer controls Wnt signaling, we generated Drosophila mutant for Vps35, an essential retromer subunit. We have examined a role of Vps35 in Wnt signaling in both Drosophila and mammalian cells. Here, we provide compelling evidence that the retromer activity is required for Wg/Wnt secretion. Importantly, Vps35 co-localizes and forms a complex with Wls in endosomes. Wls becomes unstable in the absence of the retromer activity. Furthermore, we show that Wls levels are regulated by dynamin-mediated endocytosis process. Our new findings have linked Wls and retromer in the same Wg/Wnt secretion pathway. We propose that retromer complex controls Wg/Wnt secretion via retrograde transport of Wntless from endosomes to the trans Golgi network (TGN).

391A Functional Genomic Analysis of the Wnt-Wingless Signaling Pathway. Raluca Pancratov, Emily R. Olson, Binita Changkakoty, Ramanuj DasGupta. New York University School of Medicine - Cancer Institute, Department of Pharmacology, Smilow 1111, New York, NY 10016. Wnts/Wingless(Wg) are a family of conserved signaling molecules that regulate multiple fundamental biological processes such as cell proliferation, differentiation and cell polarity. Mutations in the Wnt genes or in those that encode regulators of the Wnt/wg signaling pathway can cause birth defects, including abnormalities of the central nervous system, axial skeleton, and occasionally other organs. Aberrant Wnt signaling has also been linked to human disease such as breast, colorectal, prostate, and skin cancers. Therefore, defining the molecular regulation of the Wnt signaling pathway is fundamental to the understanding of its physiological role in development and disease. We have adopted a functional genomic approach for the systematic identification and functional characterization of all the involved components that modulate the activity of the Wnt pathway. Recently, we performed a high- throughput genome-wide RNAi screen in Drosophila cells aimed at identifying new regulators of the Wnt/wg pathway. Along with known pathway components, several other potential regulators have been identified that include genes with functions not previously linked to this pathway or genes with no previously assigned function. Candidate regulators of the pathway were further tested in secondary cell-based assays for their ability to regulate Wnt signaling, including RNAi-based epistasis analysis to investigate relationships with well-characterized components of the pathway. Selected genes have been subjected to functional analysis in vivo in Drosophila and in other model organisms as a test for cross-species validation of their function in the Wnt pathway. In this study, we present the follow-up analyses of two specific candidate genes, both of which were identified as candidate negative regulators for the Wnt pathway. These include expression analyses, misexpression and loss-of-function studies of the candidate genes in both the Drosophila and zebrafish model systems. POSTERS: Signal Transduction 221

392B Coop is a co-repressor of Pangolin that negatively regulates the Wingless pathway. Haiyun Song, Sandra Götze, Chloé Spichiger, Konrad Basler. Institute of Molecular Biology, Zurich, Zurich, Switzerland. Pangolin is a transcription factor of Tcf/Lef family that regulates the expression of Wingless target genes in Drosophila. In the absence of Wingless signaling, Pangolin is bound by co-repressors and in this context it functions as a repressor. In the presence of Wingless signaling, the co-activator Armadillo competes with co-repressors to bind Pangolin and converts it into an activator. Here we identified a new interacting partner of Pangolin by mass spectrometry and named it Coop (co-repressor of Pangolin). Coop forms a complex with Pangolin on a DNA oligo containing the Tcf/Lef/Pangolin consensus site. Over-expression of Coop represses Wingless targets, but not Hedgehog, Dpp or Notch targets, in cultured cells and in vivo. Loss of Coop has no obvious effect on Wingless targets, presumably due to gene redundancy.

393C Unexpectedly robust assembly of the Axin destruction complex regulates Wg signaling as revealed by analysis in vivo. Marcel Wehrli, Naz Erdeniz, Wynne Peterson-Nedry, Susan Kremer, Jessica Yu, Shahana Baig-Lewis. Cell & Dev Biol/L215, Oregon Health & Sci Univ, Portland, OR. Secreted proteins in the Wnt family regulate gene expression in in target cells by causing the accumulation of the transcriptional activator β-catenin/Armadillo. In the absence of Wnt, a protein complex assembled around the scaffold protein Axin targets Armadillo for destruction, thereby preventing it from transducing inappropriate signals. Loss of Axin or its binding partners APC and Shaggy/ Zw3 results in aberrant activation of the Wnt signaling response. We have analyzed the effects of mutant forms of Drosophila Axin with large internal deletions when expressed at physiological levels in vivo, either in the presence or absence of wild type Axin. Surprisingly even deletions that completely remove the binding sites for fly APC, Shaggy or Armadillo, though they fail to rescue to viability, these mutant forms of Axin cause only mild developmental defects, indicating largely retained Axin function. Furthermore, two lethal Axin deletion constructs, AxinΔRGS and AxinΔArm, can complement each other and restore viability. Our findings support a model in which the Axin complex is assembled through cooperative tripartite interactions among the binding partners, making the assembly of functional complexes surprisingly robust. 222 POSTERS: Pattern Formation

394A The role of the JAK/STAT pathway in proximo-distal (P-D) patterning in the wing. Aidee Ayala, Erika Bach. Pharmacology Dept., New York Univ School of Medicine, New York, NY. P-D axis formation in dorsal discs, such as the wing disc, differs significantly from that in ventral discs, such as the leg and antenna. We have recently reported the role of the JAK/STAT pathway in P-D patterning in the leg and antenna (Ayala-Camargo et al., Dev Dyn 2007). To extend this study, we are investigating the role of this pathway in P-D axis formation in the wing disc. This disc gives rise to the notum (dorsal thorax), pleura (ventral thorax), wing blade and hinge, which connects the blade to the thorax. We find that the JAK/STAT pathway is active early on in most cells in the wing disc primordium. Later on, its activity is restricted to the proximal domain, and by third instar, it is highly active in the presumptive hinge. Furthermore, loss of JAK/STAT signaling results in loss of hinge structures in the adult. Teashirt (Tsh), a Zn-finger domain transcription factor, is restricted early on from the presumptive wing pouch. Nubbin (Nub), a POU-domain transcription factor, is the earliest expressed marker of wing blade fate. Tsh and Nub have mutually exclusive expression domains. During late second instar, a third domain that lacks Tsh and Nub (called the Gap) forms between Tsh and Nub domains. The Tsh, Gap, Nub domains represent independent barriers to clonal growth (Zirin and Mann, Dev Biol 2007). We observe high JAK/STAT activity specifically within the Gap. These data suggest that there are negative interactions between Tsh, Nub and one or more components of the JAK/STAT pathway. Through clonal analysis, we show that JAK/STAT signaling does not repress either Tsh or Nub. However, we find that both Tsh and Nub autonomously repress JAK/STAT pathway activity. We are currently investigating at which level of the JAK/STAT signal transduction cascade Tsh and Nub act to restrict JAK/ STAT activity to the Gap domain.

395B Ectopic germ plasm assembly in the Drosophila oocyte. Agata N. Becalska, Elizabeth R. Gavis. Dept Molecular Biology, Princeton Univ, Princeton, NJ. Patterning of the anterior-posterior (A-P) axis in Drosophila is governed by the localization of maternally derived determinants to the anterior and posterior poles of the developing oocyte. Factors localized to the posterior comprise the germ plasm, and are ultimately responsible for the formation of the germ line as well as the patterning of the A-P axis. While some basic mechanisms underlying localization of germ plasm components have been elucidated, the process by which these molecules are specifically targeted to the posterior and anchored at the posterior cortex is poorly understood. To further understand this process, we are characterizing two mutations that cause ectopic assembly of the germ plasm. Preliminary evidence suggests that one of our mutations disrupts A-P patterning by affecting microtubule polarity and organization within the oocyte. We are investigating the mechanism by which these disruptions might lead to ectopic pole plasm assembly and whether our second mutation disrupts polarity via the same mechanism.

396C Generating the brinker repressor gradient in the wing imaginal disc. Gerard Campbell, Melissa Moser. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA. The transcriptional repressor Brinker (Brk) is expressed in lateral-to-medial gradients across the anteroposterior axis of the wing imaginal disc where it negatively regulates expression of genes such as spalt (sal) and optomotor-blind (omb) which are consequently expressed only in medial locations; their precise pattern of expression is determined by their sensitivity to repression by Brk so that the sal domain is narrower than that of omb largely because it is repressed by lower levels of Brk than omb. The brk gradient is established by inverse, medial-to-lateral, gradients of the secreted BMP homolog Decapentaplegic (Dpp): the intracellular Smad effectors of Dpp signaling, Mad and Medea, bind together with the repressor protein Schnurri (Shn) to silencer elements at the brk locus and repress activity of a constitutive enhancer. Consequently, the brk gradient could simply be viewed as a precise negative read-out of the Dpp gradient. However, we previously revealed that generation of the brk gradient actually requires Brk to negatively autoregulate. Here we show that this negative autoregulation appears to involve Brk interacting directly with the Mad/Medea/Shn repressor complex. We further reveal that this repressor complex alone cannot establish the endogenous graded profile of brk but that this requires an additional positive cis-regulatory element which, curiously, appears to be activated, not repressed, by Mad/ Medea. Thus, generation of the brk gradient requires simultaneous activation and repression by Mad/Medea, but with the positive and negative inputs acting via different cis-regulatory elements. In conclusion, generating the brk gradient requires at least two positive and two negative inputs: the positive being a constitutive enhancer and Dpp signaling via Mad/Medea, the negative being Dpp signaling via Mad/Medea/Shn and Brk autorepression. Generating a stable expression gradient appears to be much more complex than previously thought; this may reflect the importance of maintaining a stable gradient that is buffered against random fluctuations in any single input. POSTERS: Pattern Formation 223

397A The Hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity. Wu-Min Deng, Jianzhong Yu, John Poulton. Dept Biological Sci, Florida State Univ, Tallahassee, FL. Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the phenotypes. In mutant follicle cells Notch accumulates in late endosomes, suggesting that the Hippo pathway regulates the endocytic trafficking of the Notch receptor in the signal-receiving cells. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.

398B Doctor no and Ken and Barbie are involved in the left-right asymmetrical development of the Drosophila embryonic gut. Reo Maeda1, Shunya Hozumi1, Kiichiro Taniguchi1, Takeshi Sasamura1, Toshiro Aigaki2, Ryutaro Murakami3, Kenji Matsuno1. 1) Dept. Biol. Sci./Tech., Tokyo Univ. of Science, Japan; 2) Dept. Biol. Sci., Tokyo Met. Univ., Japan; 3) Dept. Phy. Biol. Inf., Yamaguchi Univ., Japan. Many bilateral animals show left-right (LR) asymmetry in their internal organs. In vertebrates, mutants that have LR defects have been identified, and the mechanisms of the LR axis formation are well understood. In contrast, it is suggested that different mechanisms are employed for the LR axis formation in invertebrates. To elucidate the mechanisms of LR asymmetric development in invertebrates, we studied the genetic pathway responsible for LR asymmetry of Drosophila embryonic gut. Drosophila has several organs that have stereotyped LR asymmetry, such as embryonic gut, genitalia, brain and testes. Among them, we decided to study the LR asymmetry of the embryonic gut, which is an earliest LR asymmetry during Drosophila development. The Drosophila embryonic gut is subdivided into three parts, the foregut, midgut and hindgut. Each part of the gut has stereotypical LR asymmetry. Here, we attempted to identify the genes affecting handedness of the embryonic gut. Approximately 4100 P-element insertion lines (GS lines) have been screened, and we isolated doctor no (drn) and ken and karbie (ken) mutants, which affected LR asymmetrical morphogenesis of the embryonic foregut and anterior midgut at ~20%. drn mRNA was maternally expressed, and its transcripts was detected ubiquitously during the embryonic development. Drn is orthologous to the mammalian AWP1 (associated with PRK1). ken encodes a protein with C2H2-type zinc finger motifs that are highly conserved in mammals. Embryos homozygous for both drn and ken showed more severe developmental defects than those of each single mutant homozygote, indicating that Drn and Ken function in a common pathway. We will report the roles of Drn and Ken in the left-right asymmetrical development of Drosophila melanogaster.

399C Nonmuscle Myosin II heavy chain, Zipper is required for the left-right asymmetric morphology of the proventriculus and midgut in Drosophila embryo. Takashi Okumura, Hiroo Fujiwara, Kiichiro Taniguchi, Reo Maeda, Syunya Hozumi, Kenji Matsuno. Dept Biol Sci/Tec, Tokyo Univ Science, Chiba, Japan. In both vertebrates and invertebrates, visceral organs often exhibit left-right (LR) asymmetric morphology which is genetically determined. In mouse and fish, the leftward flow of extra-embryonic fluid induced by monocilia in the node (nodal flow) triggers symmetry breaking of the LR axis. In contrast, little is known how LR axis is formed in invertebrates. In addition, it is still elusive how LR asymmetric information controls morphological asymmetries of each visceral organ in both vertebrates and invertabrates. In Drosophila, the gut is composed of three parts, the foregut, midgut, and hindgut. Each part of the embryonic gut shows stereotypic LR asymmetric morphology. We performed an EMS mutant screen to identify genes responsible for their LR asymmetry, and obtained botchan1 and botchan2 mutants whose homozygous embryos showed defects of the LR asymmetric morphology of the proventriculus (PV) and anterior midgut (AMG). In these mutants, non sense mutations was found in zipper (zip) locus, which encodes a nonmuscle Myosin II heavy chain. It is known that Zip regulates cell motility. Similar LR defects were observed in homozygous mutant embryos of spaghetti squash (sqh) and Rho-kinase (rok), encoding a myosin regulatory light chain and myosin kinase, respectively. Both proteins are known to regulate Zip activity. Rescue experiment using tissue-specific GAL4 drivers showed that a function of zip in the visceral muscle, which covers the epithelium of the gut, is crucial for the LR asymmetry of the PV and AMG. In addition, time-lapse analysis showed that LR asymmetric movement of the midgut epithelial cells was disrupt in zipbotchan1 embryos. These results suggest that a zip function in the visceral muscle is required for the LR asymmetric movement of the midgut epithelial cells and in turn generates the normal LR asymmetric morphology of the PV and AMG. 224 POSTERS: Pattern Formation

400A Distribution of the potential morphogen Upd during oogenesis. Travis Sexton, Douglas Harrison. Dept Biol, Univ Kentucky, Lexington, KY. Janus Kinase (JAK) activity controls differentiation of the follicular epithelium during Drosophila oogenesis by establishing a JAK activity gradient with highest levels at the A/P poles. Upd, a ligand for the pathway, is secreted exclusively from the polar cells potentially establishing the JAK activity gradient. Although a JAK activity gradient has been shown, the extracellular distribution of Upd is not known. We propose that Upd acts as a morphogen to directly establish the JAK activity gradient, specifying the fates of the follicular epithelium. This research aims to investigate the extracellular distribution of Upd and what factors may contribute to it’s distribution. Furthermore, upd3, a gene with sequence similarity to upd, is also expressed in the polar cells. We aim to determine what role, if any, Upd3 has in follicular development. To visualize Upd protein we have used an Upd antibody as well as an Upd-GFP construct. Conventional and extracellular staining protocols have revealed Upd protein on the basal and apical sides of the follicular epithelium. While a gradient of Upd within the follicular epithelium is not obvious, the detected distribution is consistent with that of the underlying JAK activity gradient. The distribution of Upd could occur by passive diffusion or by a more active mechanism. It has been reported that some morphogens are dependent on Heparan sulphate proteoglycans (HSPGs) for distribution. HSPGs are divided into 3 classes; glypicans, syndecans, and perlecans. Mosaic analysis using cell fate markers and JAK reporters has shown that the glypican Dally plays a role in follicular patterning but another glypican Dallylike and the perlecan Trol do not. Analysis of the effects of Syndecan and HSPG modifier genes on JAK signaling are being done and results will be presented. Another possible influence to the JAK gradient is the presence of another predicted JAK pathway ligand, Upd3. upd3 flies have small eyes and outstretched wings, however there are no visible defects in ovaries. More detailed analysis of Upd3 function in the ovaries will be presented.

401B Activation and structure-function analysis of Snake, a serine protease playing an important role in Drosophila embryonic patterning. Sufang Tian, Ellen LeMosy. Dept Cell Biol & Anatomy, Medical Col Georgia, Augusta, GA. Signaling through the receptor Toll establishes the dorsoventral axis of the Drosophila embryo after an extracellular serine protease cascade, including the proteases Nudel, Gastrulation Defective (GD), Snake (SNK), and Easter (EA), activates the Toll ligand precursor only on the ventral side of the embryo. Based on our previous studies of Nudel, GD, and EA processing, we hypothesize that SNK is a key protease whose activation or activity is spatially regulated by a ventral cue deposited during oogenesis; the generation of this cue relies on Pipe sulfotransferase. SNK processing has not been characterized in vivo even though snk was the first of these genes to be cloned. Our goals are to investigate whether SNK activation is regulated by the ventral cue by studying SNK localization and processing, and to explore the role of a cysteine-rich “clip domain” in SNK, conserved in the N-terminal pro- domains of many invertebrate proteases, by mutating charged residues to alanine within loops predicted to be on the surface of this domain, and assaying these mutants for their ability to rescue snk-mutant embryos and for their interactions with GD and EA in cultured Drosophila S2 cells. We have generated a polyclonal antibody against the SNK catalytic domain (SNK-CAT) capable of recognizing the SNK zymogen in embryo extracts, but are unable to see an activated form of the endogenous protein. Using flies expressing a myc-tagged SNK, SNK-CAT and commercial myc antibodies identify a processed form present in embryos but not in ovaries, and whose presence relies on the activity of upstream proteases Nudel and GD but not on downstream protease EA or on Pipe. If verified, this suggests that SNK processing of EA is the first ventrally restricted reaction in the cascade. In the clip domain mutagenesis, no single charged-to-alanine change results in a dramatic reduction of SNK activity in vivo, but one double mutant does; results from the S2 cell system suggest the clip domain in SNK is required for a subset of interactions with GD and perhaps also with other cofactors in the pathway.

402C Tramtrack69 controls cell fate, morphogenesis, and Notch signaling during Dorsal Appendage formation. Michael J. Boyle1,2, Celeste A. Berg1,2. 1) Molecular & Cellular Biology Program, University of Washington, Seattle, WA; 2) Department of Genome Sciences, University of Washington, Seattle, WA. The formation of the dorsal appendages (DAs) of the Drosophila melanogaster eggshell provides an opportunity to discover how cells cooperate to form a tissue. Subsets of cells within the initially flat epithelium execute a program of patterning and shape changes in order to become the roof and floor of the final tubular structure. The transcription factor Tramtrack69 (TTK69) regulates multiple aspects of this process. A hypomorphic allele of ttk, tramtracktwin peaks (ttktwk), causes correctly patterned DAs that fail to elongate. This defect was due to a failure to expand the apical surfaces of the roof cells, which had previously constricted as a part of the normal process of DA morphogenesis. In contrast, a null allele of ttk, ttk1e11, affects both patterning and morphogenesis. Cells mutant for ttk1e11 showed elevated levels of E-Cadherin and constricted their apices regardless of their position within the epithelium. This abnormal apical constriction occurred at the same time as the normal constriction of the roof cell apices, but not earlier in oogenesis. ttk1e11 also affected patterning. Roof cells normally up-regulate the transcription factor Broad (BR), which remains expressed at a low level in the rest of the “main body” follicle cells but is eliminated altogether from the floor and centripetal cells. TTK69 was required for proper regulation of BR levels in all of these cell types. ttk1e11 mutant cells expressed elevated BR outside of the roof, but failed to express the normal high levels of BR in the roof cells. Intriguingly, ttk1e11 mutant cells in stages 10 to 14 also up-regulated expression of Notch. These data suggest that TTK69 is required to mediate the response to signals that direct both cell-fate and cell- shape changes in all follicle cells, not just DA-forming cells. We hypothesize that roof cell constriction requires a transient downregulation of TTK69 expression or activity, which must then recover to allow apical expansion. POSTERS: Pattern Formation 225

403A Cell behaviour during mesoderm spreading in the gastrula. Ivan Clark, H-Arno Müller. College of Life Sciences, University of Dundee, Dundee, United Kingdom. The mesodermal germ layer is derived from cells in the ventral portion of the cellular blastoderm, which internalise before spreading over the underlying neurectoderm. Mesoderm spreading requires the fibroblast growth factor receptor encoded by heartless and its ligands FGF8-like1 and 2. The precise roles of these molecules and of downstream components of the FGF signalling pathway are unclear. There is little information regarding the morphology and behaviour of mesoderm cells as they spread, partly due the difficulty of visualising dynamic events and structures in fixed tissue. We have addressed this through imaging living embryos expressing fluorescent proteins. We find that a subset of mesodermal cells extend long, dynamic protrusions deep into the ectoderm. The pattern of protrusions is ordered along both the dorsoventral and anteroposterior axes and evolves during the spreading process. A leading edge is established by polarisation of cells in the most dorsolateral positions, which extend long, thin protrusions towards the ectoderm in the direction of migration. However, similar protrusions are also extended by more ventral cells, connecting the mesoderm to the ventrolateral neurectoderm. These are later replaced by ridge-like protrusions that form at periodic intervals along the anteroposterior axis, extending between ectodermal cells. Such an intimate contact between separated germ layers following gastrulation has not been observed in other systems, implying that mesoderm spreading in Drosophila may be accomplished by distinct mechanisms. Spreading occurs concurrently with germ band extension, so the mesodermal movements can be described as a process of “divergent extension”. We suggest that the dynamic protrusions are required in this context to maintain adhesion between the germ layers as the ectoderm undergoes extensive cell shape changes and remodelling of junctions. Our data are consistent with a model in which two major processes contribute to spreading: chemotactic movement of cells at the leading edge and intercalation of cells ventrally and ventrolaterally.

404B A role for the Small GTPase Rap1 in eye development. Eduardo Gonzalez, Layne Dylla, Stacey Lambeth, Jennifer Curtiss. Biology, NMSU, Las Cruces, NM. Cell adhesion is crucial for the proper development of multicellular organisms: it plays a major role in maintaining epithelial stability, compartmentalization and intercellular communication. Disruptions in cell adhesion can lead to pathological conditions such as metastasis. The epidermal growth factor receptor pathway (EGFR) is known to regulate the cell adhesion molecule E- cadherin in certain developmental processes. The small GTPase Rap1 is also known to regulate cell adhesion and to interact with the EGFR pathway. However, it is unknown how these three factors interact in normal development. Our goal is to look for interactions among these factors. The fly eye is an excellent system to analyze such interactions because the development of this highly organized tissue requires a complex network of intercellular communication to manage all the cell rearrangements and tissue remodeling. Both E-Cadherin and the EGFR pathway are necessary for multiple steps in eye development, such as photoreceptor recruitment and ommatidial rotation. It is also known that E-cad and EGFR signals interact in the context of eye development. To assess the importance of Rap1 in eye development, we used the FLP/FRT system to generate clones of null alleles of Rap1. In addition, we used the Gal4/UAS system to drive expression of the constitutively active RapV12. Both mutations lead to alterations of normal development such as misrecruitment of photoreceptors and accessory cells and ommatidial misrotation. These data suggest that Rap1 is necessary for proper eye development, and is also capable of disrupting normal eye development. These defects resemble those observed in loss- and gain-of-function mutants of Egfr/E-cad, supporting the idea of a possible interaction with Rap1. We are currently performing epitasis experiments that will help us understand better the possible relationships among EGFR, Rap1 and E-cadherin. This project will improve our understanding about how cell adhesion is regulated and will have major repercussions in elucidating the role of these molecules in development and cancer.

405C Function of FGF8-like1 and FGF8-like2 in mesoderm movements during gastrulation. Anna Klingseisen1, Tanja Gryzik2, H.- Arno Müller1. 1) Division of Cell & Developmental Biology, College of Life Sciences, University of Dundee; 2) Institute of Genetics, Heinrich Heine University Düsseldorf. FGF8-like1 and -2 are ligands for the FGF receptor Heartless and are required for the proper movement of cells during mesoderm spreading. After invagination through the ventral furrow mesodermal cells undergo an epithelial to mesenchymal transition and spread out to form a monolayer on the underlying ectoderm. During this process, mesoderm cells exhibit cellular protrusions towards the ectoderm and towards the direction of their migration. In embryos mutant for htl or lacking both ligands, most protrusions are absent and the cells fail to spread. Although, showing a similar gene and protein structure, the expression patterns of the two ligands suggest that their functions are not completely redundant. While their expression is overlapping at early stages, it differs at later stages of embryonic and larval development. The two genes exhibit dynamic and partially non-overlapping expression patterns in embryonic tissues that suggest requirements for these factors in the development of different mesoderm lineages. Analysis of single mutants reveals an essential function for FGF8-like2 in the development of pericardial cell clusters. FGF8-like1 has a maternal component of expression and zygotic mutants are semi-lethal. Neither of the two single mutants affects mesoderm spreading as severely as a chromosomal deletion eliminating both ligands or loss of function htl mutations. This suggests that in mesoderm spreading, the two Htl ligands might act in a partially redundant fashion. Furthermore FGF8-like2 is essential for embryonic development, while FGF8-like1 alone has no essential functions during embryogenesis. We will present our progress on studying the role of FGF8-like genes in cell shape changes during mesoderm spreading using mutant analysis and overexpression studies. 226 POSTERS: Pattern Formation

406A Identification of Eve and Runt target genes required for germband extension. Athea Vichas, Jennifer Zallen. Developmental Biology Program, Sloan-Kettering Institute, New York, NY. Elongation of the body axis during germband extension requires the coordination of individual cell behaviors across a multicellular epithelial sheet. The cell interactions that lead to these large-scale morphogenic movements are controlled by Even-skipped (Eve) and Runt, members of the pair-rule class of patterning genes. Eve and Runt are transcription factors that are expressed in a pattern of seven stripes during early embryogenesis. Previous work has shown that absence of or uniform expression of either gene causes a reduction in germband extension (Irvine and Wieschaus, 1994). Furthermore, these genes are both necessary and sufficient for the establishment of planar polarity during cell intercalation (Zallen and Wieschaus, 2004). We have performed comparative whole- genome microarray analysis to identify downstream transcriptional targets of Eve and Runt required for cell rearrangement during gastrulation. To enrich for gastrulation-specific targets, total RNA was isolated from embryos that were hand-selected at cellularization. This analysis revealed 129 putative target genes that are differentially expressed between wild-type embryos and embryos that overexpress Eve or Runt, as well as 44 putative targets that appear to be regulated by both genes in combination. Of these candidates, at least 41 are expressed in a striped pattern reminiscent of Eve and Runt and 10 appear to be expressed in the cephalic furrow. We are currently validating these candidate target genes through in situ hybridization and functional analysis. The identification and characterization of Eve and Runt target genes required for cell intercalation will provide information about how local differences between cells produce global changes at the tissue level.

407B Search for New Retinal Determination Genes. Jason Anderson, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. The retinal determination cascade in Drosophila consists of ten nuclear factors that includes the Pax6 homologs eyeless (ey) and twin of eyeless (toy), the Pax6(5a) genes eyegone (eyg) and twin of eyegone (toe), the Six family members sine oculis (so) and optix, the protein tyrosine phosphatase eyes absent (eya), a distant relative of the Ski/Sno proto-oncogene dachshund (dac), and two additional transcription factors teashirt (tsh) and homothorax (hth). Removal of these genes within the developing eye leads to a block in retinal development, while expression of all of these genes (with the exception of hth) is sufficient to induce eye formation in several non-retinal tissues. Despite the fact that additional “eye specification” genes have not been identified in recent years there is evidence to suggest that the current cadre of genes are not the only ones that regulate the earliest events in eye formation. In an effort to identify new eye specification genes we have screened a collection of mutants that drastically reduce the size of the eye field. As a first step towards determining if any of these mutants represent loss-of-function mutations in new eye specification genes we have analyzed the effects that these mutations have on eye specification, furrow initiation and photoreceptor development in mutant retinal mosaic clones. We will present our findings and discuss the impact that these mutants will have on the structure of the current retinal determination network.

408C Functionalization of Duplicate Genes - A Study of Teashirt and Tiptop During Eye Development. Rhea Datta, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. During normal eye development in Drosophila, a number of gene pairs function to specify the retina. These pairs arose through a series of duplication events and include the Pax6 homologs eyeless (ey) and twin of eyeless (toy), the Pax6(5a) genes eyegone (eyg) and twin of eyegone (toe) and the Six family members sine oculis (so) and optix. The members of each pair are similar in structure to each other. This feature is not shared by a fourth pair of genes, teashirt (tsh) and tip top (tio). The main structural difference between these two proteins concerns the number of zinc finger nucleic acid recognition motifs: Tsh has three while Tio has four such DNA binding domains. We were interested in determining if there are differences between the ability of each gene to promote eye development and if any such differences can be attributed to the zinc finger domains. Tsh is already known to be involved in retinal determination. Here we show that Tio also has a role during eye specification. Using the UAS/GAL4 system we expressed each gene within 220 different developmental patterns and can demonstrate that tio can promote eye development in several more patterns than tsh although the formation of ectopic eyes is limited to the antenna in both cases. We were also interested in determining if the structural differences between these transcription factors influences the regulatory networks that surround each gene. We have conducted a series of genetic modifier screens and yeast two-hybrid assays to determine if there are differences between the downstream transcriptional networks and binding partners of Tsh and Tio. We will present and discuss the results of our forced expression assays, genetic and biochemical screens as well as quantitative genetic data. POSTERS: Pattern Formation 227

409A Eye Development and the Six Family of Homeobox Transcription Factors. Abigail Henderson, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. The Six family of homeobox transcription factors play crucial roles in the development of insects and vertebrates. These proteins are characterized by the presence of the homeodomain (HD), which is used for nucleic acid recognition and the SIX protein-protein interaction domain (SD). In Drosophila this protein family is represented by the sine oculis (so), optix and DSix4 genes, which arose through a series of ancient duplication events that predate the emergence of the Drosophilids. A further duplication event appears to have occurred after vertebrates split away from insects thus mammals including humans have six family members (Six1-6). A number of family members are expressed and function in the insect and vertebrate retinas. In flies, the so and optix genes are required for promoting eye specification while DSix4 is neither expressed nor functions in the retina. In contrast, the DSix4 homologs (Six4, Six5) as well as the optix cousins (Six3, Six6) function in the retina while the so orthologs (Six1, Six2) do not appear to play a significant role in eye development. We are interested in understanding the molecular mechanisms that govern the function of the Six genes in Drosophila. To this end we have identified a number of potential targets of the fly Six family members through a series of genetic modifier screens. Through a set of yeast two-hybrid screens we have also identified a number of binding partners for each Six protein. Our results indicate that the regulatory networks surrounding each gene are quite different from each other with very little overlap seen amongst the downstream targets or binding partners. These unique sub-regulatory networks are predicted to form the biological basis for the functional differences between members of the Six family. We will present the putative regulatory network for each gene and will describe the role that members of each network play in the developing eye. We will also present and discuss the mechanistic relationships that selected genes have with the Six family of transcription factors.

410B A mosaic screen for X-linked mutations affecting photoreceptor differentiation identifies potentially novel components of the Wingless and EGFR pathways. Kevin Legent, Josefa Steinhauer, Magali Richard, Jessica Treisman. Skirball Institute, New York University Medical Center, New York, NY. Drosophila eye development involves signal transduction cascades that are very well conserved through evolution and often misregulated in human cancers. Photoreceptor differentiation initiates at the posterior margin of the eye disc in the third larval instar and progresses anteriorly across the disc. Initiation and progression of this wave require signaling by Hedgehog (HH) and Decapentaplegic. HH specifies the first photoreceptor (R8) within each cluster, and Epidermal growth factor receptor (EGFR) signaling recruits the remaining seven photoreceptors. The Notch pathway is required to prevent excessive R8 differentiation, and Wingless (WG) inhibits photoreceptor differentiation in the regions of the eye disc that give rise to head tissue. We have conducted a mosaic genetic screen using FLP recombinase driven specifically in the eye disc by the eyeless enhancer to identify EMS-induced mutations on the X chromosome that disrupt the normal pattern of photoreceptor differentiation. We screened 43000 flies and established 139 stocks with mutations that prevent homozygous clones of cells from differentiating as retinal tissue, but allow the cells to survive long enough to disrupt eye patterning. We have identified new alleles of the expected genes: raf, Dsor, and corkscrew, which encode components of the EGFR pathway; shaggy, a negative regulator of the WG pathway, and lozenge, which encodes a transcription factor. We have also isolated mutations with phenotypes very similar to raf and shaggy. Complementation results and on-going mapping suggest that the genes affected are very likely to encode novel regulators of the respective pathways. Our progress in cloning, phenotypic analysis and epistasis studies will be presented. The strong conservation between the human and Drosophila genomes makes it likely that these novel molecules will contribute to a better understanding of WG and EGFR signaling in human development and disease.

411C A genetic screen to identify autosomal genes that interact with the Drosophila pro-neural gene atonal. Daniel R. Marenda1, Arpit Shah1, Ginnene Middleton1, Andrew J. Gangemi1, Evan Cooke1, Alysia D. Vrailas-Mortimer2, David Melicharek1. 1) Dept of Biological Sciences, University of the Sciences in Philadelphia, Philadelphia, PA; 2) Dept of Cell Biology, Emory University School of Medicine, Atlanta, GA. We have performed a chemical (EMS) mutagenesis screen for autosomal dominant enhancers of a loss-of-function atonal rough eye phenotype in Drosophila. Atonal, a pro-neural protein in Drosophila is required for the proper formation of the founding photoreceptor cell in the developing retina (the R8 photoreceptor). Proper expression and refinement of Atonal protein is essential for the proper formation and function of the adult eye. We report here the identification of four genes that have not been previously associated with Atonal expression and/or function in the Drosophila retina. We functionally characterize two of these genes (kismet and roughened eye) with respect to eye development and Atonal expression/function, and discuss the relevance of these genes in retinal development. 228 POSTERS: Pattern Formation

412A Epitaxial patterning in a discrete reaction-diffusion system representing signaling in the Drosophila eye. Matthew W Pennington1, David K Lubensky2. 1) Biophysics, University of Michigan, Ann Arbor, MI; 2) Physics, University of Michigan, Ann Arbor, MI. R8 photoreceptors are specified in a precise hexagonal pattern behind the morphogenetic furrow as it traverses the eye imaginal disc during the 3rd larval instar. A large body of evidence suggests that the progress of the morphogenetic furrow is driven by secreted factors from the differentiating neural cells behind it, and that the spacing of individual R8 cells is established by inhibitory signals from the previously specified column of R8 cells.1 Among the genes implicated in regulating the proneural R8 marker atonal are members of the Hedgehog and Notch pathways, acting, respectively, as the (long range) activator and (short range) inhibitor.2 We have developed a mathematical model consisting of coupled differential equations incorporating auto-activation, long-range activation, and short-range inhibition interacting on a lattice in an attempt to better understand this patterning event. We have shown that this model can reproduce patterns similar to those seen both in wild-type eye discs and in several mutant phenotypes and that it makes several unexpected predictions on the effects of multiple interacting mutations. We argue that much of the model’s behavior is a consequence of the fact that atonal self-activation is cell-autonomous; this behavior represents a novel mode of pattern formation distinct from classical ideas such as Turing patterns or morphogen-dependent positional information. 1. Frankfort, B.J., and Mardon, G. Development 129, 1295-1306 (2002) 2. Hsiung, F., and Moses, K. Human Molecular Genetics 11, 1207-1214 (2002).

413B Ventral eye margin is defined by opposing interactions between homothorax and Notch pathway genes Lobe and Serrate. Amit Singh1,2, Meghana Tare1, Wonseok Son3, Krishanthan Vigneswaran3, Kwang Wook Choi3,4,5. 1) Dept of Biology, Univ Dayton, Dayton, OH; 2) Tissue Regeneration and Engineering Center at Dayton(TREND), University of Dayton, Dayton, OH; 3) Dept Molecular & Cell Biol, Baylor Col Medicine, Houston, TX; 4) Dept of Ophthalmology, Baylor Col Medicine, Houston,; 5) Developmental Biology Programme, Baylor Col Medicine, Houston, TX. During axial patterning, generation of Dorsal (D) versus Ventral (V) axis (DV patterning) is the first lineage restriction event in Drosophila eye. Drosophila eye primordium initiates with a ventral ground state on which the dorsal eye fate is established. The members of Notch Signaling pathway, Lobe (L) and Serrate (Ser) play an important role in ventral eye growth and development. Mutations in their vertebrate homologs are also responsible for developmental defects in eye. One of the interesting questions is what defines the boundary of the ventral half of the developing Drosophila eye. We identified homothorax (hth), a Meis class of gene, as a strong enhancer of the L mutant phenotype. Loss-of-function of hth, a negative regulator of eye development, results in ectopic eye enlargements only in the ventral eye margin. This phenotype is complementary to L or Ser mutant phenotype of the loss-of- ventral-eye. Ectopic induction of Hth was seen in loss-of-function (LOF) clones of L or Ser in the ventral eye. Hth form a heterodimer complex with Exd, which is required for their translocation to the nucleus. Therefore, we also studied the developmental interactions of L with exd. Ectopic nuclear localization of Exd was observed in LOF clones of L in the ventral eye. However, overexpression of Exd did not show enhancement of L mutant phenotype. Our results suggest that (a) hth and L/Ser act antagonistically to each other during ventral eye growth and development (b) fine tuning in levels of L/Ser and hth is crucial to define the ventral margin of the eye.

414C optomotor-blind inhibits cell proliferation, morphogenetic furrow initiation, and retinal differentiation during Drosophila eye development. Yu-Chen Tsai1, Stefan Grimm2, Ju-Lan Chao3, Jih-Guang Yao3, Kerstin Hofmeyer2, Jie Shen4, Y. Henry Sun3, Gert O. Pflugfelder2,4. 1) Dept Life Sci, Tunghai Univ, Taichung, Taiwan; 2) Theodor-Boveri-Institut, Biozentrum, Lehrstuhl für Genetik und Neurobiologie, Universität Würzburg, Am Hubland, Würzburg, Germany; 3) Inst. of Mol. Biol., Academia Sinica, Taipei, Taiwan; 4) 3Institut für Genetik, Universität Mainz, Mainz, Germany. During organogenesis, the positive regulators and negative regulators coordiante each other to regulate organ size and organ territory. optomotor-blind (omb) encodes a T-box transcription factor in Drosophila. omb is expressed in the lateral margins in the third instar eye imaginal disc. omb loss-of-function mutants have enlarged eyes. Ectopic expression of omb inhibits cell proliferation, morphogenetic furrow initiation, and retinal differentiation. These suggest that omb is a negetive regulator in Drosophila eye development. Eyegone (Eyg) is a Pax-like transcription factor. eyg mutant has reduced eye or no head phenotype. Omb is ubiquitously expressed in the eye disc in eyg mutants. During early eye development, expression domains of eyg and omb are not overlapped. Our results suggest eyg may play important roles to repress the negative factor omb to determinate eye territory. POSTERS: Pattern Formation 229

415A Deconstructing Pax6 in the Retina. Bonnie Weasner, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. Pax6 plays a central role in the specification of the eye in all seeing organisms. Mutations within the fly Pax6 gene, eyeless (ey) leads to flies that completely lack retinal tissue while human patients with mutations in the Pax6 gene suffer from Aniridia. Amazingly, expression of Pax6/Eyeless in non-retinal tissues is sufficient to redirect the fates of these tissues towards that of an eye. Unlike vertebrates, flies have two Pax6 genes - eyeless (ey) and twin of eyeless (toy). These genes do not appear to function redundantly during normal eye development as the eye is lost in ey loss of function mutants. Additionally, ey can induce ectopic eyes in a wider range of tissues than toy. In order to better understand the biochemical differences between the two Pax6 proteins we generated a series of deletion and chimeric proteins and forcibly expressed them in non-retinal tissues to assay their ability to induce ectopic eyes. Through this molecular dissection we are able to identify individual domains that are either required or dispensable for Pax6 function. We are able to also determine the degree to which individual domains are functionally conserved or have diverged in their function. Pax6 contains two nucleic acid recognition motifs: a Paired domain (PD and a Homeodomain (HD). Flanking these regions are stretches of nucleic acids that appear to lack any discernable motifs and are generically referred to as the N and C terminal domains. A third stretch of amino acids called the B domain lies between the two DNA binding domains. Through our dissection of the fly Pax6 homologs we have identified the minimal protein that is required to induce ectopic eyes. This molecule consists of just the PD and CT regions. A transcriptional activation assay indicates that the CT regions of Pax6 contain strong activation activity. Thus the minimal Pax6 protein (for eye development) consists of the Paired DNA binding domain and the CT activation domain. Our chimeric analysis has also identified a novel Pax6 protein that is sufficient to generate eyes within the genitals, a feat that cannot be matched by the endogenous full-length Pax6 proteins.

416B Conservation of Retinal Function Across Evolution - The Six Family of Transcription Factors. Brandon Weasner, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. In both flies and vertebrates members of the Six family of transcription factors play important roles in retinal development. In flies, the sine oculis and optix genes function within the eye specification network to promote eye formation. A third member of this protein family, DSix4, does not appear to be expressed or function within the retina. The situation in vertebrates is somewhat more complicated. While the optix homologs (Six3, Six6) continue to play critical roles in retinal development the so homologs (Six1, Six2) do not appear function in the eye. Interestingly, while DSix4 is not expressed nor functions in the fly retina, its mammalian homologs (Six4, Six5) play important roles in the vertebrate eye. The implication is that all Six genes are capable of promoting eye development but are restricted by their individual expression patterns. These results also suggest that the regulatory networks (downstream targets and binding partners) are similar. We set out to test this hypothesis by forcibly expressing each of the mammalian Six genes (Six1- 6) within the retinas of sine oculis loss-of-function mutants. We will demonstrate that the Six1, Six2, Six4 and Six5 are capable of rescuing so[1] mutants while Six3 and Six6 fail to support eye development. The failure of Six3/6 to rescue so[1] mutants is consistent with the failure of optix to rescue the same mutant. However, the rescue of so[1] by the Six1/2 pair is surprising as neither gene functions in vertebrate eye development. We were also intrigued by the rescue of so[1] by Six4/5 and DSix4 itself as DSix4 is not expressed in the fly retina. We also attempted to induce ectopic eyes in Drosophila with each of the mammalian Six genes and demonstrate that only Six5 and Six6 are capable of promoting eye formation in non-retinal tissues. We will present and discuss these data as well as a molecular dissection of the SO and OPTIX proteins.

417C Protein-Protein Interactions Between Homeodomains. Yoshitsugu Adachi1, Frederic Prince1, Serge Plaza2, Walter Gehring1. 1) Dept Cell Biol, Biozentrum, Basel, Switzerland; 2) Centre de Biologie du Développement, Université Paul Sabatier, Toulouse, France. The homeodomain (HD) was first identified as a DNA binding domain of Hox proteins and subsequently shown also to be capable of RNA binding and protein transduction (penetration). In more recent experiments we have demonstrated that competitive interactions between the Antennapedia (ANTP) and Eyeless (EY) proteins lead to the suppression eye development in Drosophila. HD-HD and HD-Paired Domain (PD) interactions were demonstrated by GST pulldown assays in vitro and supported by the isolation of mutations in the first alpha-Helix which impair protein-protein interactions, but retain normal DNA binding activity. Using Bimolecular Fluorescence Complementation (BiFC) we now can demonstrate these interactions in vivo in imaginal discs. Furthermore, we show HD-HD interactions between two different Hox genes. We have found that the HD of AbdB interacts with Antp HD in cell culture and also in imaginal discs. This might explain the basic mechanisms of posterior prevalence or “phenotypic supression”. 230 POSTERS: Pattern Formation

418A Permanent Ultrabithorax repression triggered by high Ubx protein levels. Daniel L. Garaulet, Ernesto Sánchez-Herrero. Centro de Biología Molecular Severo Ochoa (CBMSO) CSIC-UAM, Madrid, Spain. The Hox genes are responsible for segmental identity along the anterior-posterior axis in metazoans. Therefore, Hox regulation is a crucial subject deeply studied in developmental biology. Previous studies have shown that some of these genes control their own expression by autoregulatory loops. The Hox gene Ultrabithorax (Ubx) for example, negatively regulates its own expression to maintain appropriate protein levels. We have found, using the Gal4/UAS system, that in some combinations the overexpression of Ubx transforms the adult third thoracic segment into the second one, paradoxically the same transformation obtained in flies lacking Ubx. Visualization of haltere an third leg imaginal discs in these combinations shows absence of Ubx protein, and this is due to two effects: a) transitory expression of the exogenous Ubx and, b) permanent repression of the endogenous Ubx gene. Once triggered, this mechanism of Ubx inhibition becomes stable and permanent, even in the absence of the homeotic product. The involvement of Polycomb and trithorax group proteins in this mechanism will be discussed. Similar effects are observed in other homeobox-containing genes like engrailed.

419B Analysis of Drosophila Hox complex miRNAs and Hox gene expression. Derek Lemons, William McGinnis. Biology, UCSD, San Diego, CA. Hox genes are important regulators of gene expression throughout embryonic development and are themselves highly regulated. The newest layer of regulation of Hox genes is the control of protein expression by miRNAs. Very few miRNA-target gene pairs have been analyzed at a fine scale. We have explored in depth the expression of the miR-10 gene and the two miRNAs that are produced from its hairpin sequence. miR-10, produced from the 5’ arm of the hairpin, has conserved putative target sequences in the 3’ UTRs of arthropod Scr mRNAs and is expressed in a pattern complementary to that of Scr. Additionally there is evidence for repression of cytoplasmic accumulation of Scr transcripts in the ventral T1 segment. miR-10*, produced from the 3’ arm of the hairpin, has conserved putative target sequences in the 3’UTRs of Abd-B mRNAs and also exhibits complementary expression with its target.

420C Investigating the acquisition of segmentation function of fushi tarazu during arthropod evolution. Alison M Heffer1,2, Leslie Pick1,2. 1) Molecular and Cell Biology; 2) Dept. Entomology University of Maryland, College Park, MD 20742 USA. Hox genes are evolutionary conserved transcription factors, best known for their roles in determining segment identity in insects. One member of this group, fushi tarazu (ftz), is a rapidly evolving Hox gene that has changed from an ancestral homeotic gene to a pair-rule segmentation gene in Drosophila. At some point(s) during evolution, ftz has undergone three specific changes, thought to contribute to its new segmentation function in Drosophila (Lohr et. al, 2001, 2005). One: the pattern of ftz gene expression during embryogenesis changed from a Hox-like single domain to a striped pattern. The other two changes were in protein sequence: first, acquisition of an LXXLL motif, and second, loss of the YPWM motif. The addition of LXXLL gave Ftz the ability to interact with a new cofactor, the orphan nuclear receptor Ftz-F1. In Drosophila, this interaction is necessary for selection of target genes involved in segmentation. In addition, Drosophila Ftz lost the YPWM motif present in other Hox genes, which mediates interaction with Exd. This switched the protein partner/cofactor that Ftz interacts with and thus switched the DNA binding target that Ftz regulates. We aim to map these three changes through phylogeny to determine when they occurred, with the long term goal of elucidating their effects on the animal body plan. Though the homeobox region of ftz has been isolated from many species, upstream regions are unknown. Using 5’ RACE on both genomic DNA and RNA in combination with degenerate PCR, the 5’ end of ftz from several arthropods has been determined. This will be continued to determine where during evolution Ftz protein sequence switched. Studies will compare how these changes in protein sequence correlate to a change from homeotic expression patterns to a segmentation/striped pattern in Drosophila. Expression of modified ftz genes in Drosophila, as well as assays in non-model insects, will be used to assess the function of divergent ftz genes in the future. POSTERS: Pattern Formation 231

421A Regulation of spider segmentation by Wnt8. Alistair P. McGregor1, Matthias Pechmann1,2, Natália M. Feitosa1, Wim G. M. Damen1. 1) Institute for Genetics, University of Cologne, Cologne, Germany; 2) Georg-August-Universität Göttingen, Johann-Friedrich- Blumenbach-Institut für Zoologie und Anthropologie GZMB, Abteilung für Entwicklungsbiologie, Göttingen, Germany. While the Drosophila segmentation gene cascade is a paradigm for understanding pattern formation, it is still unclear if it is related to vertebrate somitogenesis. In vertebrates, a clock-like mechanism, regulated by Wnt signaling and Notch/Delta, generates new somites via waves of expression of target genes such as hairy. Remarkably it has been found that components of this system, including Notch, Delta and hairy, regulate segmentation in spiders. This mechanism of regulation of segmentation may have been lost in Drosophila. Therefore comparisons between spiders and vertebrates can be used to address important questions concerning the evolution of the regulation of animal development. To determine if Wnt signaling is also involved in spider segmentation, we sought to identify and characterize the Wnt genes from the common house spider Achaearanea tepidariorum and the wandering spider Cupiennius salei. These spiders contain members of at least nine of the Wnt gene subfamilies. Taking the Wnt genes known from crustaceans, insects and myriapods into account, this suggests that the common ancestor of arthropods contained members of all 12 recognized Wnt subfamilies, perhaps with the exception of Wnt3. Six of the spider Wnt genes, wg, Wnt4, Wnt5, Wnt7-1, Wnt8 and Wnt11, are expressed in the posterior growth zone of the spider, from which the 12 opisthosomal (abdominal) segments are sequentially added. In zebrafish, Wnt8 is required for somitogenesis, and therefore, we tested the function of the spider orthologue of this gene using parental RNA interference in Achaearanea. We found that knockdown of Wnt8 in this spider disrupted normal Delta expression and blocked segmentation, resulting in the complete absence of the opisthosoma in the most severe cases. This demonstrates that both vertebrate somitogenesis and spider segmentation are regulated by the Notch-Delta and Wnt signaling pathways, and emphasizes the genetic similarity of these two processes.

422B Apical localization of RhoGEF2 and adherens junctions during gastrulation. Verena Koelsch1,2, Maria Leptin1. 1) Institute for Genetics, University of Cologne, Cologne, Germany; 2) present adress: University of California, San Diego, Section of Cell and Developmental Biology, La Jolla, California. Gastrulation in Drosophila is driven by apical constriction of the mesodermal cells, leading to the formation of the ventral furrow and the invagination of the mesoderm. Apical constriction requires a contractile network and involves the activation of myosin II via a G protein activated pathway involving RhoGEF2, Rho1, and Rok. Whereas RhoGEF2 is absolutely essential for furrow formation, the loss of the G protein alpha subunit Concertina (cta) or the ligand folded gastrulation (fog) only results in inefficient gastrulation. Twist is the zygotic transcriptional activator essential for cell shape changes during ventral furrow formation. However, the known targets of Twist are not sufficient to explain apical constriction and ventral furrow formation. We show that the Twist target T48 acts in parallel with G protein signaling to orchestrate cell shape changes during mesoderm invagination. The apically targeted transmembrane protein T48 acts as an anchor for RhoGEF2 via a PDZ binding motif and thereby ensures the apical enrichment of RhoGEF2 during mesoderm invagination allowing rapid and efficient ventral furrow formation. Apical constriction also requires the tethering of the contractile network to the adherens junctions. During gastrulation, the adherens junctions in the mesodermal cells are moved from a subapical localization to the apical end of the lateral membranes. We demonstrate that this movement is not simply mediated by a tensile force from constricting actin cytoskeleton, but an independent step of at least partial disassembly must occur which is controlled by Snail.

423C The K50 homeodomain transcription factor Orthodenticle differentially regulates nervous system patterning through separable functional domains. Elizabeth McDonald1, Michael Workman1, Joachim Reischl2, Kerstin Meier2, Ernst Wimmer2, Tiffany Cook1. 1) Developmental Biology/Pediatric Ophthalmology, CCHMC, Cincinnati, OH; 2) Department of Developmental Biology, Georg-August-University Goettingen, Germany. From cnidarians to humans, Otd/Otx-related homeodomain transcription factors are required for anterior head patterning as well as central and peripheral nervous system development. In Drosophila, a single homolog, Orthodenticle (Otd), is required during embryogenesis for correct head segmentation and ventral nerve cord development, during metamorphosis for morphogenesis in all photoreceptor cells, and during terminal differentiation for photoreceptor subtype specification. This latter function occurs through cell-specific activation and repression of known target genes; however, the majority of Otd target genes remain unknown. Our main objective in this study is to understand how the same transcription factor can perform such diverse roles during head and neural patterning at different stages of development. In vivo and in vitro structure-function analysis has led us to identify two independent activation domains in Otd that are equally required for embryonic development but are separable for certain aspects of photoreceptor terminal differentiation. In addition, another domain is necessary for photoreceptor morphogenesis and transcriptional repression during eye development. These results reveal spatial and temporal specific mechanisms for Otd-dependent regulation of nervous system patterning. Due to the requirement for multiple Otd-related factors in regulating the development of a wide range of nervous system, future studies aimed at defining these transcriptional regulatory mechanisms in Drosophila are likely lead to an increased understanding of how nervous system diversity is achieved and maintained in other developmental systems. Support by Research Preventing Blindness, E. Matilda Ziegler Foundation for the Blind, Ohio Preventing Blindness, and NIH T23 HD046387. 232 POSTERS: Pattern Formation

424A Larval tracheoblasts as a model for cell migration. Chrysoula Pitsouli1, Norbert Perrimon1,2. 1) Genetics Department, Harvard Medical School, Boston, MA; 2) Howard Hughes Medical Institute. The Drosophila trachea constitutes an excellent system for the study of tube morphogenesis and epithelial polarity. Tracheal cells are specified in the embryo and the limited number of them migrate, elongate, fuse and form an extended network of ~10,000 interconnected branches that will allow the oxygenation of the animal. During larval stages a set of progenitor cells (larval tracheoblasts), which are clustered in various nests of the animal body, proliferate and migrate. After pupation the newly born tracheoblasts fuse and form the tracheal system of the pupa and subsequently of the adult. Although the pathways that control embryonic tracheal development are studied very extensively, little is known about the larval to pupal and adult transition. We have performed an overexpression and RNAi screen in Drosophila larvae for genes that affect tracheoblast cell number and morphogenesis. We found that the Notch and RTK pathways play important roles in regulating proliferation, differentiation and migration of these cells.

425B Regulatory interaction between Eyeless and Cut in the separation of eye-antennal identity. Cheng-wei Wang1,2, Y. Henry Sun1,2. 1) Dept Academia Sinica, Inst Molecular Biology, Taipei, Taiwan; 2) Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China. In drosophila melanogaster, the eye and antennae originate from the eye-antenna primordium of 20-30 cells, located bilaterally in the embryonic anterior end. Once formed, this cell cluster grows continuously and organizes into the eye-antennal disc during the larval instars. During late larval and pupal development, the anterior lobe of this disc gives rise to the antenna, while the posterior lobe gives rise to the eye. The eye or antennal identity of these cells is not determined until mid or late second larval instar with the restricted expression of markers such as eyeless in the eye field and cut in the antennal field. The mechanisms responsible for subdividing this epithelium into distinct eye and antennal fields are poorly understood. We tested whether Ey and Cut could antagonize each other for the restriction of cell identity respectively. We found that ectopic Cut-expressing clones can autonomously inhibit Ey. On the other hand, ectopically expressed Ey induced, instead of repressing, Cut in second instar eye disc. Since Ey was expessed in all cells of embryonic eye primordia and first instar larval eye disc until Cut was expressed, our finding raises the question whether the eye selector gene eyeless is required for inducing the antennal marker cut in fist larval stage and then becomes repressed by Cut. This reciprocal interaction will determine the eye-antennal identity.

426C Molecular fluctuations and interpreting spatial gradients, applied to Hunchback pattern formation. David M Holloway1, Alexander V Spirov2, Francisco JP Lopes2. 1) Mathematics, British Columbia Institute of Technology, Burnaby; Biology, Univ. Victoria, BC, Canada; 2) Applied Mathematics, and Developmental Genetics, Stony Brook University, NY, USA. Positional information in early anteroposterior segmentation is specified by spatial gradients of transcriptional regulators, typically at hundreds to thousands of copies per nucleus, which may be expected to display significant fluctuations. What are the effects of fluctuations on target gene expression, and what may lessen such effects? To address this, we focus on hunchback (hb) expression in response to the maternal Bicoid (Bcd) gradient. hb is activated at Bcd concentrations where roughly 4% fluctuations can be expected. More important, though, may be the random nature of binding to the hb promoter, with potential ‘bursting’ in production, as seen in yeast. From a previous deterministic model, we made a stochastic model of Hb production, with transcription, translation, decay and diffusion. Bursting, in the form of spiky activation, occurs if the production timescale is fast and closely mirrors the noisy binding states of the promoter. Slower protein reactions buffer the output from promoter noise. Initial zygotic activation can be noisy; our simulations indicate that maternal protein may reduce this. In addition, binding cooperativity must not be too high, or promoters at even low ligand number may saturate long enough to make appreciable product. We previously showed that Hb border sharpness is produced by bistable kinetics stemming from hb self-regulation. Here, we show this must be balanced against the noise amplification in self-activation. Multiple binding sites play a role in precise expression levels: simulations with high (wild-type) numbers of binding sites display less noise than those with lower (e.g. artificial promoter) numbers. This may have evolutionary implications, as other fly species display more or less binding sites than D. melanogaster. Deterministic modelling helps quantify constraints in dynamic processes like protein production; stochastic modelling reveals additional requirements for the reproducible expression of genes. POSTERS: Pattern Formation 233

427A Cis-regulatory control of sharpening and positioning in the Drosophila hunchback expression pattern. Francisco Lopes1, David Holloway2, Alexander Spirov1. 1) Dept of Applied Mathematics and Center for Developmental Genetics, Stony Brook Univ, Stony Brook, NY; 2) Mathematics, British Columbia Institute of Technology, Burnaby; Biology, Univ. of Victoria, B.C. Canada. During Drosophila embryonic development a cascade of developmental genes controls the beginning of pattern formation and later body segmentation. The first set of genes in this cascade, the gap genes, is regulated in response to maternal signals like the antero-posterior gradient of Bicoid protein. One of these genes, hunchback (hb), has strong anterior expression and a sharp on-off boundary at mid-embryo. Many efforts have been devoted to understanding the mechanism of expression of this gene. hb activation has been shown to be driven by two different promoters, distal (P1) and proximal (P2); binding within these regions, Hb activates its own production, and Bcd cooperative binding determines the position of the sharp Hb border. Using a predictive kinetic model for hb transcriptional regulation, describing ligand binding/unbinding, production, decay and diffusion, combined with a number of experimental assays, we show that the control of Hb sharpness and positioning, by hb self-regulation and Bcd cooperativity, respectively, are separate processes and can be altered independently: the sharp Hb border is produced by bistable kinetics stemming from hb self-regulation, and Bcd cooperative binding determines the position where bistability occurs. With the hb14F mutant, we show that in the absence of self-regulation, bistability vanishes and Hb sharpness is disrupted. Using Bcd cooperative mutants we show that reduction in cooperative binding shifts the position where bistability occurs, without disrupting sharpness. Taking into account the delay between transcription and translation, we analyzed the roles of the P1 and P2 promoter regions for the separation between sharpening and positioning of Hb pattern, as well as the role of the regulation of other genes on the expression of hb, like the gap gene Krüppel. The robustness of hb regulation has also been characterized and the role for bistability in this process analyzed.

428B Complex movements of segmentation gene expression domains in Drosophila wild type embryos and Kr- mutants. Svetlana Surkova1, Maria Samsonova1, John Reinitz2. 1) State Polytechnical University, St. Petersburg, Russia; 2) Stony Brook University, Stony Brook, NY, U.S.A. We have studied the dynamics of segmentation gene expression in Drosophila wild type embryos and homozygous Kr mutants at the blastoderm stage. Our data has cellular resolution in space and a resolution of about 6.5 minutes in time (http://urchin.spbcas.ru/ flyex). Here we extend results reported in Jaeger et al., 2004 and demonstrate that during cycle 14A most expression domains, located at the region of presumptive germ band, shift anteriorly, while the domains in the presumptive head region move to the posterior. Thus the cephalic furrow serves as a watershed which separates regions of anterior and posterior domain shifts. Analysis of in vivo movies of the blastoderm morphology shows that shifts in the future head region appear to arise from shifts of nuclei. On the contrary, in the presumptive germ band nuclear movements are much smaller than expression shifts and occur as a consequence of gene regulation. As it was previously reported, in Kr- embryos the gt posterior domain and eve stripe 7 are shifted to the anterior relative to their position in the wild type. We have found that there is no difference in the position of gt posterior domain and eve stripe 7 until 13 and 26 minutes from the onset of cycle 14A respectively, whereupon these domains shift by 12 and 5% embryo length as compared with wild type. As zygotic gap proteins appear at cycle 12 - 13, our results point to the existence of significant delay in the influence of the absence of Kr protein on the behavior of expression domains. This suggests that zygotic gap-gap cross-regulation does not play a role in the positioning of the segmentation gene expression domains at early times and comes into effect only during cycle 14A.

429C Positional information and the Hunchback repression gradient in Drosophila. Danyang Yu, Stephen Small. Department of Biology, New York Univ, New York, NY. Morphogen gradients are thought to contain multiple concentration thresholds that establish different cell fates during the process of embryogenesis. Despite much study, it is not clear how many different thresholds can be established by a single gradient, or how these thresholds are related to the maximum concentration produced at the source. In Drosophila, the Zn-finger protein Hunchback (Hb) is expressed as a dynamic gradient along the anterior posterior (AP) axis of the embryo. Previous work suggests that Hb function is critical for setting the anterior boundaries of seven specific RNA expression patterns. Here, we quantify the spatial and temporal relationship between the dynamic Hb gradient and the positions of these expression patterns, and use a targeted misexpression system to test the relative sensitivities of each. These experiments define five repression boundaries that are controlled primarily by threshold concentrations of Hb, and two that are controlled by Hb in combination with at least one other co-repressor. The spatial relationships between these two classes of boundaries suggest that Hb is an effective morphogen only within a specific range of relative concentrations (from 40% to 4.4% of maximum). The upper limit of this range may explain how heterozygous embryos, which contain roughly half the protein concentration found at each position in wild type embryos, can nonetheless develop normally. 234 POSTERS: Pattern Formation

430A The Ancestral morphogen otd can replace many bcd patterning functions in the early embryo. Gozde Yucel, Na-Eun Yoo, Stephen Small. Biology, New York Univ, New York, NY. One of the intriguing questions in developmental biology is how to generate a complex body plan from a single cell. The Drosophila body plan is established through a cascade of gene interactions that originates in the oocyte and subdivides the embryo into increasingly refined domains. The bicoid (bcd) gene is at the top of this cascade. Bcd protein is expressed in an anterior gradient, and turns on several target genes at specific positions along the anterior posterior axis. Embryos from homozygous bcd mothers lack all anterior structures, including cephalic, thoracic, and the anterior-most abdominal segments. In the cephalic region, several Bcd target genes are known, but how they function to pattern the head segments is still unclear. Here we focus on the target gene, orthodenticle (otd), which is expressed in a thick stripe in anterior regions. Mutations in otd cause defects in the preantennal and antennal structures in the head. To understand the role of otd in the absence of bcd, we used a transgenic approach to create an anterior maternal gradient of Otd, and crossed the transgene into bcd mutants. We find that otd rescues a significant number of head and thoracic structures, and activates several of the known “Bcd-target genes” in the absence of bcd. These results are consistent with the idea that Otd is an ancestral morphogen that patterns the head and thorax in other insects. None of the other target genes that we tested by misexpressing the same way gave similar results suggesting that otd is unique in its patterning ability.

431B A screen for modifiers of Hedgehog signaling in Drosophila identifies swm and mts. David J. Casso1, Songmei Liu1, D. David Iwaki1, Stacey K. Ogden2, Thomas B. Kornberg1. 1) Dept Biochem/Biophysics, Univ California, San Francisco, San Francisco, CA; 2) Department of Pharmacology and Toxicology Dartmouth Medical School Hanover, NH. Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its mis-regulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway, and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Four of the regions include genes that contribute constituents to the pathway- smoothened, patched, engrailed and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity.

432C The roles of knirps and abrupt in wing vein development. Orna Cook1, Hanh Nguyen1, Koen J.T. Venken2, Hugo J. Bellen2, Ethan Bier1. 1) Dept. of Biology, UCSD, La Jolla CA; 2) Program in Developmental Biology, Baylor College of Medicine, Houston, TX. The Drosophila longitudinal wing veins arise in a stereotypical pattern, making them a good model system to study the role of boundaries during pattern formation. The 2nd and the 5th veins, L2 and L5, are induced in similar but opposite positions in the anterior and posterior compartments of the wing respectively, in response to the long-range morphogene, Decapentaplegic (Dpp). The fates of both veins determined by organizing genes; knirps(kni) for L2 and abrupt (ab) for L5. These vein organizing genes are expressed along domains of different Dpp target genes. In previous studies we showed that the L2 organizer, kni, is expressed along the anterior border of the Dpp signaling target gene, spalt. In a similar way, the L5 organizer, ab, is expressed along the posterior border between two other Dpp signaling target genes, optomotor-blind and brinker. In order to understand how Dpp signaling activates expression of kni in the anterior compartment of the wing vs. ab in the posterior compartment, we first identified and cloned a genomic region that contains the L5 enhancer of ab, by using the recombineering tecnique. This enhancer region was used to compare the signals regulating the ab L5 enhancer with those regulating the kni L2 enhancer. We also compare the vein organizing activities of kni and ab by examining the ability of ab to substitute kni in the L2 primordium as well as kni to substitute ab in the L5 primordium. POSTERS: Pattern Formation 235

433A Wing membrane topography is determined by the dorsal wing epithelium. Kristy Doyle, Simon Collier. Dept Biological Sci, Marshall Univ, Huntington, WV. During pupal metamorphosis, the wing imaginal disc evaginates to form an epithelial bilayer with dorsal and ventral surfaces. Both dorsal and ventral cells secrete the wing cuticle before delaminating shortly after eclosion and migrating into the thorax. The mature wing membrane is, therefore, a cuticle bilayer with contributions from both the dorsal and ventral epithelia. Scanning electron microscopy (SEM) reveals that the wing membrane has a uniform thickness and a ridged topography. The wing membrane also has a distinct array of cell hairs. We have found that hairs on the dorsal surface of the wing are situated consistently on top of the membrane ridges, whereas hairs on the ventral surface have a variable location with respect to the ridges. This suggests that wing topography and hair positioning is coordinated on the dorsal surface, but not on the ventral surface. We have previously shown that over-expression of the Sple isoform of the prickle gene product during wing development results in a specific change in ridge orientation and a reversal of hair polarity. When the Sple isoform is over-expressed only on the dorsal surface of the developing wing, both the dorsal and ventral wing surface display ridge orientation that is characteristic of Sple over-expression even though the ventral hair polarity remains normal. We conclude that the dorsal wing epithelium determines wing membrane topography. We are currently investigating the adult wing structure of other members of the Endopterygota to see if this observation holds true across this insect group.

434B Coregulation of a neighboring EST with crossveinless-c (cv-c). Tia Hughes, Jeffrey Marcus. Dept Biol, 11080, Western Kentucky Univ, Bowling Green, KY. It has been reported that the crossveinless-c locus corresponds the RhoGAP88C gene. This determination was based on three criteria: proximity in genetic map position, complementation tests, and similarity of phenotype between RhoGAP88C RNAi constructs and the phenotype of hypomorphic cv-c1 mutant homozygotes. At the time this report was published, we had been studying the group of mutations that produce crossveinless phenotypes for several years, and had already tentatively identified cv-c as a poorly annotated PDZ-domain containing gene corresponding to Clot 13975 (approximately 60 kb upstream from the RhoGAP88C transcriptional start). A P-element insertion l(3)06951 is located immediately adjacent to Clot 13975, but the homozygous lethal phenotype of the insertion appears to be unrelated to the Clot. The data showing that l(3)06951 fails to complement cv-c1 is relatively weak; only about 35% of l(3)06951/ cv-c1 heterozygotes have a broken crossvein phenotype. This is in spite of the fact that l(3)06951 is homozygous lethal and cv-c1 homozygoetes have a completely penetrant broken/missing crossvein phenotype. We have mobilized l(3)06951 and recovered lethal insertion mutants 500 bp away from the original insertion that complement l(3)06951 and fail to complement cv-c1 with a completely penetrant crossveinless phenotype. We suspect based on our data and that of Denholm et al. (2005) that l(3)06951 and RhoGAP88C are allelic with one another and distinct from the cv-c locus. We have used RealTime PCR to show that mutations that fail to complement cv-c and mutations that fail to complement RhoGAP88C result in lower levels of both RhoGAP and Clot 13975. This suggests that the mutations in this region may be affecting transcription levels of multiple mRNAs and may be causing some of the difficulty in assigning mutations to specific genetic loci. We are now working to use UAS constructs carrying full-length RhoGAP or Clot 13975 transcripts to try to rescue the crossveinless phenotype in cv-c mutants and definitively determine which of these transcripts should be assigned to cv-c.

435C The Glucose Transporter (GLUT4) Enhancer Factor is Required for Normal Wing Positioning in Drosophila. Jonathan Terman, Umar Yazdani, Zhiyu Huang. Departments of Neuroscience and Pharmacology, The University of Texas Southwestern Medical Ctr, Dallas, TX. Many of the transcription factors and target genes that pattern the developing adult remain unknown. In the present study, we find that an orthologue of the poorly understood transcription factor, Glucose Transporter (GLUT4) Enhancer Factor (Glut4EF (GEF)) (also known as the Huntington’s Disease gene regulatory region-binding protein (HDBP) 1), plays a critical role in specifying normal wing positioning in adult Drosophila. Glut4EF proteins are zinc finger transcription factors named for their ability to regulate expression of GLUT4 but nothing is known of Glut4EF’s in vivo physiological functions. Here, we identify a family of Glut4EF proteins that are well-conserved from Drosophila to humans and find that mutations in Drosophila Glut4EF underlie the wing positioning defects seen in stretch mutants. In addition, our results indicate that previously uncharacterized mutations in Glut4EF are present in a number of publicly available fly lines and on the widely used TM3 balancer chromosome. These results indicate that previous observations utilizing these common stocks may be complicated by the presence of Glut4EF mutations. For example, our results indicate that Glut4EF mutations are also present on the same chromosome as two gain-of-function mutations of the homeobox transcription factor Antennapedia (Antp) and underlie defects previously attributed to Antp. In fact, our results support a role for Glut4EF in the modulation of morphogenetic processes mediated by Antp, further highlighting the importance of Glut4EF transcription factors in patterning and morphogenesis. 236 POSTERS: Pattern Formation

436A Impact of the sulfation state of heparan sulfate proteoglycans on Hedgehog signalling. Alexandre Wojcinski1, Takuya Akiyama2, Cathy Soula1, Hiroshi Nakato2, Bruno Glise1. 1) Centre de Biologie du Développement, CNRS, Université Paul Sabatier, UMR-5547, Toulouse, France; 2) Department of Genetics, Cell Biology and Development, The University of Minnesota, Minneaopolis, USA. The Hedgehog (Hh) family of morphogen plays a pivotal role in vertebrate and invertebrate development. Over the past decade, intensive biochemical and genetic studies have elucidated the central components of the Hh signalling pathway. However, several important issues remain to be resolved concerning the mechanisms by which the distribution and movement of Hh is regulated in morphogenetic fields. Newly synthesized Hh protein undergoes several post-translational modifications that lead to the presentation at the cell surface of a mature and active lipid-modified Hh. These lipid moieties have been shown to be necessary for the normal distribution and diffusion of Hh. It has also been clearly demonstrated that Heparan sulphate proteoglycans (HSPGs) play crucial roles in regulating Hh movement during development. However the function and mode of action of these extracellular matrix macromolecules are still not well understood. HSPGs are molecules that are comprised of a core protein to which heparan sulphate (HS) glycosaminoglycan chains are attached. HS chains are initially synthesized by HS polymerases on core proteins as linear polysaccharides composed of glucuronic acid N-acetylglucosamine repeating units. These moieties are subsequently subjected to marked structural modification by sulfation implicating N-, 2-O-, 6-O-, 3-O-sulfotransferases and epimerization, through the activity of C5-epimerase in the Golgi and finally by desulfation (6-O-sulfatase) at the cell surface. We will present evidence showing the importance of the sulfation state of HSPGs in modulating Hh signalling during Drosophila wing development. POSTERS: Organogenesis and Gametogenesis 237

437B Obstructor, a novel Megatrachea interactor protein, is essential for tracheal tube maturation. Matthias Behr, Birgit Stümpges, Tina Radtke, Christian Wingen, Michael Hoch. Molecular Developmental Biology, LIMES Institute, University of Bonn, Germany. Many epithelial organs are composed of tubular networks transporting gases or liquids. The mechanisms controlling size and shape of tubes are poorly understood. Previous studies on the Drosophila tracheal system have demonstrated that core components of septate junctions (SJ) such as the claudin Megatrachea, not only seal the tracheal epithelium but also regulate its size and shape. The underlying mechanisms are poorly understood. In a screen we have recently identified novel regulators for tube maturation and liquid clearance, including the wurst gene (Behr et al, 2007, Nat Cell Biol) and the obst-A gene (CG17052; Behr and Hoch, 2005, FEBS). obst-A is a member of a novel evolutionary conserved gene family encoding secreted proteins with chitin binding domains. Immunohistochemical analysis shows that the Obst-A protein is localized in a vesicular pattern and at the apical cell surface of tracheal cells. Furthermore, we find the protein in the tracheal lumen consistent with a role in chitin secretion and/or luminal organization. In obst-A mutants, tracheal tube size is increased and the chitinous extracellular matrix is severely disorganized. In addition, the epithelial barrier of the tracheal cells is affected. Biochemical analysis demonstrates that Obst-A is a direct interaction partner of Megatrachea. We further find that Obst-A accumulates intracellularly in megatrachea mutants and that the secretion of the protein is abolished. Our results show a direct molecular link between a core component of SJ and a putative chitin binding protein required for extracellular matrix organization and tube size control in Drosophila.

438C The transcription factor SoxNeuro directs trichome formation in the ventral embryonic epidermis upstream of and in parallel to Shavenbaby. Marita Buescher, William Chia. Drosophila development, Temasek Lifesciences Laboratories, 1 Research Link, Singapore 117604. Trichomes are cytoplasmic extrusions of epidermal cells. Within the Drosophila epidermis specialized cells produce trichomes while other cells retain a smooth surface. The binary decision to differentiate into trichome-producing versus non-trichome-producing cells is effected by the transcription factor Shavenbaby (Svb;Ovo). Svb directs trichome formation via the transcriptional control of cellular effectors which in turn modulate epidermal cell shape. We have previously shown that the transcription factors SoxNeuro (SoxN) and Dichaete (D) act genetically upstream of svb: SoxN/D are necessary and sufficient to activate the expression of svb in the epidermis. A linear relationship between SoxN/D, svb gene expression and trichome formation implies that the loss of either SoxN/D or the loss of svb should have the same effect on trichome formation. However, this is not the case. The concomitant loss of SoxN and D results in the loss of all trichome formation in the ventral epidermis. By contrast, in svb mutants, trichome formation remains unaffected in a small, distinct subset of epidermal cells. These data suggest that SoxN/D have a function in trichome formation which is independent of svb. This interpretation is corroborated by our observation that misexpression of SoxN in the ventral epidermis results in the formation of small cytoplasmic extrusions even in the absence of svb function. In addition, misexpression of either SoxN or svb in the epidermis of wildtype embryos leads to the formation of ectopic denticles which are morphologically distinct: while trichomes resulting from the misexpression of svb are atrophied, trichomes resulting from the misexpression of SoxN more closely resemble wildtype trichomes. Our results suggest that SoxN directs trichome formation in ventral epidermal cells upstream of and in parallel to Svb. Our current research aims at the identification of SoxN target genes which function in the modification of epidermal cell shape.

439A Serrano (Sano) is a novel planar cell polarity regulator that controls tube length in Drosophila trachea. Se-Yeon Chung1, Melissa Vining1, Chih-Chiang Chan2, Pamela Bradley1, Keith Wharton2, Deborah Andrew1. 1) Dept Cell Biol, Johns Hopkins Univ, Baltimore, MD; 2) Dept Pathol and Mol Biol, UT Southwestern Medical Center, Dallas, TX. Tubes are required in metazoans to transport the liquids and gases that sustain life. Proper tube geometry is crucial for organ function, and recent studies have revealed that the genes encoding septate junction proteins or chitin modifying enzymes play a role in tube-size control in the Drosophila trachea. Here we identify a novel gene, serrano (sano), loss of which causes lethality and an extended, convoluted trachea. Sano was identified in an enhancer trap screen for genes expressed in the developing salivary gland and trachea. Expression of sano in the salivary gland and the trachea is regulated by the early transcription factors Sex combs reduced (Scr) and Trachealess (Trh), respectively. Sano encodes a novel protein that is conserved in arthropods, and localizes to the cytoplasm with enrichment in apical regions. Interestingly, ectopic expression of Sano caused planar cell polarity (PCP) defects manifested by misorientation of the hairs of the adult wing and bristles on the thorax. In Sano-overexpressing wing cells, core PCP proteins such as Flamingo (Fmi), Strabismus (Stbm) and Prickle (Pk) were mislocalized in a non cell-autonomous fashion. Yeast two-hybrid analysis revealed that Sano physically interacts with Dishevelled (Dsh), a cytoplasmic protein downstream of Frizzled (Fz), a serpentine receptor in the PCP signaling pathway. Our data suggests that PCP signaling may be linked to the tube length control in the early embryo. 238 POSTERS: Organogenesis and Gametogenesis

440B Making and shaping subcellular tracheal tubes. Amin Ghabrial1, Boaz Levi2, Mark Krasnow2. 1) Dept Cell/Developmental Biol, Univ Pennsylvania, Philadelphia, PA; 2) Dept of Biochemistry/HHMI, Stanford University School of Medicine, Stanford, CA. During larval life, tracheal terminal cells extend dozens of new branched tubes that ramify on surrounding tissues and promote efficient gas exchange. These new tubes originate as cellular protrusions, and are then converted into subcellular “seamless” tubes by a process believed to involve fusion of vesicles. We have isolated mutants that are defective in various aspects of making and shaping seamless tubes. Progress in identifying the cellular and molecular steps of seamless tube formation will be presented.

441C The role of VHL in tracheal development. Anita Hsouna, Tien Hsu. Hollings Cancer Ctr, Medical Univ South Carolina, Charleston, SC. The human von Hippel-Lindau (VHL) tumor suppressor gene acts as an E3 ubiquitin ligase involved in protein ubiqitination and degradation. Germline mutations in VHL gene in humans cause highly vascularized tumors in kidney, retina and central nervous system that characterize the inherited VHL disorder. VHL null cultured tumor cells exhibit increased cell migration and over- accumulation of cell surface FGF Receptors. In Drosophila, previously, it has been shown in our lab that loss of function of VHL by RNA interference resulted in ectopic migration defects during tracheal development (Adryan et al., 2000). In order to study further the role of VHL in the trachea, we generated genomic knock-out mutants of VHL via homologous recombination. The VHL mutant flies are homozygous lethal and they exhibit tracheal defects at the embryonic and in larval stages. VHL mutant embryos with ectopic migration defects in the trachea also display some changes in the level and distribution in other proteins, such as Breathless (Btl), the Drosophila homolog of FGFR, Pointed (Pnt) and Trachealess (Trh). The VHL function in the epithelial systems will also be examined.

442A Plexin-Semaphorin signaling is necessary for proper salivary gland migration. Afshan Ismat1, Melissa S. Vining1, Alex Kolodkin2, Deborah J. Andrew1. 1) Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD; 2) Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD. The salivary gland (SG) is a unique system in which to learn how tubular epithelia undergo regulated migration to achieve their final correct positions in the developing embryo. During posterior migration, the SG contacts a number of tissues, including the visceral mesoderm, somatic mesoderm and fat body clusters; signals from all of these tissues are likely to play key roles in guiding migration. The sema2a gene, which has previously been implicated in axonal guidance, is expressed in a muscle in the third thoracic segment (T33) that the SG directly contacts during late embryonic stages. Correspondingly, the gene encoding a receptor for the Sema2a protein, known as plexinB, is expressed in the migrating SG. Mutations in both genes lead to misoriented SGs, with the distal portion of the SG being randomly arrayed instead of lying along the anterior-posterior axis. We are currently testing if tissue- specific expression of the ligand (in only muscle T33) and receptor (in only the SG) can rescue the defects, and if misexpression of the ligand in ectopic sites can redirect SG movement. Finally, we are testing which of the components known to function downstream of receptor activation in axonal migration are required for correct SG guidance. Our data, in combination with studies revealing roles for both VEGF and Netrin in SG positioning, suggest that correct tissue placement is a consequence of activating sequential, combinatorial signaling pathways. POSTERS: Organogenesis and Gametogenesis 239

443B Identification and characterization of Fkh targets in Drosophila salivary gland. Rika Maruyama, Elizabeth Grevengoed, Deborah J. Andrew. Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD. The Drosophila FoxA homolog Fork head (Fkh) plays important roles in multiple events essential for salivary gland development and function. For example, Fkh is required for the maintenance of expression of CrebA, a transcription factor essential for the expression of secretory pathway genes in salivary glands. Although a few targets of Fkh have already been discovered, the question of what kinds of genes and functions Fkh regulates in salivary gland morphogenesis remains unclear. To obtain a comprehensive picture of the Fkh targets that function in salivary gland development and physiology, we examined the expression of 193 known salivary gland expressed genes in fkh mutants by whole-mount in situ hybridization. Loss-of-function mutations in fkh altered the expression of 67 salivary gland genes (35%), completely eliminating salivary gland expression of only 22 of the 67 (11% of the total). The genes whose expression was altered in fkh mutants encode proteins involved in various biological phenomena, including transcriptional regulation, secretion, endocytosis, signal transduction, cytoskeletal organization, metabolism, catabolism, and protein biosynthesis and modification. These data indicate that although Fkh regulates a wide range of activities in salivary gland development and function, it is not the sole factor specifying this organ since expression of 65% of salivary gland genes are unaffected by the loss of Fkh function. To begin to learn the function of newly identified Fkh targets, we examined the morphologies of salivary glands from embryos either deficient or mutant for a subset of target genes, selected based on their homologies to known proteins and their early expression patterns. Among the several deficiency lines and homozygous mutants that resulted in interesting defects was the cation transporter Nramp homolog Malvolio, which resulted in delayed salivary gland invagination. We will report on our progress in characterizing the specific role of Malvolio in tissue invagination.

444C From regulatory networks to cell morphology: Control of epidermis differentiation. Francois Payre1, Jennifer Zanet1, Philippe Valenti1, Serge Plaza1, Alistair McGregor2, David Stern2. 1) Biologie du Development, University ToulouseIII CNRS, Toulouse, France; 2) Department of Ecology and Evolutionary Biology, Princeton University, USA. It is well established that regulatory networks control morphogenesis during development, but how they produce cell shape changes remains largely elusive. We address this question by studying the morphological differentiation of epidermal derivatives. It is governed by a large circuit of regulators, leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). Although laboratory-induced mutations in dozens of these regulators modify the trichome pattern, multiple changes at a single gene, Shavenbaby, underlies all examined cases of evolutionary diversification of the trichome pattern between species. We found that shavenbaby (svb) encodes a transcription factor that directly activates the expression of numerous cellular effectors, being collectively responsible for remodelling the shape of epidermal cells. The pattern of shavenbaby expression in epidermal cells thus appears as an ultimate determinant of the trichome pattern. We thus undertook a systematic analysis of the cis-regulatory elements that direct svb expression. We found that epidermal expression of svb is controlled by several elements, scattered over a large genomic region (>50kb). Transgenic analyses allowed distinct svb cis-regulatory elements to be delineated. We show that different elements contribute to complementary aspects of svb expression, in specific subsets of epidermal cells along the body. In addition, a given svb control element behaves as an independent regulatory module, with respect of upstream signaling activities and positional information. All together, these data show that the control of svb expression relies on a complex array of regulatory modules, which individually respond to specific developmental cues and collectively define the set of epidermal cells that form trichomes. The svb promoter thus acts as an integrator to translate multiple outputs from upstream regulatory cascades to a coherent pattern of cell shape changes.

445A Identification and Characterization of Genes Involved in Subcellular Lumen Formation. Oscar E Ruiz, Mark M Metzstein. Department of Human Genetics, University of Utah, Salt Lake City, UT. Branched tubular networks, such as the vascular and respiratory systems, employ a common structural design that permits the transport of liquids and gases throughout the body. The cellular cues required for generating these complex structures are not well understood. To identify components involved in tube and network formation, we previously conducted a genetic screen for mutations affecting branching and lumen formation in the terminal cells of the Drosophila tracheal system. Terminal cells are specialized tracheal cells that undergo subcellular branching and tubulogenesis, and are responsible for transporting and exchanging gases in hypoxic target tissue. To characterize the distinct branching and lumen phenotypes, mutagenized animals were analyzed using a combination of GFP labeling, to analyze the branching pattern, and brightfield microscopy, to analyze the formation of subcellular tubes. 32 lines with mutations affecting different aspects of branching and lumen formation were identified. Of these 32, we are focusing on 8 lines in which tracheal terminal cells undergo essentially normal branching, but are unable to generate a functional lumen. These mutants have been mapped to discrete intervals using a multipoint visible marker recombination mapping strategy. The mapping has been further refined for 2 of these 8 lines by using a combination of single nucleotide polymorphisms (SNP) and P-element recombination frequencies. We have identified a small number of candidate genes for each of these mutations. Molecular analysis of the role these genes play in tracheal development will be presented. 240 POSTERS: Organogenesis and Gametogenesis

446B Genetic control of imaginal hindgut development in Drosophila. Shigeo Takashima, Marianna Mkrtchyan, Amelia Younossi- Hartenstein, John Merriam, Volker Hartenstein. Univ California, Los Angeles, Los Angeles, CA. During metamorphosis, most larval organs degenerate and are replaced by adult organs. Not only outer body parts, such as wings and legs, the digestive tract is also reconstructed during the pupal period. The gut of Drosophila consists of major three parts; the foregut, the midgut, and the hindgut. Despite of the accumulating knowledge of embryonic gut development, little is known about late development of the gut during metamorphosis. In this work, we mainly focused on hindgut and studied its reconstruction process during metamorphosis. The hindgut occupies posterior part of the gut, which is derived from ectoderm. A cluster of the adult precursor cells of hindgut resides at the junction between the midgut and the hindgut, and is called the hindgut imaginal ring. First, we observed a gross morphology of pupal hindgut, and also observed the development of the hindgut imaginal ring during larval period. Also we investigated the change of visceral muscle during metamorphosis.

447C Multiple requirements for Rho1 GTPase during salivary gland development. Na Xu1, Benison Keung2, Monn Monn Myat1. 1) Dept Cell & Developmental Biol, Weill Medical Col of Cornell, New York, NY; 2) New York Medical College, Valhalla, NY 10595. We are investigating how the small GTPase Rho1 regulates coordinated movements of the embryonic salivary gland. The salivary gland is a pair of elongated epithelial tubes that form by invagination of the ventral ectodermal primordial cells and migration of the epithelial tube along the circular visceral mesoderm (CVM). Here, we report that Rho1 function is required for different aspects of salivary gland invagination and migration. By analyzing zygotic loss of function mutants and the tissue specific expression of dominant negative Rho1 GTPase, we show that Rho1 is required to maintain apical polarity of salivary gland cells during their invagination. This function of Rho1 is mediated through the transcriptional regulation of the apical determinant, Crumbs. During salivary gland migration, Rho1 is required for cell contraction at the proximal tip and at the distal tip of the migrating gland. In addition to a cell- autonomous requirement for Rho1 in gland migration, Rho1 is also required for proper formation of the CVM upon which the gland migrates. In Rho1 mutant embryos, a continuous band of CVM cells is not formed and instead the CVM consists of isolated clusters of cells. Our studies demonstrate that Rho1 maintains apical polarity in invaginating salivary gland cells through Crumbs and mediates contraction of migrating gland cells. Moreover, Rho1 is also required for proper formation of the CVM.

448A Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential Notch signaling. Benjamin Ohlstein. Department of Genetics and Development, Columbia University HHSC 1130, New York, NY 10032. The adult Drosophila midgut contains multipotent intestinal stem cells (ISCs) scattered along its basement membrane that have been shown by lineage analysis to generate both enterocytes and enteroendocrine cells. ISCs containing high levels of cytoplasmic Delta-rich vesicles activate the canonical Notch pathway and down-regulate Delta within their daughters, a process that programs these daughters to become enterocytes. ISCs that express little vesiculate Delta, or are genetically impaired in Notch signaling, specify their daughters to become enteroendocrine cells. Thus, ISCs control daughter cell fate by modulating Notch signaling over time. Our studies suggest that ISCs actively coordinate cell production with local tissue requirements by this mechanism. POSTERS: Organogenesis and Gametogenesis 241

449B Identification of the ov and v subloci and the analysis of complementing nonsense mutants in the dumpy gene. Amber Carmon, Ross MacIntyre. Dept Molec Biol & Genetics, Cornell Univ, Ithaca, NY. The complex dumpy gene of Drosophila melanogaster was intensively studied with regard to its mutational phenotypes and its genetic fine structure. There are seven kinds of mutants singly affecting wing (o), the thorax (v), or viability (l), or combinations of two or three of the phenes, ov, ol, lv or olv respectively. Grace (1980) mapped the v, ol, lv, and ov mutants to single subloci in the gene. o and olv mutants mapped at many different places. We cloned and analyzed the structure of the Dumpy protein (Wilkin et al, 2000) and subsequently induced and recovered 97 mutants in an isogenic background. With these mutants, we found the ol and lv subloci and mapped the positions of 6 olv mutants (Carmon et al, 2006). Interestingly, almost all of these are nonsense mutants or affect splice sites. Here we report the positions of ov and v mutants in the gene. With the only two EMS induced mutants in our collection, we found the ov sublocus in exon 11. These mutants are changes in Cys residues, presumably affecting the structures of EGF and DPY modules in the extracellular component of this matrix protein. The oft used dpov1 mutant, originally recovered in the Morgan lab 90 plus years ago, appears to be due to the insertion of a blood transposon in an intron adjacent to exon 11. Dumpy vortex mutants also appear to be due to transposable elements in the large intron between the 5’ UTR and first exon, consistent with Grace’s fine structure map. The olv nonsense mutant ,dp6, is located in an exon (exon 15) near the PIGSFEAST sequences in the gene which are evolving by unequal crossing over (Carmon et al, 2007). We are determining which of our other olv nonsense mutants complement dp6. These mutants will presumably mark exons which can form trans-spliced transcripts with exon 15. We will present evidence for the presence of these trans-spliced messages in dumpy heterozygotes.

450C Characterization of slowmotion, a novel modulator of cell adhesion in Drosophila melanogaster. Eliezer Gilsohn, Talila Volk. Molecular Genetics department, Weizmann Institute of Science, Rehovot, Israel. In a microarray screen aimed at finding tendon specific genes in Drosophila melanogaster we have identified slowmotion, a previously uncharacterized gene, coding for an ortholog of the vertebrate EGFL7 protein. It has been shown that vertebrate EGFL7 is secreted out of endothelial cells of developing embryos and is essential for blood vessel formation. The molecular mechanism underlying EGFL7 activity, however, is currently unknown. Slowmotion (Slomo) is produced in tendon cells and additional sites in the embryo. Using immunostaining, we have found that it is a secreted protein which is highly enriched at the muscle/tendon junctions. We have generated a fly strain deleted for slowmotion, and found that the deletion is semi-lethal. Only ~25-35% of the slomo homozygous flies are viable and appear unable to fly. We noticed that slomo homozygous 1st instar larvae move significantly slower than heterozygous larvae. The embryonic muscle pattern, as observed using confocal microscopy, appears normal. However, in the 3rd instar larvae of the slomo mutant, a variable number of muscles appear broken or detached. These observations imply that slomo mutants have impaired muscle function. We further discovered that Slowmotion interacts with Thrombospondin, a conserved ECM protein that has been shown to be crucial for muscle/tendon adhesion through interaction with integrin receptors. Ectopic overexpression of Slowmotion and Thrombospondin together in embryos led to abnormal muscle adhesion. All of the above results have led us to hypothesize that Slowmotion affects adhesion properties at the myotendinous junction.

451A Dead man walking encodes a conserved cathepsin B-like ECM protein that interacts genetically with PS3 integrin. Ellen LeMosy, Michael Dinkins. Dept Cell Biol & Anatomy, Medical Col Georgia, Augusta, GA. The Drosophila gene CG3074 (dead man walking, dmw) encodes a secreted protein containing an N-terminal cysteine-rich domain similar to von Willebrand factor type C and a C-terminal cathepsin B-like domain, which contains a serine where typical cathepsin B active sites contain a cysteine. Conserved in bilaterians, Dmw is orthologous to the mammalian Tubulointerstitial Nephritis Antigen related proteins. dmw is expressed by centripetal and dorsal appendage-forming floor cells during Drosophila oogenesis. mRNA in situ hybridization shows dmw is maternally supplied to the oocyte and is expressed at low levels during gastrulation, germband movements, and throughout embryonic development in various tissues. dmw is highly expressed in a subset of maxillary cells and prothoracic precursors of the ring gland. To better understand the function of Dmw during development, we deleted dmw by homologous recombination. We recovered and verified a single homozygous lethal dmw allele (P[w+]dmw) that failed to complement Df(2R)7171 as well as two other alleles produced by imprecise excision of P(XP)d09601. P[w+]dmw embryos develop normally and survive to the L3 stage where larvae die peri-pupation. Those that do pupate are delayed by at least 24 hours suggesting a possible disruption of ecdysteroid synthesis in the ring gland. L3 P[w+]dmw larvae also develop melanotic tumors, suggesting altered antigenicity of ECM structures. Maternal null dmw embryos fail to complete embryogenesis and exhibit a stochastic range of developmental defects in germband extension and retraction, dorsal closure and segmentation, all suggestive of ECM disorganization. Since maternal null dmw embryos displayed phenotypes similar to those reported for integrins, we tested for a genetic interaction of dmw with scab (alphaPS3 subunit). scab embryos developed with 4.2% penetrance a mild herniation with the gut tissue located dorsally to the embryo. While dmw embryos develop normally, scab, dmw embryos developed with 13.5% penetrance severe herniation where gut tissue was located ventroposteriorly in the vitelline membrane. 242 POSTERS: Organogenesis and Gametogenesis

452B cut and slow border cells act antagonistically to regulate adherens junction polarity during follicle cell migration. Benjamin Levine, Truesdale Angela, Bergen Andrew, Dobens Leonard. Dept Molecular Biol, Univ Missouri Kansas City, Kansas City, MO. Stage 10 of oogenesis can be subdivided into stages 10A and 10B based on the transition of the centripetal FC from a columnar epithelial shape to an elongated shape. From stages 11-13, the centripetal FC ingress between the nurse cells and oocyte in a process known as centripetal migration. We have shown previously that centripetal migration requires Notch activation of the gene slow border cells (slbo) in the centripetal FC. slbo expression is high at 10A in the centripetal FC and subsequently decreases due to autorepression as centripetal migration proceeds. Here we show that expression of the transcription factor CUT increases in a complementary pattern due in part to cross repression with slbo: (1) FLP-out expression of CUT or SLBO, respectively, was sufficient to repress each other’s expression; and (2) cut mutant clones recovered in the centripetal FCs at 10B led to increased slbo expression. Notch activation of slbo at 10A did not apparently influence the timing of cut expression at 10B: (1) slbo mutant clones recovered at 10A did not result in precocious cut expression; and (2) increased Notch signaling at 10B did not repress cut expression in the centripetal FC. CUT-expressing FLP-out clones recovered in the columnar FC at 10B were associated with increased apical accumulation of adherens junction proteins and noticeable apical constrictions so that CUT-expressing clones elongate to resemble stage 10B centripetal FC. Reduced CUT levels, either in cut mutant clones or FLP-out clones expressing SLBO, resulted in mislocalization of apical adherens junction proteins to the basolateral locations. We propose that cut and slbo levels in the centripetal FC tightly regulate adherens junction polarity required for centripetal migration, and their antagonistic interactions mediate a Notch- dependent switch from epithelial to invasive cell shapes at the 10A/B transition.

453C Control of apical cell shape: role of Zona Pellucida proteins in epidermal cell morphogenesis. Francois Payre1, Isabelle Fernandes1, Helene Chanut-Delalande1,2, Pierre Ferrer1, Serge Plaza1. 1) Biologie du Development, University ToulouseIII CNRS Toulouse, France; 2) Bizentrum der Universitat Basel, Basel, Switzerland. How developmental programs act to ultimately modify the form of individual cells remains poorly understood, particularly little is known on the mechanisms that remodel the apical compartment during epithelial cell differentiation. We addressed this question through studying epidermal cell morphogenesis, which produces apical protrusions of specific shape, collectively referred to as trichomes. We have previously shown that trichome formation is triggered by a transcription factor, Shavenbaby, and identification of its downstream targets provided access to cellular effectors. In addition to cytoskeletal regulators, we show here that the shape of trichomes relies on modifications of the apical Extra-Cellular Matrix (aECM). We demonstrate that a family of 8 membrane proteins, characterized by an extracellular Zona-Pellucida (ZP) domain, provides localised cues for shaping cell extensions. We generated null alleles for each of the ZP genes and analysed their respective contribution to apical cell remodelling. First, we found that differential expression of a subset of ZP proteins contributes to the distinct shape of trichomes, at different positions along the body. Second, ZP proteins that are expressed in the same cells are individually targeted to different subapical domains, fulfilling specialized roles in shaping the cell extension. Ultrastructural analyses reveal that a given ZP protein accumulates in a limited region of the aECM, where its activity is required for sculpting the shape of cellular extensions. All together, our results reveal a specific assemblage of Zona Pellucida proteins that structure aECM in microdomains within the apical cell compartment, in order to finely tune cytoskeleton- driven remodelling of the cell shape. These data might help understanding human pathologies that result from alteration of this interplay between actin regulators and ZP proteins.

454A The role of Tissue Inhibitor of Metalloproteases (Timp) in Drosophila oogenesis. John Pearson, Acaimo Gonzalez-Reyes. Centro Andaluz de Biologia del Desarrollo (CABD),CSIC Universidad Pablo Olavide, Seville, Spain. The Extracellular Matrix (ECM) plays a critical role in a range of developmental processes, including tissue remodelling, cell migration and cell signalling. Extracellular proteases and protease inhibitors play an important role in determining the integrity and composition of the ECM. Drosophila has emerged as an important model for the study of interactions between the cell signalling pathways, ECM components and their regulators and the dynamics of epithelial cells. Metalloproteases (MMPs) are a family of extracellular proteases capable of degrading ECM components, whose misexpression has been linked to the invasiveness of cell clusters. The activity of MMPs is thought to be regulated in part by Tissue inhibitor of metalloproteases (Timp) proteins. Loss-of- function studies have suggested that Drosophila Timp is required for adhesion between the two epithelial layers of the Drosophila wing. Ectopic expression of Timp has also been shown to block the ability of MMP expressing epithelial cells to degrade the basement membrane. However, little is known about the in vivo function of Drosophila Timp in other tissues, or its involvement in processes such as cell migration, cell-signalling and the control of ECM composition. We found that ovaries of Timp mutant females are abnormally small and often highly disorganised, suggesting that Timp is required for normal oogenesis. We have performed an analysis of Timp mutant flies and Timp mutant clones as part of a comprehensive study into the in vivo role and molecular mechanisms of this important ECM regulator. POSTERS: Organogenesis and Gametogenesis 243

455B Cohesive migration of the embryonic salivary gland. Carolyn Pirraglia, Monn Monn Myat. Cell & Developmental Biology, Weill Medical College of Cornell University, New York, NY. Cell migration is a fundamental process important in embryogenesis and pathological conditions. While some cells are known to migrate individually, others migrate as a cohesive unit. The embryonic salivary gland is a pair of elongated epithelial tubes that migrates cohesively along the circular visceral mesoderm (CVM) as an intact organ. Previous studies demonstrated a requirement for integrins and Rac GTPases in gland migration; however, it is not known how these key regulators of single cell migration control cohesive migration of the salivary gland. Here, we show that integrin-dependent and integrin-independent events guide cohesive migration of the salivary gland. At the advancing distal tip of the gland, integrin adhesion and signaling is required for attachment of the distal tip of the gland to the CVM and subsequent activation of Rac1 GTPase through membrane recruitment. We previously showed that Rac regulates gland migration at least in part through downregulation of E-cadherin (E-cad)-mediated cell-cell adhesion (Developmental Biology, 290, 435-446). Here, we demonstrate that the p21-activated kinase (Pak) also regulates gland migration downstream of or in parallel to Rac to control E-cad mediated adhesion. Furthermore, Rac and Pak are required in an integrin- independent manner for cell contraction at the proximal tip that follows the advancing distal tip. Based on our studies, we propose a model where integrins, Rac GTPases and Pak control cohesive migration of the salivary gland through coordinated contraction of the proximal tip and transient downregulation of E-cad-based cell-cell adhesion at the distal tip. Our data provide novel insight into the poorly understood process of whole tissue/organ migration and reveal that while certain features, such as contraction at the rear and protrusion at the front, are conserved between single cell and whole organ migration, other features, such as spatial and temporal regulation of cell-cell adhesion are unique to cohesive cell migration.

456C Slit and Roundabout regulate E-Cadherin-mediated cell adhesion required for Drosophila heart tube lumen formation. Edgardo Santiago-Martínez1,2, Nadine H. Soplop1,2, Rajesh Patel1, Sunita G. Kramer1,2. 1) Dept of Pathology & Laboratory Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ; 2) Prog in Cell & Developmental Biology, Rutgers University & UMDNJ-Graduate School of Biomedical Sciences, Piscataway, NJ. The formation of a lumen is an essential step during the development of vascular tissues. The Drosophila embryonic heart, a linear tube structurally similar to a vertebrate capillary, provides a simple and straightforward genetic model for studying lumen formation. The fly embryonic heart is composed of two rows of cardioblasts that migrate from lateral sides of the embryo toward the dorsal midline where they establish cell-cell contacts and form a lumen at the apical surface between them. Several models, including apical vesicle targeting have been proposed for lumen formation in vitro and in vertebrates, but the mechanism that drives lumen formation in the Drosophila embryonic heart remains to be explored. In this study, we show that the extracellular guidance molecule Slit and its receptor Roundabout (Robo) are required for regulating cardioblast cell adhesion during formation of the heart tube lumen. Slit protein accumulates at the apical surface of cardioblasts prior to lumen formation. Ultra-structural analysis shows that embryos mutant for slit and robo disrupt lumen formation between cardioblasts, while expression of the Slit protein outside its normal apical domain induces ectopic lumen formation. In these embryos, we found that cardioblasts were correctly polarized, indicating that Slit and Robo are not responsible for establishing initial cell polarity. Meanwhile, we did observe mis-localization of two adherens junction proteins E-Cadherin and Enabled (Ena), away from their normal apical domains. Furthermore, we show that Robo and E-Cadherin are co-expressed in the heart and genetically interact in this process. We propose that activation of Robo by Slit inhibits E-Cadherin mediated cardioblast cell adhesion specifically at the apical membrane, permitting the formation of a lumen in this region.

457A Guidance Molecules in Epithelial Tube Maintenance: Slit Signaling in the Drosophila Hindgut. Nadine Soplop1,2, Edgardo Santiago-Martínez1,2, Sunita G. Kramer1,2. 1) Department of Pathology and Laboratory Medicine; 2) Cell and Developmental Biology Program, Graduate School of Biomedical Sciences; Rutgers University and University of Medicine and Dentistry of New Jersey at Robert Wood Johnson Medical School, Piscataway, NJ. The presence of guidance cues that lead migrating cells to their destinations have been shown in the CNS and somatic muscle systems in the Drosophila developing embryo. Specifically, the ECM protein, Slit, and the Roundabout (Robo) family of receptors function in both systems to guide migrating axonal growth cones or myotubes during neuronal and muscle patterning. Here we report that Slit, Robo and Robo2 are also expressed in the Drosophila hindgut, an epithelial tube that represents the most posterior section of the alimentary canal, at a late stage in embryonic development. The central part of the hindgut, the large intestine, consists of three cell types: dorsal, ventral, and the boundary cells. Each cell type expresses a unique complement of genes to make up the single cell layer epithelial tube. Nutrient-absorbing microvilli are found on the apical surfaces of the boundary cells in the large intestine and not along the entire luminal surface. We show, using hindgut-cell specific markers, that mutations in slit or robo do not affect the overall patterning of the three cell types. However, electron microscopy experiments viewing the large intestine in cross section reveal lumen defects, as well as defects in cell shape and adhesion in slit loss of function mutants. Furthermore, we observe ectopic microvilli in robo gain of function embryos in which the microvilli are no longer restricted to the boundary cells, but are present on the apical surface of all cell types. We suggest a novel role for Slit signaling in the maintenance of cell and lumen shape, as well as apical microvilli formation in the Drosophila hindgut’s large intestine. 244 POSTERS: Organogenesis and Gametogenesis

458B Shaping the “ball and socket” joints. Reiko Tajiri, Shigeo Hayashi. RIKEN Center for Developmental Biology, Kobe, Japan. An animal’s body is supported by the skeleton, which consists of modified extracellular matrix. The skeleton forms internally in vertebrates, whereas in many invertebrate species the cuticle on the body surface serves the role. The skeleton possesses flexible parts called joints, which enable movement. For efficient and controlled motion, neighboring bones or integuments must be shaped in a precisely reciprocal manner at the point of articulation to form a tightly interlocking structure. Clarifying how the shapes of the skeleton are regulated would thus be valuable to understanding animal development. Arthropods are characterized by their jointed limbs. A Drosophila leg comprises nine segments, each separated from the next by a joint containing a socket-shaped piece of cuticle and a ball-shaped one that fits into it. Although previous studies have shown that Notch signaling is required for the specification of joint cells, the manners in which it regulates subsequent morphogenesis and formation of the cuticlular structures are poorly understood. To address this question, we first made thorough observation of how the joint cells undergo morphogenesis and secrete cuticle. Next we examined spatiotemporal distribution of Notch activity during joint development. Using both gain- and loss-of-function approaches, we tested which aspects of cellular behaviors and cuticle formation in joint development are controlled by Notch signaling. Based on the results, we shall discuss the mechanism that accurately shapes the skeleton, and function of Notch signaling in it.

459C Highly conserved cysteines in the vitelline membrane domain of the sV23 eggshell protein are functionally distinct. Tianyi Wu, Gail Waring. Dept Biological Sci, Marquette Univ, Milwaukee, WI. We are using the multi-layered Drosophila eggshell, a specialized extracellular matrix, to study molecular mechanisms and motifs that are used to regulate the assembly of a complex extracellular structure in vivo. Proteins in the vitelline membrane (VM), the oocyte proximal layer, become incorporated into a large disulfide linked network during late oogenesis. All major VM proteins have three cysteine residues that fall within a highly conserved block of 38 amino acids termed the “VM domain”. Remnants of this domain are found in mosquito vitelline envelope proteins, with the number and spacing of the cysteines being conserved (CX7CX8C). To investigate the importance of the number and positions of the cysteines in eggshell assembly we made a series of cysteine substitution mutations in the sV23 VM protein. To test the effects of the mutations we introduced the mutant sV23 transgenes into an sV23 protein null female sterile mutant. Single substitution mutations indicate that the cysteines are not functionally equivalent. A transgene with a serine substitution at the third cysteine rescued the fertility of the sV23 null mutant, whereas a transgene with a substitution at the second cysteine did not. While specific single cysteine substitution mutations were tolerated, double and triple sV23 cysteine mutants were sterile. sV23 failed to accumulate in the triple mutant and was released from the eggshell during late oogenesis in the double mutants. Although sV23 was released in the latter, other VM proteins remained integrated in large disulfide linked complexes. While this result suggests VM proteins incorporate into different disulfide linked networks, non-reducible cross-link formation of other vitelline membrane proteins was compromised in the absence of sV23 suggesting some molecular interdependence. In wild type eggshells sV23 appears to be integrated into a disulfide network that includes other VM proteins as several vitelline membrane proteins bound and co-eluted with histidine tagged sV23 on nickel affinity columns under denaturing conditions.

460A An O-glycosyltransferase is required for proper cell adhesion in Drosophila. Liping Zhang, Ying Zhang, Kelly G. Ten Hagen. NIDCR/NIH, Bethesda, MD. Cell-cell adhesion and the factors that govern it are crucial in many aspects of eukaryotic development. Here we demonstrate that an enzyme responsible for the initiation of protein O-linked glycosylation is involved in epithelial cell adhesion in the Drosophila wing blade. Mutations in a member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family result in a blistered wing phenotype, characteristic of genes regulating cell-cell and cell-ECM interactions. The frequency of this phenotype is exacerbated in certain genetic backgrounds. Expression of the wild-type O-glycosyltransferase in mutant flies rescues the wing blistering phenotype. RNAi to the O-glycosyltransferase in wild type flies results in wing blistering. RNAi to this transferase in Drosophila cell culture causes Golgi apparatus misorganization and cell adhesion defects. We postulate that this transferase is involved in proper Golgi organization as well as processing/secretion of components involved in maintaining cell-cell adhesion. Lectins that recognize the O- linked glycans are being used as affinity reagents on wild type wing disc extracts to identify proteins that are normally glycosylated by this transferase and are therefore candidates for components mediating proper cell adhesion. This study demonstrates the role for a new class of genes in the regulation of cell-cell interactions in this system. POSTERS: Organogenesis and Gametogenesis 245

461B Fusion Regulates Myoblast Proliferation and Fiber Numbers during Adult Myogenesis in Drosophila. Krishan Badrinath. Dept Zoology, Miami Univ, Oxford, OH. Myogenesis in Drosophila occurs twice, once during embryogenesis, and again during metamorphosis, when adult muscles are formed. All embryonic muscles, and most adult muscles are formed by the fusion of myoblasts to specialized founder cells that express the transmembrane protein Dumbfounded (Duf), which is essential for fusion. The skeletal muscles of vertebrates, and the adult Indirect Flight Muscles (IFMs) of Drosophila share the feature of being dependent on neuronal influence for their development. Denervation leads to loss of duf expression in founder cells of the Dorso-ventral muscles that are part of the IFMs, and to a reduction in myoblast proliferation. This is similar to the effects of denervation in vertebrates, where the primary fiber degenerates, and secondary myoblasts stop proliferating. We have investigated the relationship between the founder cells and myoblasts, and find that the founder cell regulates myoblast proliferation. The rate of myoblast proliferation is dependent on fusion, and is temporally linked to a surge in MAPK signaling. We have also found that the disruption of fusion results in the formation of supernumerary fibers, which suggests that some myoblasts have the ability to seed fiber formation independent of the founder cells. We are investigating the signaling mechanisms that may play a role in the acquisition of founder cell like properties by myoblasts, and how fusion impinges on these mechanisms.

462C Fly Muscle LIM Protein, Mlp84B, cooperates with Sallimus to maintian muscle structural integrity. Kathleen Clark1,2, Mary Beckerle1,2. 1) Huntsman Cancer Inst, Univ Utah, Salt Lake City, UT; 2) Dept. of Biology, Univ Utah, Salt Lake City, UT. Muscle LIM Protein is a cytoskeletal “LIM-only” protein found at the intercalated disc and Z-line in cardiac muscle. Genetic ablation of murine MLP produces dilated cardiomyopathy (DCM) and heart failure. Mutations in human MLP are also associated with cardiac hypertrophy and DCM; however, the molecular mechanism by which MLP functions in normal and diseased muscle is still not established. We developed the fly system to probe the role of MLP in a genetically tractable model. We generated null mutations in mlp84B and found that the protein is essential for post-embryonic muscle function. The mlp84B mutants cannot fully contract their body wall muscles during pupariation, and make a long, thin pupal case. Most animals arrest development at this point, and exhibit only limited muscle contractions. A few mutants eclose as adults, but have impaired flight and cardiac dysfunction. While the mlp84B mutant muscles on their own do not have any observable structural defects, flies null for mlp84B and heterozygous for a mutation in sallimus (Titin) display a severe disruption of muscle structure, with most fibers displaying loss of sarcomeric organization and/or tearing. This enhancement of the mlp84B phenotype provides us with a method to screen for modifiers of the mlp84B pupal phenotype. We are also testing candidate mutations as mlp84B modifiers. For example, mutations in components of the Toll signaling pathway and the integrin ligand tiggrin affect pupal case shape suggesting that Mlp84B may participate in these molecular pathways. Other data indicate a role for Mlp84B in directing actin filament assembly. Forcing Mlp84B into the nucleus can result in the production of intricate actin filaments that also contain Mlp84B. A report from Montana and Littleton demonstrates that Mlp84B, the related protein Mlp60A and proteins that regulate actin dynamics are upregulated in damaged muscle. Together these observations suggest a model by which the fly MLPs promote normal muscle function by regulating actin dynamics during muscle repair.

463A The role of Myocyte enhancer factor-2 in controlling adult skeletal muscle development. Richard Cripps, TyAnna Lovato, Melanie Adams, Phill Baker. Dept Biol, Univ New Mexico, Albuquerque, NM. Myocyte enhancer factor-2 (MEF2) is a MADS domain transcription factor which is expressed at high levels in the musculature throughout development, and which is critical for embryonic muscle differentiation. We have recently utilized a temperature-sensitive combination of two Mef2 alleles to address the roles of MEF2 in the formation of the adult musculature. We found that reduction in MEF2 function affects adult skeletal muscle patterning but does not affect general aspects of differentiation. In order to define how temperature-sensitivity arises in these mutants, we are analyzing in detail the molecular defects engendered by each mutant allele, one of which arises from a mutation in the MADS domain, and one of which affects a more C-terminal residue. In addition, we are analyzing further Mef2 mutant lines in order to define their effects upon adult myogenesis in the multiple muscle types of the adult. These studies will provide insight into the functions of critical residues with the MEF2 protein, and will also indicate how MEF2 controls the formation of the complex adult muscles. 246 POSTERS: Organogenesis and Gametogenesis

464B Requirement for the Zn-finger transcription factor CF2 in larval blood and indirect flight muscle. Kathleen M. Gajewski1, Richard P. Sorrentino2, Tien Hsu3, Robert A. Schulz4. 1) Dept Biochemistry & Molec Biol, MD Anderson Cancer Ctr, Houston, TX; 2) Department of Biology, University of Guyana, Georgetown, Guyana; 3) Laboratory of Cancer Genomics, Medical University of South Carolina, Charleston SC; 4) Dept of Biological Sciences, University ofNotre Dame, South Bend IN. The function of the transcription factor CF2 has been characterized in the Drosophila ovary, but it is expressed in many other tissues. Reduced CF2 function causes a significant decrease in the larval circulating hemocyte count, reduced lymph gland size, and in certain alleles, the appearance of large numbers of lamellocytes. In the indirect flight muscle, loss of CF2function can cause an increase in transcription of muscle structural genes and an increase in myofibril size. In larval blood isoform II is required, while in developing flight muscles isoform I and a putative new isoform are expressed. CF2 may be needed in blood for cell survival, in lymph gland to prevent a too early maturation (and exit from the cell cycle) and in flight muscle to help maintain proper filament stoichiometry.

465C Hemangioblast Differentiation: Pathways Interacting with the Notch/Delta Signal. Melina Grigorian, Lolitika Mandal, Volker Hartenstein. Dept MCDB, Univ California, Los Angeles, Los Angeles, CA. The discovery of a hemangioblast precursor in Drosophila (Mandal, 2004), has led us to investigate the mechanisms involved in its differentiation. The two theories that are the most probable for the distinction of vascular cells from blood cells from a single precursor include asymmetric cell division or the presence of an asymmetrically distributed signal. Our data supports the latter of the two theories. Notch signaling has been found to be the main factor deciding hemangioblast cell fate and studies have shown a localization of Delta expression in cells flanking the blood precursors. Currently, we are studying different signaling pathways that may play a role in spatially restricting the expression of Delta and thereby effecting hemangioblast cell fate. Detailed genetic analysis will be presented regarding the combinatorial signaling cascades that give rise to the hemangioblast in Drosophila.

466A The cytokinesis protein Tumbleweed/RacGAP50C plays an essential role in postmitotic myotube guidance. Colleen M Guerin1,2, Robert Connacher1, Sunita G Kramer1,2. 1) Dept Pathology, RWJMS, Piscataway, NJ; 2) Cell & Dev Bio, GSBS-UMDNJ & Rutgers, Piscataway, NJ. Establishment of the intricate muscle patterns found in higher organisms occurs as migrating muscles are guided towards specific sites of attachment. Little is known about the guidance cues or mechanisms that govern migrating muscle fibers. Development of the Drosophila somatic musculature provides a simplified system for studying muscle attachment site selection. Following muscle cell specification and myoblast fusion, multi-nucleated myotubes must select their appropriate attachment sites in the epidermis. This complex process involves myotubes searching their environment using actin-rich filopodia at their leading edge. At the same time, muscle attachment cells secrete guidance cues to direct muscles to their correct positions. We performed an EMS screen to identify genes required for muscle attachment site selection. Genetic mapping and sequencing of one of our mutant lines revealed a point mutation in the GAP domain of tumbleweed/RacGAP50C (tum). tum encodes a GTPase Activating Protein that functions with Pavarotti to coordinate the cytoskeleton and GTPase signaling during cytokinesis. Here we show that tum plays a previously uncharacterized role in attachment site selection of postmitotic myotubes. Tum is expressed in muscle fibers and their attachment cells. In embryos expressing the mutant form of the protein, we observe specific defects in muscle patterning. Although early mesodermal markers reveal that myoblast specification and fusion are normal, we observe that myotubes extend filopodia that are not restricted to the leading edges of the migrating fiber. Furthermore, these muscle fibers often form epidermal attachments at the wrong sites. We plan to further examine myotube dynamics in our mutants using live imaging techniques. Our data provides evidence that tum plays a role in coordinating guidance machinery in migrating myotubes and suggests that regulation of the cytoskeleton during cell division and cell migration share a common mechanism. POSTERS: Organogenesis and Gametogenesis 247

467B Live imaging reveals that myoblast fusion requires dynamic remodeling of the actin cytoskeleton by SCAR/WAVE and Arp2/3. Brian Richardson1, Mary Baylies1,2. 1) Program in Biochemistry, Cell and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY; 2) Developmental Biology Program, Sloan-Kettering Institute, New York, NY. Muscle formation in both Drosophila and mammals relies on the reiterative fusion of myoblasts. The molecular mechanisms underlying this fusion process, however, are not well understood. Using new methods for fixed and live imaging, we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. We have identified a dynamic F-actin accumulation (actin focus) that forms at the site of fusion prior to cytoplasmic mixing. Actin focus formation is dependent on molecules that modulate myoblast adhesion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components localize with and regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged actin foci that do not dissolve, consistent with the observed block in fusion. We show that Kette positively regulates SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and enlarged actin foci, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for actin focus dissolution. Our data provide insight to the roles and dynamics of the actin cytoskeleton and its regulators during myoblast fusion, leading to a revision of the existing model.

468C Expression and functional analysis of a novel Fusion Competent Myoblast specific GAL4 driver. Kate Rochlin1, Karen Beckett1, Hong Duan2, Hanh Nguyen2, Mary Baylies1. 1) Dept Developmental Biol, Sloan-Kettering Inst, New York, NY; 2) Dept of Medicine, Developmental and Molecular Biol, Albert Enstein School of Medicine, Bronx, NY. In the Drosophila embryo, fusion of two distinct cell types is essential for the formation of skeletal body wall muscle. In the current model, body wall muscles are seeded by Founder Cells (FCs), which contain all the information to direct the formation of a specific muscle with a particular size, shape, and orientation. Founder cells fuse to surrounding naïve Fusion Competent Myoblasts (FCMs) to achieve their final muscle size. However, research in Drosophila myogenesis has focused primarily on the role of fusion proteins in FCs due to a lack of reagents that manipulate gene expression specifically in the FCMs. Previous studies have identified enhancers responsible for the expression of Dmef2 in FCs and FCMs. Further dissection of an FCM-specific enhancer (I-E) identified a 55bp sequence ([C/D]*) that is necessary and sufficient to direct expression of a lacZ reporter gene in FCMs. To examine the role of fusion proteins in FCMs, we used this enhancer to generate the first GAL4 driver specifically expressed in FCMs. We have determined that this GAL4 driver causes expression in a subset of FCMs and subsequently, upon fusion, in the developing myotube from stage 14 onwards. Furthermore, we have demonstrated using our Dmef2-5x[C/D]*- GAL4 that overexpressing dominant negative Rac, which is known to block fusion when expressed throughout the mesoderm, causes a partial fusion block. This novel GAL4 driver will provide a useful and powerful tool to further understand the role of fusion genes in Drosophila myoblast fusion and muscle differentiation.

469A SNS and DUF mediate cell interactions in the development of the garland cell cluster in the Drosophila embryo. Shufei Zhuang, Huanjie Shao, Jeffrey McDermott, Susan Abmayr. Stowers Institute for Medical Research, Kansas City, MO. Two types of nephrocytes are present in the embryo, garland cells and pericardial cells. The bincleate garland cells are responsible for removal of waste material from the larval hemolymph, and have a rapid rate of fluid phase endocytosis. They localize to the anterior end of the proventriculus at its junction with the esophagus, in a structure called the subesophageal body. Although they are known to derive from the late secondary head mesoderm, their development is poorly understood. sns (stick-and-stones) and duf (dumbfounded) both encode members of Ig superfamily of cell adhesion molecules and are essential for myoblast fusion. Our data show that both of these proteins are expressed on the surface of the garland cells but that they are not highly co-localized. Promoter- reporter constructs for sns and duf exhibit overlapping patterns of expression suggesting that, in comparison to the musculature, sns and duf may be co-expressed in the same cells. The overall organization of the subesophageal body is perturbed in embryos mutant for either sns or a deficiency that removes both duf and rst. SNS is necessary for the integrity and adhesion of cells in the cluster, but cells remain tightly associated in the absence of duf/rst. Surprisingly, the majority of the garland cells are still binucleate in the absence of sns or duf. Studies are now in progress to characterize the early development of these cells, to determine the role of SNS and Duf/Rst, and to examine how they become binucleate. 248 POSTERS: Organogenesis and Gametogenesis

470B Structure and function of ring canals in somatic cells. Stephanie Airoldi, Lynn Cooley. Genetics, Yale University, New Haven, CT. Ring canals in the somatic cells of Drosophila are similar to germline ring canals in that they results from arrested cleavage furrows, and allow for direct contact of cytoplasm between bridged cells. EMs have revealed the presence of ring canals in the follicle cells and imaginal discs. These somatic ring canals contain actin, but little else is known about their structure or function. Previous reports have indicated that Anillin and Pavarotti-KLP localize to follicle cell ring canals. Ani and Pav-KLP localize to cleavage furrows, and retain their punctate localization in post-mitotic follicle cells. A protein trap in Visgun show localization to both female germline ring canals and puncta in post-mitotic follicle cells. Pav-KLP and Vsg also show localization to puncta in imaginal discs. Co-staining of Pav-KLP and Vsg with markers for septate junctions, gap junctions, and adherens junctions shows no colocalization. We have confirmed the localization of Pav-KLP and Vsg to follicle cell ring canals using ImmunoEM. Using Pav-KLP as a marker for ring canals, we can count the number of ring canals per follicle cell, and are gathering this data across many egg chamber stages. Because Ani and Pav-KLP have essential roles in the cleavage furrow, we are currently using RNAi to disrupt protein function post-mitotically. Concurrently, we are generating mutations in Vsg in order to study its function. A major question that remains to be answered is how many follicle cells are in a syncytium, and if that number is variable. We are using a FLP-out Gal4 construct to address this question. We have also noted that GFP traps in ribosomal proteins, as well as some Gal4 drivers expressed in follicle cells, exhibit mosaic patterns of expression. Incorporation of BrdU in post-mitotic cells also occurs in a mosaic fashion. We are currently investigating whether these mosaic staining patterns are all marking the same cells, and whether they are marking cells in a syncytium. Through these methods we hope to elucidate the structure and function of somatic ring canals.

471C Clonal analysis using a follicle cell dependent dominant female sterile mutation. Phoenix Bouchard Kerr, Aliaa Eleiche, Laura Nilson. Department of Biology, McGill University, Montréal, QC, Canada. Establishment of the body axes of the Drosophila embryo depends on maternally expressed genes, some of which function in the follicular epithelium of the developing egg chamber. Many such maternal-effect genes were identified in genetic screens for homozygous mutant females that produce abnormal embryos. However, this approach cannot identify patterning genes that also have lethal homozygous mutant phenotypes. This problem can be overcome by generating heterozygous females with mosaic ovaries containing homozygous mutant follicular epithelia, through Flp/FRT-mediated mitotic recombination. Definitive analysis of such mosaic females depends on the ability to distinguish which eggs and embryos are derived from mutant follicular epithelia. To achieve this goal, we have developed a novel genetic marker system using a transgene bearing a dominant negative form of an eggshell structural protein gene (decDN; gift of Gail Waring). Females with a single copy of decDN are sterile and lay collapsed eggs. Induction of mitotic recombination in females that are heterozygous for an FRT-decDN chromosome and an FRT chromosome bearing a mutation of interest results in clones of homozygous mutant follicle cells that lack the decDN transgene. Some of the eggs produced by these mosaic females do not collapse, and all non-collapsed eggs bear the expected mutant phenotype, confirming that they are the products of egg chambers with mutant follicle cell clones. Moreover, the non-collapsed eggs produced by females mosaic for mutant windbeutel or pipe alleles bear embryos that are completely dorsalized. This observation indicates that non-collapsed eggshells are the product of completely mutant follicular epithelia, since previous characterization of windbeutel and pipe mosaics has shown that smaller clones lead to localized dorsalization. This technique thus allows us to identify unambiguously eggs derived from egg chambers with homozygous mutant follicular epithelia, and will be useful for assessing the somatic requirement for known candidates or novel genes in eggshell and embryo patterning.

472A Studies on Tis11 in Drosophila melanogaster. Robert Fedic1, Perry J. Blackshear2, James M. Mason1. 1) Lab Molecular Genetics, NIEHS/NIH, Res Triangle Park, NC; 2) Laboratory of Neurobiology, NIEHS/NIH, Res Triangle Park, NC. The control of gene expression is an essential mechanism that allows a cell to respond rapidly to extracellular and intracellular changes. One crucial part of this process is regulation of mRNA stability. Actual mRNA levels in the cell are the result of mRNA turnover as well as synthesis. mRNA turnover is controlled by a complex network of cis and trans acting elements. One of the best studied examples are AU-rich elements (AREs) in the 3’ UTR region of certain mRNAs. TIS11 binds to these elements and targets mRNAs for rapid degradation through the exosome. Tis11 was discovered in mouse where it is a member of a family of four tandem zinc-finger proteins. Tis11 homologues are found in vertebrates as well as in invertebrates. While the homology is relatively high in vertebrates, in invertebrates homology is restricted to the tandem zinc finger region. The Drosophila Tis11 homologue is a single copy gene with four messages present throughout development, but their levels fluctuate. Using imprecise excision of P-element EP147 we created several deletions varying in length and exhibiting a spectrum of phenotypes, including embryonic and first instar larval death, third instar misshapen imaginal discs, and adult leg and wing defects. Using TIS11 tagged with eGFP we were able to follow time and space localization including missexpression driven by several wings and legs drivers. Overexpression using actin 5C and Tubulin drivers is embryonic or larval lethal, depending on the insertion site. We also raised antibody against TIS11 and performed immunostaining studies on eggs and larvae. Our data suggest a possible role of TIS11 in the regulation of early Drosophila development, and wing and leg imaginal disc morphogenesis. POSTERS: Organogenesis and Gametogenesis 249

473B Ecdysone Receptor and Ultraspiracle are Required for Chorion Gene Amplification. Jennifer Hackney, Leonard Dobens. Dept Molecular Biol & Biochem, Univ Missouri, Kansas City, Kansas City, MO. During late Drosophila oogenesis, the follicle cells (FC) coordinately subject the chorion protein loci to repeated, localized DNA replication. Chorion protein loci amplification affords a means to rapidly produce large amounts of chorion protein needed for eggshell production. Recently, we have shown that misexpression of a dominant negative version of the Ecdysone Receptor (EcR- DN) at stage 10B-14 blocks amplification of the chorion gene clusters, leading to reduced chorion protein gene expression and thin eggshell phenotypes. Here we demonstrate two separable requirements for EcR in chorion gene amplification. At stage 9-10A, both Flp-out misexpression of EcR-DN and mutant clones of a null allele in usp, which encodes the RXR partner of EcR, result in precocious amplification of the chorion genes, as measured by ectopic incoporation of BrdU. This indicates that EcR represses chorion gene amplification prior to stage 11. At later stages, usp null and EcR-DN clones do not incorporate BrdU, consistent with our previous observation using tissue-specific drivers that EcR is required for late chorion loci amplification. Using antisera, we show that EcR protein accumulates in a punctate pattern in the FC specifically in late stage egg chambers and several of these foci correspond with sites of BrdU incorporation. Consistent with a direct role in chorion gene amplification, ChIP analysis reveals that EcR binds at or near ACE3 (Amplification Control Element on 3), which is required for amplification to occur. Together, these data suggest that EcR/Usp bind to the chorion clusters to mediate a hormonal signal that coordinates gene amplification.

474C Making inroads into the Drosophila female reproductive system. Yael Heifetz1, Anat Kapelnikov1, Einat Zelinger1, Vidya Nagalakshmi1, Paul Mack2, Michael Bender3, Kahn Rhrissorrakrai4, Kristin C. Gunsalus4, Patricia Rivlin5, Ronald Hoy5. 1) Dept. of Entomology, The Hebrew Univ, Rehovot, Israel; 2) Dept. of Sciences and Mathematics, Mississippi Univ. for Women, Columbus, MS, USA; 3) Dept. of Genetics, Georgia Univ., Athens, GA, USA; 4) Center for Comparative Functional Genomics, Dept. of Biology, New York Univ., NY, USA; 5) Dept. of Neurobiology and Behavior, Cornell Univ., Ithaca, NY, USA. Reproduction involves interaction between males and females on several levels. At the cellular and molecular levels, male-derived molecules and sperm interact with female reproductive cells and molecules, culminating in the union of male and female gametes. Studies of fertilization across species suggest that mating sensitizes the female reproductive tract (RT) at the cellular and molecular level to ensure a successful reproductive event. We hypothesize that mating triggers the final maturation of the female RT, switching it from an unmated to mated physiological state. To uncover the mechanisms that underlie mating-induced maturation, we conducted comparative microarray, proteomic, and morphological studies to characterize the RT of unmated and mated Drosophila females. We subdivided the RT into two functionally different regions: the upper-RT, consisting of oviducts and the lower-RT consisting of sperm storage organs and accessory glands. We found that mating elicits distinct molecular changes in the female RT as it transitions from an unmated to a mated physiological state. Each region of the RT appears to bear a unique molecular signature that reflects its specialized function. Our combined transcriptional and proteomic profiling show that some post-mating changes in expression may be mediated by translation initiation or by post-translational mechanisms. Moreover, mating induced distinct morphological changes in the epithelium, musculature, and innervation of the RT. Together, our results suggest that mating triggers molecular changes and active tissue remodeling in the female RT that mediate its progression to a mature developmental stage.

475A Juvenile hormone controls entry to metamorphosis through Methoprene-tolerant and Broad-complex. Marek Jindra, Barbora Konopova. Biology Center ASCR, Ceske Budejovice, Czech Republic. Insect metamorphosis is an elaborate change between larval, pupal and adult forms, and a prime example of how development is regulated by hormones. Growth and differentiation, tissue remodeling and death are orchestrated by the morphogenesis-promoting ecdysteroids and the morphostatic juvenile hormone (JH), whose presence precludes metamorphosis. How tissues interpret this combinatorial effect of the two hormonal signals is poorly understood, mainly because the JH receptor is unknown. Genetic studies of JH regulation of metamorphosis are hindered by the lack of a robust effect of JH on Drosophila: unlike in other insects JH cannot prevent larval-pupal transition or induce extra larval instars in the fly. We therefore chose to study metamorphosis in the flour beetle, Tribolium castaneum, which is both amenable to systemic RNAi and sensitive to the typical anti-metamorphic JH effect. Here, we show that JH controls the entry of beetle larvae to metamorphosis through the ortholog of the Drosophila PAS domain protein Methoprene-tolerant (Met). Loss of Met has been known to cause resistance to JH but no developmental defects in Drosophila. In contrast, Tribolium larvae deficient in Met function enter the pupal program prematurely, before reaching their final instar. This finding defines Met as an essential component of the anti-metamorphic JH signaling, encouraging the idea that Met might be the missing JH receptor. Next, we examine the role of the beetle ortholog of Broad-Complex (BR-C), known to be essential for Drosophila metamorphosis. We show that BR-C is necessary for differentiation of pupal characters and for temporal coordination of the metamorphic changes in Tribolium. Importantly, we demonstrate that regulation of BR-C expression requires Met, because (i) BR- C is expressed upon Met RNAi in precocious prepupae, and (ii) ectopic JH induces BR-C in normal beetle pupae but not in pupae deficient in Met. Thus Met and its downstream target BR-C play a critical role in JH regulation of insect metamorphosis. 250 POSTERS: Organogenesis and Gametogenesis

476B Oviduct under construction: Post-mating changes in Drosophila reproductive tract. Anat Kapelnikov1, Patricia Rivlin2, Ronald Hoy2, Yael Heifetz1. 1) Dept. of Entomology, The Hebrew Univ., Rehovot, Israel; 2) Dept. of Neurobiology and Behavior, Cornell Univ., Ithaca, NY, USA. Mating induces molecular and morphological changes in the female reproductive tract. To understand the mechanisms that underlie these changes, we used microarray, proteomic, and comparative morphological analyses. We find that morphological changes in innervation, muscle and epithelium, as well as region-specific differences in cytoskeleton-related genes are distinguishable as early as 6 hours after mating. For example, proteins involved in epithelial cell polarization and communication, such as alpha and beta spectrin, and alpha actinin are highly up-regulated in the oviduct post-mating, but show no change or are down-regulated in the lower reproductive tract. At the electron microscopic level, we observed changes in the oviduct that are likely mediated by these molecules, such as increased differentiation of cellular junctions and remodeling of apical and basolateral membranes. Our results suggest that mating is essential for the final maturation stage of the reproductive system, switching it to a functional “mated state”. These findings lead us to hypothesize that a sensitizing event occurs during the first mating and its effect is fixed cellularly and molecularly, and that later mating events enhance processes that normally decline with time post-mating. To determine if this sensitizing event occurs only during the first mating we focused on cytoskeleton-related proteins that were highly up-regulated post- mating in the oviduct and tested the effect of different mating regimens (i.e. unmated, once mated, twice mated) on their expression level. We observed differences in protein expression level between unmated, once-mated, and twice-mated females. To determine the physiological significance of these protein differences, we examined the effect of different mating regimens and silencing of these cytoskeleton-related-genes on female fertility. Our results suggest an efficient and specific developmental program necessary for reproduction.

477C A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles. Stuart Newfeld1, Denis Bulanin2, Aaron Johnson1, Teresa Orenic2, Norma Takaesu1. 1) Sch Life Sci, Arizona State Univ, Tempe, AZ; 2) Dept Bio, Univ Illinois, Chicago. A previous genetic analysis of a reporter gene carrying a 375bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Here we report that within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first with the ability to integrate temporally distinct positive (TCF and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, our results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung.

478A Antagonistic role of Notch and Ecdysone signaling in regulating the endocycle/gene amplification switch in Drosophila follicle cells. Jianjun Sun, Alexander Armento, Wu-Min Deng. Dept Biological Sci, Florida State Univ, Tallahassee, FL. The cell-cycle programs must be precisely controlled to ensure proper development of multi-cellular organisms. An excellent model system for study of the regulation of cell-cycle programs is the epithelial follicle cells , which go through three rounds of endocycle to produce 16 copies of the genomic DNA content and then switch to synchronous chorion-gene amplification to meet the dramatic demand for chorion products for rapid eggshell formation during late oogenesis. No developmental cues, however, have been reported to regulate this endocycle/amplification (E/A) switch. In the work presented here, we found that Notch signaling was downregulated in follicle cells during the E/A transition, and this downregulation was important for follicle cells to leave the endocycle and enter the chorion-gene-amplification stage. Misexpression of an active form of Notch (Notch intracellular domain, NICD) kept the cells in the endocycle, as revealed by BrdU incorporation, ORC2 and E2F1 staining, and FACS analysis of the genomic DNA content. We also found that activation of the Ecdysone receptor (EcR) pathway at around stage 10 was required for the E/A switch and that ectopic Notch activity could suppress ECR activity. Furthermore, we found that the zinc-finger protein Tramtrack (Ttk) at stage 10 acted downstream of ECR and Notch in regulation of the E/A switch. Removal of ttk function in follicle cells kept the cells in the endocycle even after stage 10B, as happened in the NICD-misexpressing cells. Overexpression of Ttk before stage 10 was sufficient to stop DNA replication. In addition, Ttk overexpression alleviated the endocycle-exit defect caused by ectopic expression of NICD after stage 10B. Our results reveal a developmental pathway that includes downregulation of Notch activity, activation of the EcR pathway, and upregulation of Ttk to execute the E/A switch. Our data illustrate, for the first time, the antagonistic interaction between Notch and Ecdysone signaling in the regulation of cell-cycle programs and differentiation. POSTERS: Organogenesis and Gametogenesis 251

479B Identification of Genes Required for Adult Muscle Development in Drosophila. Chrisna V Thomas, Richard Cripps. Biology, University of New Mexico, Albuquerque, NM. Identifying genes necessary for skeletal muscle development in fruit flies is important because similar genes are required for muscle development in humans. Recent research has focused on identifying these genes required for adult myogenesis in Drosophila. Genes critical to muscle formation are Myocyte-enhancer factor-2 (Mef-2), twist (twi), and Broad complex (BR-C). These genes act as transcription factors in the muscle formation pathway and are expressed during the development of the dorsal longitudinal indirect flight muscles (DLMs). Twist and BR-C act together to regulate adult myogenesis by activating Mef-2, so we tested what happens when we reduced the expression of two or more of these factors. The goal of our research is to develop multiple mutant flies that will be crossed with deficiencies around the fly genome and then analyzed for muscle patterning. When compared to wildtype (wt), we found that the muscle patterning in the mef-2 twi double mutant heterozygotes is not greatly affected (6.0 pairs DLMs in wt vs. 5.0 pairs DLMs in double mutant heterozygotes). The mef-2 twi BR-C triple mutant heterozygotes however, have reproducibly fewer muscles (4.0 pairs DLMs). This result indicated that the dosage of myogenic genes is indeed important for normal adult myogenesis. To test whether other genes are involved in the muscle differentiation pathway, we performed a screen using a deficiency kit on the third chromosome to look for a more exacerbated phenotype. We found that three deficiency lines enhanced the phenotype from the double mutant heterozygotes suggesting the presence of three new genes which contribute to the normal muscle patterning in adult flies.

480C Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation. Tina L Tootle, Allan C Spralding. Embryology, Carnegie Institution, Baltimore, MD. Prostaglandins are local, transient hormones that mediate a wide variety of biological events, including reproduction. The study of prostaglandin biology in a genetically tractable invertebrate model organism has been limited by the lack of clearly identified prostaglandin-mediated biological processes and prostaglandin metabolic genes, particularly analogs of cyclooxygenase (COX), the rate-limiting step in vertebrate prostaglandin synthesis. Here, we present pharmacological data that Drosophila ovarian follicle maturation requires COX-like activity and genetic evidence that this activity is supplied in vivo by the Drosophila peroxidase Pxt. pxt mutants act early to alter follicle production and block multiple actin-dependent steps in follicle maturation, including nurse cell dumping, border cell migration, follicle elongation and dorsal appendage formation, ultimately resulting in sterility in the severest case. Maturation of pxt follicles in vitro is stimulated by prostaglandin treatment and fertility is restored in vivo to pxt mutants by expressing mammalian COX1 protein. Our experiments suggest that prostaglandins promote Drosophila follicle maturation by modulating the actin cytoskeleton and establish Drosophila oogenesis as a model for understanding these critical biological regulators.

481A Determining the Role of 18-Wheeler in Morphogenesis of the Ovarian Follicle Cell Epithelium. Erika Vielmas, Crissy Danusastro, Elizabeth D. Eldon. Biological Sciences, California State University Long Beach, Long Beach, CA. In Drosophila melanogaster, the follicle cell epithelium (FCE) of the ovary is a great structure to study epithelial morphogenesis. The epithelial cells provide signaling components that determine embryonic axes and secrete the eggshell. Epidermal growth factor receptor (EGFR), decapentaplegic (Dpp), Broad Complex/EcR, and Notch signaling pathways all act to specify cell fate and migration in the FCE during oogenesis. We are studying 18-wheeler (18w), which is a Toll-like receptor that is expressed in the FCE. 18w mutant follicle cells fail to migrate properly during oogenesis, leading to eggshell patterning defects. Our goal is to determine the molecular mechanism underlying 18w’s activity in epithelial migration and morphogenesis. We are using immunofluoresence and confocal microscopy to investigate whether the epidermal growth factor receptor (EGFR) pathway, Dpp pathway, or Broad Complex/ Ecdysone signaling interact with 18w. Recently published work demonstrated a role for 18w in salivary gland invagination, and identified a genetic interaction between 18w and Rho-GTPase signaling. We are currently testing whether 18w-Rho-GTPase interactions also play a role in FCE morphogenesis. This work is supported by NIH-RISE 5R25GM071638. 252 POSTERS: Organogenesis and Gametogenesis

482B The Rho-GTPase Cdc42 is required for Drosophila heart morphogenesis. Georg Vogler1, Li Qian2, Jiandong Liu3, Rolf Bodmer1. 1) Burnham Institute for Medical Research, San Diego, CA; 2) Gladstone Institute of Cardiovascular Disease, San Francisco, CA; 3) University of California, San Francisco, CA. The morphogenesis of the Drosophila heart is a multiple step process which begins with the specification of myocardial and pericardial cells. Later on, cells of both types migrate towards the dorsal midline where the adjacent rows of myocardioblasts of both hemisegments align to form a tube. During this process myocardioblasts undergo changes in cell shape and establish cell-cell contacts in a highly orchestrated manner. Within these cells, a number of molecules are relocalized with respect to polarity which is necessary for proper heart cell alignment. For example, the epithelial polarity marker Discs-large is localized baso-laterally before and apico-laterally after myocardial alignment. Similarly, the transmembrane receptor Toll also becomes apico-laterally localized during later stages in morphogenesis. How this change in localization is achieved is unknown. The small Rho-GTPase Cdc42 is a key regulator of cell polarity and actin filament dynamics in several developmental contexts and our preliminary data suggests a role for Cdc42 during multiple steps of heart morphogenesis. Heart-specific overexpression of dominant-negative and constitutively active Cdc42 during heart morphogenesis disturbs cell polarity and alignment of myocardioblasts in distinct ways, which indicates a role for Cdc42 in this process. In addition, Cdc42 function is also required for adult heart function and interacts genetically with the homeobox transcription factor Tinman. We are currently analyzing these distinct roles of Cdc42 during different steps of cardiogenesis.

483C The variable nurse cells gene encodes Ard1, an N-terminal acetyltransferase subunit implicated in sister chromatid cohesion and cell cycle progression. Ying Wang, Tolga Turan, Michelle Mijares, Megan Gall, Kevin Manage, Anna Javier, Rahul Warrior. Dept of Developmental & Cell Biol, Univ California, Irvine, Irvine, CA. During Drosophila oogenesis, wildtype egg chambers invariably consist of an oocyte and 15 nurse cells that are of germline origin. These germline cells arise from a single precursor cystoblast that undergoes four mitotic divisions with incomplete cytokinesis. Mutations that affect levels of cyclin/Cdk activity result in extra rounds of division and egg chambers with excess nurse cells while mutations reducing activity of components of the fusome or the cytoplasmic dynein complex results in egg chambers with abnormally few germline cells. Mutations in the variable nurse cells (vnc) gene were first isolated almost 30 years ago, in the course of a screen for female sterile mutations that mapped to the 67 A-E region of chromosome 3 (Rees, 1990). Homozygous vnc females are sterile and produce egg chambers with too few, normal numbers, or too many nurse cells. We have used high resolution recombinational mapping, imprecise P-element excision and direct sequencing of mutant alleles to determine that vnc encodes arrest defective1 (ard1). Yeast Ard1p is the catalytic subunit of NatA holoenzyme, an N-Acetyltransferase complex. In Drosophila, disruption of 2 different acetyltransferases, Separation anxiety (San) and Drosophila eco1 (Deco), induces precocious sister chromatid separation, and spindle checkpoint activation, resulting in mitotic delay or arrest (Williams et al, 2003). The San protein has been shown to associate with Nat1 and Ard1. Taken together these data suggest that vnc/ard1 is likely to play a role in sister chromatid cohesion and the germline phenotype may result from defects in mitotic progression in germline cells.

484A Arginine methylation of SmB is needed for its localization in the pole plasm and formation of germ cells. Joel Anne, Bernard M. Mechler. Dept Developmental Genetics, DKFZ, Heidelberg, Germany. Sm proteins, such as SmB, are components of the small nuclear ribonucleoprotein particles and are methylated by the Capsuléen (Csul) arginine methyl-transferase. However, the biological relevance of Sm arginine methylation remains to be determined. For this purpose we first used a GFP-SmB+ transgene and determined the distribution of the fusion protein during oogenesis. GFP-SmB was detected in nurse cell nuclei and at the posterior pole plasm of the growing oocyte. The relevance of the methylation for the subcellular localization was then investigated by examining GFP-SmB distribution in csul or valois egg chambers, in which no arginine methylation can be immuno-detected. Although GFP-SmB was found in nurse cell nuclei of the mutant egg chambers, no GFP-SmB could be found at the posterior pole of the oocyte, indicating that csul-mediated arginine methylation is needed for SmB positioning in pole plasm, but not for nuclear localization. To ascertain the role of the arginine residues in pole plasm targeting we generated a GFP-SmB7xRL transgene in which seven potentially methylated arginine residues in RG motifs at the C-terminal tail of SmB were substituted by leucine residues. Expression of this transgene during oogenesis resulted in the absence of GFP-SmB7xRL at the posterior pole of the oocyte. To determine the phenotype resulting from the leucine substitution we expressed UAS::SmB+ and UAS::SmB7xRL transgenes under the control of a tub::Gal4 driver in SmB mutants. Under these conditions SmB+ expression could fully restore the development of the mutant animals and their fertility, whereas SmB7xRL expression gave rise to agametic animals. In conclusion our analysis shows that arginine methylation of SmB is required for SmB localization in the pole plasm and needed for the maintenance of Drosophila primordial germ cells. POSTERS: Organogenesis and Gametogenesis 253

485B The Regulation of Germ Cell Sex Determination in Drosophila. Abbie Casper, Mark Van Doren. Dept Biol, Johns Hopkins Univ, Baltimore, MD. The establishment of sexual identity is critical for the formation of male or female gametes and the continuation of a species. Previous work has shown that the decision to develop as male or female in the germline is regulated by non-autonomous signals from the somatic gonad, and by germ cell autonomous cues regulated by germ cell sex chromosome constitution. However, little is known about how and when these signals interact to establish and maintain germ cell sexual identity. To identify genes involved in germline sex determination and development, a molecular screen was conducted looking for genes expressed sex specifically in the embryonic germ cells. From this screen, eight genes were found to be expressed sex-specifically in the male germ line. Sex-specific expression of some of these genes begins as early as stage 15 of embryogenesis; suggesting that germ cell sexual identity is being established during this time. We are using these genes as indicators of germ cell sexual identity to investigate how somatic signals and germ cell autonomous cues control germ cell sex determination. We have shown that the male soma can turn on male-specific gene expression in XX germ cells and the female soma can repress male-specific gene expression in XY germ cells (using loss of function and gain of function of transformer). We also see that germ cells outside the somatic gonad autonomously know there sex chromosome constitution (using serpent mutants). Lastly, we are investigating the role of these genes in germ cell development. Viable mutant alleles of one gene, no child left behind (nclb), affect fertility in both sexes and males show a severe depletion of germline stem cells. The homolog of nclb in yeast, PWP1, is involved in chromatin regulation. We are investigating how nclb may control the chromatin state in Drosophila to maintain male germline stem cells. In conclusion, we are learning more about how and when initial germ cell sexual identity is established, as well as how germline sexual development is regulated at the molecular level.

486C DSXM and DSXF expression in subsets of Drosophila somatic gonad cells. Leonie Hempel, Brian Oliver. LCDB/NIDDK/NIH, Bethesda, MD. In Drosophila melanogaster a pre-mRNA splicing hierarchy controls sexual identity and leads to transcription of sex-specific Doublesex (DSX) transcriptionfactor isoforms. While male specific DSXM activates genes involved in male development and represses genes involved in female development, female specific DSXF acts in the opposite way. We generated an antibody specific for DSXM as well as an antibody recognizing both sex-specific DSX isoforms. Through immunofluorescence studies with sex sorted embryos we show that DSXM is specifically expressed in subsets of male somatic gonad cells that are closely associated with the germ cells during embryogenesis as early as stage 13. Following testis formation during embryogenesis, germ cells remain in contact with DSXM-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes. We also show that DSXF is expressed in subsets of female somatic gonad cells that are closely associated with the germ cells during embryogenesis just as seen in males. Thus, our results suggest that each somatic gonad cell of an embryo establishes its sexual identity before overt sexual development of the gonads is morphologically evident in stage 17. Furthermore, our results indicate that dsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The sex of the soma influences the sex of the germ cells. DSX expressing somatic gonad cells are in intimate contact with germcells. This contact may enable the somatic sex determination pathway to influence germ cell development in the embryonic gonad. To discover DSX target genes in embryos we are performing chromatin immunoprecipitation in combination with microarray technology. Further, we are also examining global gene expression at different stages of male and female embryonic development using microarrays. Our initial results show sexual dimorphic expression beginning with 10-16 hour old embryos correlating with the onset of embryonic DSX expression.

487A Mjl expression depends on somatic and germline signals. Rasika Kalamegham, Brian Oliver. LCDB, NIDDK/NIH, Bethesda, MD. We have previously reported on mojoless (mjl), a gene required for male fertility in Drosophila. Mjl is exclusively expressed within the male germline. Depletion of Mjl through RNA interference (RNAi) leads to male sterility due to loss of germ cells (Kalamegham et al., 2007). We examined expression of Mjl in the germline of flies with sex-transformed somas to determine if Mjl expression was germline autonomous or directed by somatic cues. Mjl is expressed in germcells of XX flies with a soma transformed from female to male (tra2B/Df(2R)trix and dsxswe/Df(3R)dsxM+15 mutants). Interestingly, Mjl is also expressed in germcells of XX otu (ct otu1 v 24/ y wa otu 17) as well as XX Sxl (y cm Sxl 7BO/ y Sxl fs3) mutant flies. otu and Sxl are components of the female germline sex determination pathway and otu acts upstream of Sxl. In XY germcells Sxl is not expressed whereas Mjl is. Mutations that affect Sxl expression appear to be capable of inducing Mjl expression even in XX germcells suggesting that Mjl may be repressed by Sxl expression in wild-type XX germcells. We also examined EMS mutant alleles of mjl generated by the Seattle tilling project. Four missense mutations lead to male sterility when tested over a deficiency that lacks the mjl locus out of a total of nine mutations that were tested. All four mutations leading to sterility are within the canonical kinase domain of the protein and one of the four is within the activation loop. We are pursuing more detailed analyses of these EMS induced mutations. Reference: Kalamegham, R., et al., 2007. Drosophila mojoless, a retroposed GSK-3, has functionally diverged to acquire an essential role in male fertility. Mol Biol Evol. 24, 732-42. 254 POSTERS: Organogenesis and Gametogenesis

488B JAK/STAT regulation of germ cell sex determination. Gretchen H. McConnell, Carla M. Wood, Mattew J. Wawersik. Biology, The College of William and Mary, Williamsburg, VA. Germ cells are the only cells in the body that develop into sperm or eggs. Correct germ cell sex determination is essential for healthy gonads and fertility. In Drosophila, this process is regulated germ cell autonomously and by signals from somatic cells in the somatic gonad. Within the germline, genes such as ovarian tumor (otu), sans fille (snf) and ovo promote female development, and loss of function of these genes results in ovarian germ cell tumor formation. Additionally, initiation of male germ line development is controlled by activation of the JAK/STAT signaling pathway from the male somatic gonad. However, the extent to which the JAK/ STAT pathway controls germ cell sex has not yet been determined. Ectopic expression of the JAK/STAT activating ligand upd in adult Drosophila leads to unrestrained proliferation of germ cells in ovaries. This phenotype is similar to tumors formed due to loss of ovo, otu, and snf gene function; suggesting that the JAK/STAT pathway is sufficient to masculinized genetically female (XX) germ cells. To directly test whether these tumors are in fact masculinized, we are examining whether they express spermatogenic markers including the LacZ enhancer trap M5-4 and escargot-GFP. We are also testing for genetic interactions between the JAK/STAT pathway and genes that regulate female germ cell sex. Finally, to elucidate additional mechanisms that regulate germ cell sex, we have designed a gain of function screen for signaling pathways that regulate initial germline sexual dimorphism. Data from our initial analysis of candidate pathways will be presented.

489C Identification of targets of the germline sex determination pathway. John Smith, Brian Oliver. Lab. of Cellular & Developmental Biology, National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda, MD. Germ cells integrate their cell-autonomous karyotypic information with sex-specific signals from the soma. Within a female soma 1X germ cells or 2X germ cells mutant for germline sex determination genes produce a phenotype which has historically been called ovarian tumors — many germ cells are found with a follicle or extended germarium as single cells or small clusters of interconnected cells similar to germline stem cells, cystoblasts or cystocytes. Some germ cells in these ovarian tumors also exhibit characteristics of male germ cells such as expression of male germ cell reporters, male-specific splicing and primary spermatocyte morphology. Through microarray analysis of gonads from otu1/otu17, Sxlfs3/SxlfP7B0 or sex-transformed 1X hs-tra flies we have identified a number of genes up- or down-regulated in ovarian tumors relative to ovaries. Some of the up-regulated genes reflect a more testis-like gene expression profile. Up-regulated genes could also include those normally repressed by the germline sex determination pathway or genes expressed in immature mitotically-active germ cells. Down-regulated genes could include those normally activated by the germline sex determination pathway or even direct mRNA targets of Sxl-binding.

490A The octopamine receptor OAMB regulates ovulation through Ca2+/Calmodulin-dependent protein kinase II. Hyun-Gwan Lee1, Kyung-An Han1,2. 1) The Huck Institute Genetics Graduate Program; 2) Department of Biology, Pennsylvania State University, University Park, PA 16802. The monoamine octopamine is a major neuromodulator in invertebrates. Octopamine neurons in the abdominal ganglion innervate the female reproductive system and plays crucial roles in fertility. However, the underlying mechanism is unclear. We identified two octopamine receptors, OAMB-K3 and OAMB-AS, which are produced by alternative splicing of oamb transcripts and have distinct capacities to activate cAMP and intracellular calcium increases. Both receptors are expressed in the central nervous system and the reproductive system. The females lacking both isoforms are impaired in ovulation. Here, we investigated the tissue type(s) and intracellular effectors that OAMB mediates ovulation. The transgenic oamb females with ubiquitous expression of either OAMB-K3 or -AS only at the adult stage were fecund, indicating that OAMB plays a physiological, as opposed to developmental, role in ovulation. When OAMB expression was targeted in the nervous system, the transgenic oamb females remained infertile; thus, neural OAMB expression is insufficient or dispensable for ovulation. To address whether the OAMB’s functional site is the reproductive system, we generated the enhancer GAL4 line oambRS-GAL4, in which GAL4 was specifically expressed in the reproductive system. In particular, strong GAL4 expression was detected in the oviduct epithelial cells where endogenous OAMB is normally found. Notably, the oamb females with transgenic OAMB-K3 or -AS expression driven by oambRS-GAL4 were fully fertile, suggesting that OAMB is required in the oviduct epithelial cells for ovulation. Moreover, constitutively active Ca2+/Calmodulin-dependent protein kinase II (CaMKII) expressed in the adult oviduct epithelial cells rescued the ovulation defect of oamb females whereas CaMKII inhibitor peptides caused sterility in the oamb heterozygous background. Therefore, CaMKII is a major downstream signaling molecule of OAMB for regulating ovulation. POSTERS: Organogenesis and Gametogenesis 255

491B Proteomics of Drosophila reproductive system 1: Profiling of Drosophila seminal fluid proteins by two-dimensional gel electrophoresis. Nobuaki Takemori1, Masaki Yamada2, Takashi Ohsako1, Masa-Toshi Yamamoto1. 1) Drosophila Genetic Resource Center, Kyoto Institute of Technology, Kyoto, JAPAN; 2) Life Science Laboratory, Shimadzu Corporation 1, Kyoto, JAPAN. Seminal fluid of Drosophila plays important roles in the reproductive processes. To determine protein components of the seminal fluid, we conducted a proteomic analysis of Drosophila male reproductive organs (accessory gland, ejaculatory duct, and ejaculatory bulb) using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. After dissection of the reproductive tissues from pre-/post-mating males (Canton-S), each tissue was homogenized in lysis solution containing urea, CHAPS and β-mercaptoethanol. The extracted proteins were separated by two-dimensional gel, stained with Coomassie Blue, and identified by peptide mass fingerprinting using MALDI time-of-flight mass spectrometer. Protein sequences and post-translational protein modifications were analyzed by MALDI quadrupole-ion-trap time-of-flight mass spectrometer. To date, we displayed ~2000 protein spots on our gels, establishing the two-dimensional protein reference map of Drosophila male reproductive tissues. After identification of protein spots, we have collected bioinformatics of their biological/physiological functions using FlyBase. Seminal fluid proteins in the male reproductive tissues were explored by comparing two-dimensional gel images. Our comparative proteomic analyses identified several novel seminal fluid proteins expressed differentially in accessory gland, ejaculatory duct, and ejaculatory bulb. Processing of seminal fluid proteins (e.g., Acp36DE) during mating have also been detected on our gels. Our comparative proteomic analysis would provide valuable information toward comprehensive understanding of the nature of seminal fluid proteins in Drosophila reproductive system.

492C Mat89Bb is required for proper coupling of centrosomes/basal body to the nucleus during spermatogenesis. Michael Anderson, Laura Lee. Dept Cell & Developmental Biol, Vanderbilt Univ, Nashville, TN. Mat89Bb is a novel, maternally expressed gene that is also present at low levels in the male germline of Drosophila. We have previously shown that knockdown of Mat89Bb gene function in Drosophila embryos results in a block in nuclear division, and knockdown of the Mat89Bb homolog in Xenopus embryos by morpholinos similarly results in both cell-cycle defects and developmental arrest. Furthermore, we have shown that human Mat89Bb is required for cell-cycle regulation in cultured cells. These findings have revealed that Mat89Bb plays critical, conserved roles in regulating both the cell cycle and development. Our recent analysis of a Mat89Bb mutant (Mat89Bbf02815) has revealed that Mat89Bb also plays a crucial role during Drosophila spermatogenesis. Mat89Bbf02815 spermatids contain a variable number of irregularly sized nuclei associated with a single large nebenkern body (a mitochondrial aggregate) indicative of defects in both cytokinesis and chromosome segregation during meiosis. During meiosis in wild-type Drosophila spermatocytes, centrosomes migrate from the cell membrane to the nucleus during prophase of meiosis I. Once they have attached, they migrate to opposite poles of the nucleus and organize the formation of the meiotic spindle. In Mat89Bbf02815 mutant spermatocytes, however, the centrosomes fail to migrate to and/or maintain their attachment to the nucleus and spindle. Furthermore, while the centrosomes are able to organize meiotic spindles, the spindles are often monoastral and chromosomes often fail to properly segregate. In postmeiotic Mat89Bb spermatids, normal coupling between the basal body (a derivative of the centriole) and the nucleus is not observed. Our preliminary data indicate that these defects are a consequence of a loss of proper Dynein localization during spermatogenesis in Mat89Bb mutants. These findings support a model in which Mat89Bb is required for regulating centrosome/ basal body behavior during spermatogenesis.

493A Translational control of boule in spermatogenesis. Catherine Baker, Margaret Fuller. Developmental Biology, Stanford University School of Medicine, Stanford, CA. In Drosophila spermatogenesis, entry into the meiotic divisions is coordinated with the transcription program in spermatocytes, so that male germ cells in meiotic G2 prophase do not undergo division until the mRNAs needed for terminal differentiation have been transcribed. This cross-regulatory pathway connects the transcription program (mediated by the testis TAFs, or tTAFs) with the core cell cycle machinery via a cascade of translational control. Translation of the Twine/Cdc25 phosphatase, essential for meiotic division, requires the Boule RNA-binding protein. The boule mRNA, in turn, is translationally repressed in the absence of the tTAFs. Thus tTAF activity and the initiation of the transcription program allow boule translation, then twine translation and meiotic division. To investigate the mechanism underlying positive and negative translational regulation of boule, we created GFP and Boule-HA reporter lines. Expression data from these transgenes indicated that both the 5’ and 3’ untranslated regions (UTRs) of boule were required for translational repression of boule in the absence of the tTAFs. In addition, when the tTAFs were present, sequences in the boule coding sequence were needed for release of UTR-mediated translational repression. Experiments are underway to fine- map the positive and negative cis-acting sequences, and to identify putative trans-acting factors. 256 POSTERS: Organogenesis and Gametogenesis

494B yuri gagarin is required for actin, tubulin and basal body functions in spermatogenesis. Kathleen M. Beckingham, Michael J. Texada, Ravi P. Munjaal, Rebecca A. Simonette, Cassidy B. Johnson, William J. Deery. Biochemistry and Cell Biology, Rice University, Houston, TX. Males of the Genus Drosophila produce sperm of remarkable length. In D. melanogaster, the cell biological processes underlying the elongation and individualization of the giant sperm have been investigated. Specialized functions of both the Actin and Microtubule cytoskeleton are known to play key roles. The gene yuri gagarin, which encodes a novel protein, was previously identified through its role in gravitaxis. A new, male sterile, mutation of yuri has revealed roles for Yuri involving the Actin and Tubulin structures found in late spermatogenesis. Yuri protein is a component of the investment cones, motile fibrillar Actin structures that individualize the syncytial spermatids, and is essential for their formation. Further, Yuri is concentrated on the sperm nuclei in the “dense complex”, a microtubule-rich structure thought to strengthen the nuclei during their dramatic elongation. In the yuri mutant, the nuclei are deformed and disorganized. As a component of the dense complex, Yuri also has a role in positioning the basal body and its associated centriolar adjunct (which contains gamma tubulin) on the sperm nuclei. In the yuri male sterile mutant, the centriolar adjunct is detached and axonemal chirality and structure are affected.

495C Studying of the expression and protein-protein interactions of Stellate protein in testes of Drosophila melanogaster. Ksenia Egorova, Ludmila Olenina, Oxana Olenkina, Vladimir Gvozdev. Department of Molecular Genetics of Cell, Institute of Molecular Genetics RAS, Moscow, Russian Federation. The crystal-Stellate system is one of the best-studied examples of interactions between euchromatine and heterochromatine loci in D. melanogaster. Heterochromatic locus crystal represses the expression of tandem repeats Stellate genes via RNA-interference mechanisms. In the absence of crystal locus high-level expression of Stellate occurring in germinal tissues causes the accumulation of needle- or stellate-like crystals in testes of D. melanogaster and also meiotic abnormalities which leads to partial or total male sterility. The pathogenesis mechanism of Stellate protein acting is still poorly known. The main task of our studies is the search and identification of the putative partners of Stellate protein in testes of D. melanogaster to understand the mechanism of Stellate- induced pathogenesis. To this purpose we have obtained polyclonal anti-Stellate antibodies in mice. Using the methods of subcellular fractionation we have shown that soluble Stellate protein is nearly absent in cytoplasmic fraction; the major part of it can be found in the nuclear fraction. The following confirmation of the nuclear localization of Stellate protein was received using the double immunofluorescent staining of squash-preparations of testes of D. melanogaster, line cry1. We have detected crystalline Stellate in cytoplasm and no Stellate crystals, but disperse stained zones of Stellate associated with chromatine regions in nuclei. We have also performed the immunoprecipitation of soluble Stellate from the lysate from testes of D. melanogaster, line cry1, and revealed the main putative partner of Stellate.

496A Characterization of the hypomorphic male sterile nmdry4 allele and analysis of the nmd paralog CG4701’s putative role in spermatid mitochondrial shaping. Bevin C. English, Sarah D. Durnbaugh, Kara M. Koehrn, Sara H. Holmberg, Sheena E. Favors, Karen G. Hales. Department of Biology, Davidson College, Davidson, NC. During Drosophila spermatogenesis, mitochondria undergo dramatic morphogenesis, aligning along the meiotic spindle, aggregating near the nucleus, fusing into a structure called the Nebenkern, and elongating along the developing flagellar axoneme. A hypomorphic allele of the essential gene nmd is associated with defective mitochondrial aggregation in developing sperm cells and results from insertion of P element in the 5’ UTR of the gene. Inconclusive results from immunofluoresence and immunoblotting experiments with anti-Nmd polyclonal antibodies led to the hypothesis that a cryptic promoter in the P element may be driving gene expression in a subset of cells. Preliminary RT-PCR results suggest that there is no cryptic promoter. To determine the subcellular location of both Nmd and the product of its uncharacterized paralog CG4701, we are generating transgenic flies carrying GFP-tagged versions of each gene. To determine the molecular function of CG4701, whose expression is highly enriched in the testis based on EST and microarray data, we performed a chemical mutagenesis screen in the paralog’s genomic region. Deficiency mapping suggested that two allelic male sterile mutants with mitochondrial and cytokinetic defects may represent CG4701 mutants. After comparing CG4701 sequences from both mutant strains and the background stock, we determined that neither strain appears to carry a mutation in the coding region of the paralog. Further experiments will determine whether CG4701 is affected at the transcriptional level in the two mutants generated. POSTERS: Organogenesis and Gametogenesis 257

497B Separation of Nebenkern anchoring and mitochondrial elongation phenotypes in germ line clones of a milton allele. Sheena E. Favors, Karen G. Hales. Department of Biology, Davidson College, Davidson, NC. During Drosophila spermatogenesis, mitochondria gather along the meiotic spindle and later aggregate, fuse, and interwrap to form the Nebenkern, whose two mitochondria derivatives unfurl and finally elongate along the sperm tail. Work by others in the fly brain and ovary demonstrated that the essential gene milton encodes a protein important for mitochondrial transport in Drosophila neurons; at least one Milton isoform of Milton competes with kinesin light chain to associate with the kinesin heavy chain to mediate plus end-directed movement of mitochondria on microtubules, while at least one other isoform of Milton may mediate minus end- directed movement. We previously generated testis germline clones with a milton null allele and showed that Milton is required both for proper anchoring of the Nebenkern to the nucleus and for proper mitochondrial elongation. In the current study we made mutant germline clones of miltonK06704, an allele that disrupts only a subset of isoforms, to ask whether the phenotypes we previously observed are separable. Our preliminary data indicate that miltonK06704 mutant spermatids show abnormal anchoring of the Nebenkerne similar to the null mutant cells but that sperm tail mitochondrial elongation appears more normal.

498C Condensin II catalyzes chromosome individualization to enable meiosis I segregation and separation of polytene chromosomes. Tom Hartl, Helen Smith, Giovanni Bosco. Dept Molec & Cellular Biol, Univ Arizona, Tucson, AZ. A variety of nuclear processes are mediated by non-random interactions between chromosomal loci. Homologous chromosome associations are necessary for proper meiotic chromosome segregation, DNA repair, and dosage compensation. There are also instances where chromosomal associations occur in somatic tissues, dependent or independent of DNA homology, and are inferred to influence each loci’s respective transcriptional status. Here we demonstrate that the Drosophila condensin II complex is a chromosome individualization factor, functioning to resolve chromosomal linkages between sister chromatids, homologous chromosomes, and non-homologous sites. In ovarian nurse cells, condensin II is necessary to enable the transition from polyteny to non-polyteny as flies with loss-of-function mutations in condensin II subunits lead to persistence of polytene chromosomes. This result alone demonstrates that condensin II is necessary to resolve linkages between homologous chromosomes and sister chromatids. Condensin II is also sufficient to induce chromosomal individualization as over expression of Cap-H2 leads to dramatic separation of salivary gland polytene chromosomes. Furthermore, due to failure in chromosome individualization during male meiosis of Cap- H2 mutants, linkages between homologous and non-homologous chromosomes persist into anaphase I and lead to substantial chromosome loss. We propose a model that homologous and non-homologous interactions occur normally and can be considered the default state of the Drosophila nucleus. In preparation for chromosome separation, these interactions must be actively disrupted through condensin II chromatin binding and subsequent chromosome individualization. Finally, it may be that condensin II mediated individualization is not solely to enable large-scale chromosome movements such as segregation. Preliminary data show that transvection via Cbx1 Ubx1 is dominantly enhanced by Cap-H2 mutations, possibly through an increased frequency of homologous chromosome interactions at the bithorax complex.

499A The ped gene and delayed translation of dhod RNA during spermatogenesis. David Keesling, Jianyuan Luo, John Rawls. Department of Biology, University of Kentucky, Lexington, KY 40506. Pre-meiotic spermatogenesis includes the formation of numerous mRNAs which have their translation delayed until post-meiotic spermiogenesis, at which time they are translated in tightly choreographed programs. Translation of the spermatogenesis-specific transcript of the dhod gene is normally delayed until late spermiogenesis, due to a translation control element (dTCE) within its 5'- UTR. A dTCE-controlled reporter construct was used to survey approximately 200 male-sterile mutant lines, one of which contained a mutation that resulted in pre-meiotic expression of the dTCE construct. Subsequent mapping and sequencing revealed that the ped1 mutation results from a nonsense mutation within the CG6091 gene. Additional ped alleles have been isolated, all of which result in precocious expression of dhod as well as spermatogenesis arrest during spermatid elongation. We will describe PED protein expression patterns and experiments to determine interactions between PED and dTCE-containing RNA. 258 POSTERS: Organogenesis and Gametogenesis

500B Mutants in the Drosophila SUN gene dspag uncouple individualization complex progression from membrane investment in gametogenesis. Martin Kracklauer1, Heather Wiora1, Xin Chen2, Janice Fischer1. 1) Dept MCD Biol, Univ Texas, Austin, Austin, TX; 2) Dept Dev Biol, Stanford U School of Medicine, Stanford, CA. In the Drosophila testis, after meiotic divisions generate cysts of 64 round spermatids, dramatic morphological changes and membrane remodeling result in elongated spermatids, each invested in its own plasma membrane. Membrane investment starts at the sperm nuclei, where 64 actin-rich investment cones that comprise the individualization complex (IC) assemble. The IC moves caudally down the cyst as a coordinated front, pushing along an increasing amount of cytoplasm that is visible as the cytoplasmic bulge (CB). As the end of the cyst is reached, the CB is sloughed off, becoming the waste bag (WB); the cyst with individualized spermatids coils, the surrounding cyst cells degenerate, and mature spermatids are deposited into the seminal vesicle. The Drosophila gene dspag encodes a protein with a C-terminal SUN domain. dspag is expressed only in males, and null mutations generated by ends-out homologous recombination result in complete male sterility. The meiotic steps of gametogenesis occur in an apparently normal manner in dspag mutant testes, and the post-meiotic morphological changes are also unaffected. However, the seminal vesicle fails to fill up with mature, motile sperm even after sequestering males away from females for several days. In addition, only small CBs are observed in the mutant, and WBs of wild type size are not found. While phalloidin staining of dspag mutant testes suggests ICs form and advance normally, TEM on cross-sections of mutant cysts indicates that cytoplasmic elimination and membrane investment are severely compromised compared to wild type. Interestingly, in a handful of axonemes in each mutant cyst, the nine outer doublets appear normal and are regularly spaced, the central doublet however is missing. We are currently using tagged dspag cDNA transgenes, along with transgenes derived from Spag4, the putative rat ortholog of dspag, to rescue the dspag phenotype and to establish a mechanism for Dspag function in gametogenesis.

501C Developmental control of testis-TAF expression and male germ cell differentiation. Chenggang Lu, Xin Chen, Margaret Fuller. Department of Developmental Biology, Stanford University School of Medicine, Stanford , CA. When Drosophila male germ cells differentiate from spermatogonia into spermatocytes, they must turn on a dramatic and cell- type specific transcription program that drives expression of terminal differentiation genes required for spermatid formation and maturation. Recent studies in our lab revealed that these terminal differentiation genes are silenced in earlier stages of the stem cell lineage by Polycomb. We have also found that the expression and function of 5 testis specific homologs of TBP associated factors (tTAFs) is crucial for releasing gene repression by Polycomb. The tTAFs may bind to promoters of terminal differentiation genes and displace or prevent binding of Polycomb to allow transcription of terminal differentiation genes. To investigate how the developmental program directs the cell-type and stage specific transcription of the tTAFs in early spermatocytes, we are going to search for trans- acting factors that bind to cis-acting sequences at the promoters of the tTAFs, and then study in vivo how expression and function of these regulators are controlled by the developmental program of male germ cell differentiation. We have already defined, by reproter analysis, minimal cis-acting sequences that drive spermatocyte-specific expression of 4 of the 5 tTAFs to 20 base pairs near their transcription start sites. I am now searching for trans-acting regulators that bind to these cis-acting motifs. Once these protein factors are found and their function on tTAF transcription confirmed, I will explore how these protein regulators are themselves regulated in the context of differentiation of the male germ cells. Updated progresses will be presented at the meeting.

502A Suppressors and Enhancers of Segregation Distorter. Janna McLean, Reid McLean, David Sisneros, Hollie Vigil. Dept of Biol, Colorado State Univ-Pueblo, Pueblo, CO. Segregation Distorter, SD, is a meiotic drive system that does not allow for equal transmission of each chromosome from the males to the progeny. Abnormal chromatin condensation in developing spermatids appears to interfere with the transmission of about one-half of these cells. The Segregation distorter gene (Sd+) encodes RanGAP, which is involved in nuclear transport. Kusano et al. (2001, 2002) have shown that the presence of active Sd-RanGAP is responsible for distortion, and that this distortion can be suppressed by the over-expression of Ran or RanGEF. Thus, distortion seems most likely to occur because of a reduction in nuclear transport in developing spermatids. To further test this hypothesis we have obtained a number of nuclear transport mutations, as well as mutations that affect chromatin condensation more directly. We have tested the ability of these mutations to suppress or enhance distortion, and will present the results of this functional analysis. POSTERS: Organogenesis and Gametogenesis 259

503B Di-(2-ethylhexyl) phthalate (DEHP) affects Drosophila melanogaster development possibly through meiotic defects during spermatogenesis. Janet Rollins1, Kwesi Blackman3, Penel Joseph3, Thomas Onorato2. 1) Division of Science, College of Mount St. Vincent, Riverdale, NY; 2) Department of Natural Applied Sciences, LaGuardia Community College, Long Island City, NY; 3) Department of Biology, Kingsborough Community College, Brooklyn, NY. Phthalates are infused with polymers to increase flexibility of plastics. Exposure to phthalates is ubiquitous, e.g., food containers, toys, baby bottles, IV tubing, blood storage bags, PVC flooring, and household dust. Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used phthalates, has adverse effects on the male reproductive system. Moreover, DEHP may shorten the lifespan of the fruit fly, Drosophila melanogaster, as well as increase lipid peroxidation, an indicator of oxidative stress. Therefore, this study determined whether DEHP affects Drosophila development. Adult wild-type Canton S flies were grown in glass vials containing insect medium (control) or insect medium with 500 μM DEHP. After 7 days the Parental generation was transferred to new vials and exposed to DEHP (500 μM) for an additional 14 days. The resulting F1 generations were counted on day 21. Acute exposure (up to 21 days) to 500 μM DEHP was found to be non-lethal to the P generation. No differences in the number of flies of the F1 generation were observed after short-term exposure (7 days) to DEHP when compared to the matched control. Interestingly, longer exposure (21 days) to DEHP reduced the number of flies in the F1 generation. Testicular squashes revealed abnormal association of nuclei with the nebenkern (mitochondrial structure) in round spermatids, which is indicative of a meiotic defect. Our preliminary findings suggest that acute exposure to DEHP, although not lethal to adult flies, appears to affect the development of the fly; possibly through meiotic defects in spermatogenesis. Further studies will be designed to elucidate whether DEHP exposure results in increased oxidative stress.

504C A role for basigin (bsg) in late-stage Drosophila spermatogenesis. Kathryn Vecomnskie, Meaghan Crook, James Fabrizio. Biology Department College of Mount Saint Vincent 6301 Riverdale Ave. Bronx, NY 10471. During Drosophila spermatogenesis, the development of the maturing germline takes place within a spermatogenic cyst encapsulated by two somatic cyst cells. Although the roles of the cyst cells in the early stages of spermatogenesis have been extensively characterized, little is known of their roles in post-meiotic spermatid development. A collection of P-element GFP-tagged protein traps was screened in order to identify novel somatic regulators of late-stage spermatogenesis. One particular protein trap exhibited expression in the cytoplasm of the cyst cells, including late-stage cyst cells. Inverse PCR followed by cycle sequencing and BLAST analysis confirmed that this GFP-tagged protein trap was a P-element insertion into the basigin (bsg) gene, suggesting that Bsg is expressed in the cyst cell cytoplasm during post-meiotic spermatid development. Interestingly, analysis of testes from male-sterile bsg mutant flies using rhodamine-conjugated phalloidin revealed a paucity of individualization complexes, and rare individualization complexes in bsg mutant testes appear unstructured as compared to wild-type complexes. Taken together, these results suggest a novel role for bsg in spermatid individualization. Moreover, since GFP-tagged protein trapping reveals Bsg expression in the somatic cyst cells, these results may ultimately uncover a role for the somatic cyst cells in the spermatid individualization process.

505A Cloning and characterization of mitoshell, a gene required for normal mitochondrial aggregation during Drosophila spermatogenesis. Kathleen H. Wood, Laura M. Bergner, Francis E. Hickman, Michael C. Beaucaire, Amanda C. Aldridge, Sheena E. Favors, Karen G. Hales. Department of Biology, Davidson College, Davidson, NC. In Drosophila melanogaster spermatogenesis, mitochondria undergo dramatic morphological changes. After meiosis, the mitochondria in spermatids aggregate and fuse into the Nebenkern and then elongate along the sperm tail. Mitochondria in mitoshell homozygotes aggregate prematurely and form a shell around the nucleus of primary spermatocytes. The nuclear divisions of meiosis occur, but meiotic cytokinesis fails. Homozygous male flies are sterile while homozygous females are fully fertile. To characterize the molecular defect more fully, we are examining the localization in mitoshell testes of several GFP-tagged proteins important for cytokinesis; preliminary data have yet revealed no obvious protein localization difference between wild type and mutant cells. The mitoshell gene has been mapped by deficiency to a region of the second chromosome containing 23 candidate genes. Four genes in this region show enriched expression in the testes relative to other tissues. We sequenced two candidate genes in two alleles of mitoshell and its background strain for comparison. For one candidate gene, each allele had a unique point mutation causing a nonsense mutation in the coding sequence of the gene, consistent with this gene representing mitoshell. The mitoshell gene encodes a novel protein, conserved in insect lineages, with no recognizable protein motifs. 260 POSTERS: Organogenesis and Gametogenesis

506B The Ubiquitin Specific Protease Scrawny is Required in Diverse Drosophila Stem Cells. Michael Buszczak1,2, Shelley Paterno2, Allan Spradling2. 1) Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX; 2) Department of Embryology, Carnegie Institution, Baltimore, MD. Stem cells within diverse tissues need a chromatin configuration that promotes self-renewal, yet few chromatin proteins are known to regulate multiple types of stem cells. We describe a Drosophila gene, scrawny (scny), encoding a ubiquitin-specific protease, that is required in germline, epithelial and intestinal stem cells. Like its yeast relatives UBP8 and UBP10, Scrawny deubiquitylates histone H2B and functions in gene silencing. Consistent with previous studies of this conserved pathway of chromatin regulation, scny mutant cells have elevated levels of H3K4 methylation, and over-express Notch target genes. Our findings show that H2B ubiquitylation plays important roles in diverse stem cells, and suggest that scny controls the activity of key target genes that are fundamental to cell division and differentiation.

507C Sex-lethal is required for asymmetric fate specification of ovarian germ cells. Johnnie Chau, Laura Shapiro Kulnane, Helen K. Salz. Department of Genetics, Case Western Reserve University, Cleveland, OH. Sex-lethal (Sxl) encodes a female-specific RNA binding protein that is the “master regulator” of sexual cell fate in the soma. Sxl is also required for ovarian germ cell development, but its role remains unclear. Here, we characterized the snf148 ovarian tumor phenotype, which is due solely to the absence of SXL in the germline. The snf mutant ovary accumulates proliferating germ cells with abnormal fusome-like structures of varying shapes. Nevertheless, our analysis of both snf, CycB and snf, zpg double mutants indicates that the tumor cells continue to require at least some of the same essential growth-control mechanisms used by wild type germ cells. Interestingly, we find that in tumor cells the distinction between germline stem cells (GSCs) and the mitotic cyst cells is disrupted. Normally, the transition from GSC to its differentiated daughter cell requires the coincident down regulation of piwi and pum, two GSC self-renewal genes, with the upregulation of bam, a differentiation gene. Following the expression of these molecular markers, we show that the proliferating snf tumor cells express all three molecular markers. In particular, we detect bam expression in the most apical tumor cells where transcription is normally repressed by dpp signaling. Because snf tumor cells can receive dpp signals, as assayed by activation of a dad-lacZ reporter construct, we propose that SXL functions in the GSCs to interpret (either directly or indirectly) the signals that silence bam transcription. Our observation that precocious BAM expression is not sufficient to drive differentiation in a snf mutant background, combined with the phenotype of snf, bam double mutants, indicates that SXL is also required for BAM protein function. In summary, our data lead to a model in which at least two different programming events required for asymmetric cell fate specification in the germline are under Sxl control. Studies to understand the molecular mechanism by which SXL controls these pathways are underway.

508A The role of NURF301 in the Drosophila testis stem cell niche. Christopher Cherry, Erika Matunis. Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD. Various signaling pathways are required for the maintenance of different stem cell types and emerging data also suggests that the state of chromatin within a stem cell is a key contributor to the ability of the cell to self-renew or differentiate. In the Drosophila testis JAK/STAT signaling is required for the maintenance of both germline (GSCs) and somatic stem cells (SSCs). We are interested in understanding the role of NURF301, a component of a chromatin remodeling complex and a putative JAK/STAT regulator, in the maintenance of both of these stem cell populations. NURF301 is the largest subunit of the Drosophila Nucleosome Remodeling Factor (NURF), an ISWI-containing ATP-dependent chromatin remodeling complex. Previous work has shown that ISWI is needed for GSC maintenance in the Drosophila ovary, however, it is not clear which of the three known ISWI containing complexes is responsible for stem cell maintenance, nor the pathway or mechanism by which it occurs. By targeting NURF301, a unique member of the NURF complex, we are able to selectively hamper its function while leaving the remaining two ISWI-containing complexes unaffected. The role of NURF301 in the Drosophila spermatogonial stem cell niche will be discussed. POSTERS: Organogenesis and Gametogenesis 261

509B A circuit discriminating somatic stem cells from niche cells in the testis. Stephen DiNardo, Natalie Terry, Sarah Freilich, Colin Palmer. Dept Cell & Developmental Biol, Univ Pennsylvania Sch Medicine, Philadelphia, PA. Due to the difficulty in identifying stem cells in tissues, most work has centered on intrinsically-acting factors that control stem cells, or on identifying culture conditions to maintain undifferentiated stem cells. Thus, there is significantly less known about the stem cell microenvironment (niche), even though it provides many of the signals that govern stem cells, including their self-renewal and inhibition of differentiation. Understanding stem cell-niche interactions is pivotal as recent work showed that neural stem cells and human ES cells can each generate their own supporting niche cells. The ovary and testis of the fruitfly have been exquisitely revealing about niche-stem cell interactions. We conducted expression profiling in the testis to identify novel niche and stem cell factors. We find that the protein, Lines, is an essential somatic stem cell factor. When one depletes a stem cell factor, we usually expect differentiation or death of the cell. We do not expect the stem cell to switch and take on niche fate. Yet this is what we find: lines-deficient somatic stem cells express multiple hub (niche) markers. Furthermore, by mosaic analysis we find that lines mutant somatic cells recruit adjacent wild-type cells to act like somatic stem cells: nearby wild-type cells express high levels of Zfh1 (as do stem cells), 2) exhibit activation of the Hh pathway (as do stem cells), 3) and continue to cycle and express Wingless (as do stem cells). These data suggest strongly a close molecular relationship between a stem cell and its niche support cell. We suspect that somatic stem cells and hub cells derive from the same precursor pool during gonadogenesis. Le Bras & Van Doren (2006) have shown that hub cells are organized in late stage embryos, after coalescence of germ cells with somatic gonadal precursors. Our preliminary work shows that gonads in lines mutant embryos have excess hub cells. We are testing whether these excess cells come at the expense of somatic stem cells, as our model predicts.

510C Brat is required for correct differentiation of Drosophila ovarian stem cells. Robin Harris, Hilary Ashe. Faculty of Life Sciences, University of Manchester, Manchester, Lancashire, United Kingdom. Germline stem cells (GSCs) in the Drosophila ovary continually divide to both self-renew and give rise to a differentiated lineage. These differentiated cells mature into a germline cyst that includes the oocyte, which ultimately forms the unfertilised egg. Although basic signalling elements involved in this system have been discovered, the downstream factors involved in differentiation remain largely unknown. We have characterised the role of the translational repressor Brat in the differentiation of germline cells in this system. Confocal immunofluorescence indicates Brat is strongly expressed in differentiating germline cells, but absent from renewing GSCs. When Brat is overexpressed, cell growth and division are retarded, eventually causing complete differentiation of the germline and stem cells, rendering the organism sterile. Conversely, mutation of Brat causes greatly increased expression of the major growth promoting factor dMyc. Genetic analysis also suggests that Brat downregulates Decapentaplegic (Dpp), the main signal required for self renewal of GSCs. Together, these data suggest a model in which Brat encourages differentiation through both downregulation of a cellular growth factor to limit cell growth and division, and attenuation of the external self-renewal signal Dpp. This model shares interesting parallels with the role of Brat in cell differentiation of the neural stem cell system in the larval brain, and therefore could suggest a common role for Brat in at least two stem cell systems in Drosophila.

511A Socs36E mediated JAK/STAT signal attenuation regulates the balance of germline and somatic stem cells in the Drosophila testis niche. Melanie Issigonis1, Natalia Tulina2, Margaret de Cuevas1, Crista Brawley1, Laurel Mellinger1, Erika Matunis1. 1) Cell Biology Department, Johns Hopkins University School of Medicine, Baltimore, MD; 2) Genetics and Gene Regulation Program, University of Pennsylvania Medical School, Philadelphia, PA. Stem cells are maintained in microenvironments, or niches, that control their behavior, but little is known about the extrinsic cues and intrinsic signaling that regulate stem cell niches. Furthermore, niches can contain more than one type of stem cell, but the coordinate regulation of stem cell behavior is poorly understood. The Drosophila testis niche is a tractable model to study the coordinate regulation of stem cells because there are two known stem cell types: the germline stem cells (GSCs) and somatic stem cells (SSCs), which both require cell autonomous JAK/STAT pathway activation for their maintenance. Although factors required for stem cell identity have been characterized for many different niches, little is known about signal attenuation. Proteins of the mammalian Suppressor of Cytokine Signaling (SOCS) family are the best-characterized negative regulators of the JAK/STAT pathway. Like its mammalian orthologues, several studies have indicated that Drosophila socs36E negatively regulates JAK/STAT signaling, but loss- of-function studies of socs36E have not been reported. We have found that socs36E is expressed specifically in the testis niche and is a STAT target. We have also identified a strong loss-of-function allele, socs36EPZ1647, which causes testes to have significantly fewer GSCs. Mosaic analysis indicates that socs36E is not directly required in GCSs in order to prevent GSC loss but rather is needed to attenuate JAK/STAT activity in SSCs to prevent them from displacing GSCs from the niche. We are currently investigating the molecular basis for this observation. This work demonstrates how the coordinate regulation of two stem cell populations in a single niche maintains the proper balance of each stem cell type in the tissue. 262 POSTERS: Organogenesis and Gametogenesis

512B GFP-NRE sensor directly monitors Pumilio activity in germline stem cells. Sung Yun Kim, Jiyoung Kim, Changsoo Kim. School of Biological Sciences and Technology, Chonnam National University, Gwangju-Si, Korea. Stem cells possess the unique ability to self-renew as well as to generate differentiated progeny. In Drosophila ovary, extrinsic signals from adjacent niche and intrinsic factors govern the self-renewal of germline stem cells (GSC). The pumilio as an intrinsic factor and a translational repressor is required for the self-renewal and inhibits premature differentiation of GSC. Genetic analysis suggests that there are a number of upstream components that regulate pum gene function; however, it remains unclear how pumilio activity is regulated in GSC and later germ cells during differentiation. To visualize pumilio activity in vivo, we have developed a reporter GFP containing NRE sequence on its 3’UTR. We show that the GFP expression of the ‘NRE sensor’ is dependent on endogenous pumilio activity. The sensor transgene reveals dramatic patterns of pumilio activity in germarium. Various known mutations involved in GSC self-renewal affect the pumilio activity. We show that Bam inhibits pumilio activity prior to GSC differentiation. We propose that the ‘NRE sensor’ provides outstanding opportunity of identifying factors that regulate pum activity in GSC maintenance.

513C Dietary Regulation of Normal and Tumorous Germline Stem Cells Occurs Via both Insulin-Dependent and -Independent Mechanisms. Leesa M. LaFever, Hwei-Jan Hsu, Daniela Drummond-Barbosa. Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN. How external factors, such as diet, affect stem cell biology is not well understood. Our previous data have shown that a protein-rich diet upregulates germline stem cell (GSC) proliferation and cyst growth via neural Drosophila insulin-like peptides (DILPs) received directly by the germline. However, it was unclear how diet and insulin signaling control GSC division at a mechanistic level. To address these questions we performed mosaic analysis of chico mutants lacking the ability to activate either the phosphoinositide- 3 kinase (PI3K) or MAPK branches of the insulin signaling pathway. We found that diet-induced DILP-mediated signals absolutely require the PI3K branch of the insulin pathway to stimulate GSC division, but do not require MAPK. We also performed a detailed analysis of cell cycle markers in GSCs, showing that diet regulates both the G1 and G2 phases of the cell cycle. In contrast, the analysis of insulin pathway mutants indicates that this pathway is predominantly involved in the control of G2. These findings suggest that a separate, unknown, diet-induced mediator is acting at the level of G1. Finally, we find that tumorous GSC-like stem cells, induced by mutation of bam, retain their ability to respond to diet even though they escape normal niche control, indicating that diet-induced signals act independently of the niche. Our results document the effect of diet and insulin-like signals on the cell cycle of stem cells within an intact organism, and demonstrate that the response to diet requires multiple signals. Moreover, the retained ability of GSC tumors to respond to diet parallels the long known connections between diet, insulin signaling, and cancer risk in humans.

514A Coordination between two stem cell populations in the Drosophila ovary. Lucy Morris, Allan Spradling. Carnegie Institution of Washington, Baltimore, MD. Many tissues are established and maintained by more than one stem cell population. One such tissue is the Drosophila ovary, which contains three different stem cells. How these stem cell populations coordinate to ensure continuous egg production is not understood. Escort stem cells are the least well-understood of the three stem cell populations in the ovary. These somatic stem cells have long, thin cytoplasmic extensions that completely enclose the germline stem cells. Together the escort and germline stem cells coordinate to build a germline cyst, offering a unique opportunity to study interactions between two stem cell populations. The ratio of escort cells to germline cysts is constant, indicating that coordination between the two stem cell populations is tightly controlled. We have shown that the mechanism of regulation is not simply coordinated stem cell division. Interestingly, whilst lineage tracing experiments demonstrate that escort stem cells divide frequently. Escort cells in mitosis have not been identified at the expected frequency, suggesting that this population may have a rapid or unusual cell cycle. The observation that escort stem cells have incredibly long cytoplasmic projections raises the possibility that they may require a streamlined cell cycle. To investigate what other mechanisms may be involved in stem cell-stem cell coordination we have 1) begun using markers to further characterize the cell cycle of the escort stem cells. And 2) we are currently undertaking a screen for mutants that perturb either the escort stem cell cell cycle and/or the coordination between the escort and germline stem cells. POSTERS: Organogenesis and Gametogenesis 263

515B Elucidating the Role of the Notch Pathway in the Drosophila Testis Stem Cell Niche. Tishina C Okegbe, Natalie Terry, Tim Kelliher, Steve Dinardo. Cell and Developmental Biology, UPenn, Philadelphia, PA. Stem cells are multipotent cells which have the potential to produce daughter cells that either self-renew or differentiate. The stem cell biology field has recently garnered much interest in the hopes to use stem cells to aid in regenerative medicine. Although this is true, the exact mechanism by which stem cells balance self-renewal with differentiation is unknown. To this end, the Drosophila testes, which contains both germline and somatic stem cells, has been studied as a model system to identify the contribution made by specific signaling pathways in determining stem cell fate. Stem cells reside in microenvironments, known as niches, that help to maintain stem cell character by signaling both directly and indirectly to the stem cells. We recently conducted a gene expression profiling approach to identify components enriched in testis stem cells and/or their niche. Several genes and targets of the Notch pathway were enriched (Terry et al, 2006). We report here that ectopic activation of the Notch pathway influences hub (niche) cell number, and that this is dependent on Serrate- but not Delta- induced signaling. We are currently determining Notch’s role both in the adult testes as well as the developing gonad by a series of loss of function experiments. Identifying Notch’s role in the Drosophila testes stem cell niche may shed light onto the workings of other stem cell systems and ultimately aid in the progress of using stem cells in regenerative medicine.

516C Germline stem cells are resilient during Drosophila development. Halyna R Shcherbata, Ellen J Ward, Karin A Fisher, Jenn- Yah Yu, Hannele Ruohola-Baker. Biochemistry, University of Washington, Seattle, WA. Disruption of microRNA processing in Drosophila germline stem cells (GSC) during pupal development delays the G1/S cell transition through up-regulation of CDK inhibitor, Dacapo/p21/p27. Here we report that in addition to regulating the cell cycle, microRNAs are also required for Drosophila GSC maintenance. If GSCs lose functional Dicer-1 during adult life, GSCs are lost, while loss of Dicer-1 during development does not result in a maintenance defect. Since Dicer-1 and, therefore, microRNA function is required for adult GSC maintenance, we analyzed which microRNA(s) is/are responsible for this phenotype. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam is a key microRNA required for GSC self-renewal. We also show that Mad, a component of the receiving end of the TGF-β pathway, behaves similarly: adult GSC maintenance requires Mad if it is lost during adult life, but not if lost already during pupal development. Preadult stem cells have a youthful resiliency that is lost at adulthood. To identify the latest stage of development when this youthful resiliency can be activated, we introduced Mad mutations in GSC of 3rd instar larva/early pupae, late pupae and 1-4 days old adult flies. Interestingly, Mad GSCs were lost only after adult clonal induction, suggesting that the resiliency of preadult GCS maintenance extends through late pupal stages, but not into adulthood. Previous studies have shown that GSCs already reside in a niche at late pupal stage, suggesting that this resiliency is not a result of major differences in the morphological environment of GSCs during development and adulthood. These results suggest that GSC maintenance is governed by robust, redundant mechanisms during development: the adult requirement for either Dicer-1 or Mad for GSC maintenance is compensated if either pathway is compromised during development. Through genetic interaction studies we find that Dicer-2 is required for this robust stem cell behavior.

517A integrin-dependent anchoring of a stem cell niche. Guy Tanentzapf1,2, Danelle Devenport2,4, Dorothea Godt3, Nicholas H. Brown2. 1) Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada; 2) The Gurdon Institute and Department of Physiology, Development, and Neuroscience,University of Cambridge, Cambridge, UK; 3) The Department of Cell & Systems Biology University of Toronto Toronto, ON., Canada; 4) Laboratory of Mammalian Genetics and Development, Rockefeller University, New York. USA. Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem cell properties. The ‘hub’ cells in the adult Drosophila testis are a discrete morphological stem cell niche that provides an excellent model for the study of niche development. The small cluster of non-dividing, somatic hub cells at the anterior tip of the fly testis is contacted by the germline stem cells (GSCs) that retain their stem cell character through the direct association with the hub. We have demonstrated that integrin-mediated adhesion is important for maintaining the correct position of embryonic hub cells during gonad morphogenesis. The misplaced hub in integrin mutant embryos directs the orientation of cell divisions in the presumptive GSC, a hallmark of the active germline stem cell niche. A reduction of integrin-mediated adhesion in adult testes, which resulted in a loss of hub cells and the stem cell population, revealed the importance of hub cell anchoring. Finally, we show that an ECM is present around the gonad in late embryogenesis and that this ECM is defective in integrin mutant gonads. Based on our data we propose that integrins are required for the attachment of the hub cells to the ECM, which is critical for maintaining the stem cell niche. 264 POSTERS: Organogenesis and Gametogenesis

518B Regulation of somatic stem cells in the Drosophila ovary through multiple signaling pathways. Cynthia Vied, Daniel Kalderon. Dept Biological Sci, Columbia Univ, New York, NY. The somatic stem cells (SSC) of the ovary have been shown to be directly regulated by the Hedgehog (Hh) signaling pathway. Ectopic Hh signaling results in SSC duplication and consequent somatic cell over-proliferation. We have identified mastermind (mam) mutations as dominant suppressors of the Hh phenotype. By visualizing the SSCs we determined that this results from preventing SSC duplications. In addition, complete loss of Mam results in decreased persistence of SSC clones, suggesting that Mam also regulates SSC longevity. Since Mam functions as a component of the Notch (N) signaling pathway, we examined other members of the N pathway to determine their role on SSC behavior. Loss of N pathway components, other than Mam, did not greatly reduce SSC maintenance. We suggest that Mam regulates SSCs through a mechanism distinct from the Notch signaling pathway. We have also tested other signaling pathways and their role in regulating SSC behavior. We have tested components of the Wingless, Jak/Stat and BMP pathways to compare their effects on SSC longevity to Mam and Hh signaling components. Members of Wingless and BMP pathways have previously been shown to regulate SSCs as has DE-Cadherin, which was shown to be important for adherence of the SSCs to the niche cells. By inducing mutant SSC clones at different times in development, we obtained evidence for a SSC establishment phase that differs from SSC maintenance in its genetic requirements for a subset of these components. While Mam and DE-Caderin had a similar effect on the apparent establishment and maintenance phases, decreased Hh signaling and increased Wingless signaling impaired establishment more than maintenance. Surprisingly, loss of Wingless signaling appears to favor SSC establishment even though it impairs SSC maintenance. Our data and previously published data show that multiple signaling pathways are critical for SSC behavior, but exactly how they integrate to regulate SSC establishment and maintenance remains to be uncovered.

519C Somatic stem cells contribute to the apical hub in the Drosophila testis. Justin Voog, Leanne Jones. Laboratory of Genetics, The Salk Institute, La Jolla, CA. Adult stem cells are necessary for the replenishment of many tissue types during the lifetime of an individual. These stem cell populations are regulated in part by the local microenvironment (niche). However, little is known of factors that regulate the size and number of niches or maintain niche function. Here we investigate mechanisms utilized to maintain a functional stem cell niche in the Drosophila testis. At the apical tip of the testis, germline and somatic stem cells contact the hub, a cluster of approximately 10-15 somatic cells that is required for stem cell self-renewal and maintenance. We observe that somatic stem cells contribute to the apical hub and this property is dependent upon the transcriptional repressor escargot. We propose that somatic stem cell contribution to the hub is one mechanism by which the stem cell niche is maintained throughout adulthood. Identifying factors regulating stem cell niche dynamics may aid in the implementation of regenerative therapies.

520A Genetic interactions suggest a role for chromatin modification in maintenance of germline stem cells. Lin Yu1, Yan Song2, Tammy Lee1, Robin Wharton1. 1) Department of Molecular Genetics and Microbiology/Cell Biology, Duke University/HHMI, Durham, NC; 2) Department of Pathology, Stanford University School of Medicine, Stanford, CA. The translational inhibitor Nanos (Nos) is required for a number of processes, including segmentation of the embryo and maintenance of germline stem cells. Nos governs abdominal segmentation by repressing translation of maternal hunchback mRNA; the mechanism by which Nos prevents differentiation of germline stem cells is currently unclear. To identify factors that act in conjunction with Nos, we have performed a genetic screen for modifiers of the segmentation defects in embryos with limited levels of Nos activity. One such genetic modifier was mapped to the gene encoding CG4699. The CG4699 locus encodes 210 and 150 kDa proteins, that are ubiquituously expressed and localize to nuclei throughout oogenesis and embryogenesis. Oogenesis is superficially normal in germline clones of a lethal P-element allele of CG4699. However, we observe a synthetic, age-dependent female sterile phenotype in animals homozygous for a nos hypomorph and hemizygous for CG4699. Analysis of the ovaries in these flies suggests the sterility results from the failure to maintain germline stem cells. Recent work has shown that CG4699 is a member of a large protein complex that includes both histone methylase and acetylase activities. Taken together, these results suggest that Nos and chromatin modification factors may act synergistically in maintenance of the germline stem cell fate. POSTERS: Chromatin and Gene Expression 265

521B Role of Su(var)3-9 in preserving genome integrity. Irene Chiolo, Jamy Peng, Gary Karpen. Genome Biology, LBNL, Berkeley, CA. Heterochromatin contains many repetitive DNA elements and some protein-encoding genes, and it has essential functions in controlling chromosome organization and inheritance. HP1 and its partner Su(var)3-9 H3K9 methyltransferase are typically located in heterochromatin and are required for its formation and maintenance. With experiments in fly mutants and in S2 cells, we observe that Su(var)3-9 is important for the DNA damage response and it is required for preventing the accumulation of spontaneous and induced DNA damage events in heterochromatic regions. Our results suggest that H3K9 methylation is important for preserving genome integrity by stabilizing the heterochromatic regions.

522C HIRA-dependent chromatin assembly during Drosophila development. Benjamin Loppin, Cécile Doyen, Tzvetina Brumbarova, Guillermo Orsi, Pierre Couble. Centre de Genetique Moleculaire et Cellulaire, CNRS, University of Lyon, Lyon, France. In eukaryotes, the bulk of nucleosome particles is assembled at DNA replication forks using core histones that are synthesized in S phase. This Replication Coupled nucleosome assembly pathway involves the conserved CAF-1 (Chromatin Assembly Factor-1) complex. The male pronucleus is the only nucleus during development whose chromatin is totally assembled by a replication- independent nucleosome assembly pathway. Indeed, de novo paternal chromatin assembly occurs after the removal of sperm specific protamines and before the first zygotic S phase. Functional studies of the histone chaperone HIRA have established its essential and presumably conserved role for the assembly of paternal chromatin at fertilization. Maternal HIRA specifically enters the decondensing sperm nucleus immediatly after fertilization and is necessary for the deposition of core histones on paternal DNA. Moreover, de novo assembled nucleosomes on paternal DNA contain the H3.3 replacement histone and not the replicative H3 histone. Because of its histone H3.3 chaperone activity, HIRA is expected to play additional (although not essential) roles during fly development. Accordingly, we have observed that Hira mutant alleles behave as suppressors of variegation, suggesting a role for HIRA in the maintenance of heterochromatin. We are interested to determine the other chromatin factors that cooperate with HIRA for the RI assembly pathway. In addition, to better understand the biology of this histone chaperone, we have generated a series of transgenic lines expressing various mutant alleles of Hira. This transgenes will help determine the important domains of HIRA responsible for its targeting to the fertilizing sperm nucleus and for its general nucleosome assembly activity.

523A An inducible over-expression screen identifies new factors that alter heterochromatic gene silencing. Jonathan I Schneiderman, Kami Ahmad. BCMP Dept, Harvard Medical School, Boston, MA. Heterochromatic gene-silencing leads to discrete active ‘on’ or inactive ‘off’ states, which are inherited through cell division, but also randomly switch in dividing cells. A number of chromatin proteins that are characteristic of euchromatic or heterochromatic states have been identified, but it remains poorly understood how chromatin switching occurs. Here we report a genetic overexpression screen for dominant modifiers of bwD, a heterochromatic allele of the brown gene, which leads to silencing in trans of a functional allele placed opposite of it. bwD and bwD -containing elements thus provide a robust and reliable system in which levels of bw activity are a sensitive assay for switching in response to over-expression of different factors. The screen was performed using the late eye driver GMR-GAL4, but set up to enable any GAL driver to be used. We identified new insertions of the P{Mae-UAS.6.11} element, and sequenced off the ends to identify the genes involved. In addition, we tested the effect of over-expressing candidate genes from nearby insertions, encoding for previously characterized chromatin factors. In total, categories of genes found to effect switching included: (a) known modifiers of position-effect variegation, (b) chromatin associated proteins, (c) non-coding RNAs, (d) transposons, (e) transcription factors, (f) nucleosome assembly factors, and (g) the H3 histone variant, H3.3. We used the eye- specific eyGAL4 and GMR-GAL4 drivers to control over-expression of flanking genes to test the significance of developmental timing. In addition, we address the requirement for DNA replication in chromatin switching, by expressing the cyclin-dependent kinase inhibitor p21, under the same GMR promoter. We expect that some modifiers require DNA replication in order to switch silencing (and thus will be implicated in propagation of chromatin states), while others act anytime. This test will define the role of chromatin duplication in maintaining chromatin states. 266 POSTERS: Chromatin and Gene Expression

524B The role of Scaffold Attachment Factors in nuclear organization and gene expression. Catalina Alfonso, Keith Maggert. Dept Biochemistry/Biophysics, Texas A&M Univ, College Station, TX. In the eukaryotic nucleus, chromosomes are organized into thousands of topologically independent loop domains, which are fixed at their bases to an intranuclear frame composed of proteins and RNA known as the nuclear matrix or scaffold. These loops are important not only for the compaction of the chromatin fiber but also for gene expression and DNA replication. Attachment of the chromatin to the nuclear scaffold occurs via specific long A-T rich DNA sequences called scaffold- matrix attachment regions (S/ MARS). In order to elucidate the function of the loops in vivo, several groups have described proteins with specific binding to S/ MARS in human cells, such as Topoisomerase I, lamin B1, nucleolin, SAF- A (scaffold attachment factor A, hnRNP- U) and SAF-B (scaffold attachment factor B). Human SAF- A and SAF-B contain a DNA- binding domain called the SAP - box, and a RNA binding domain. Both proteins have been shown to precipitate with the nuclear matrix and bind to S/MARS as wells as to RNA. Some phenotypes have been described for SAF-A and SAF-B associated with apoptosis, cell viability and differentiation, and gene regulation. There are homologues to SAF- A and SAF- B in Drosophila. These genes include the characteristic SAF- box and RNA binding domains. Drosophila SAF-A and SAF- B are expressed throughout development. To verify and expand the knowledge of the scaffold attachment factors in nuclear organization and gene regulation, we are characterizing the Drosophila SAF- A and SAF-B homologues. We are performing in situ hybridization, immunofluorescence, RT-PCR and quantitative RT- PCR in S2 cells and whole flies to describe expression profiles and localization. In order to address the biological role of these SAF proteins, we are taking two approaches. First we are generating knockouts by Ends out gene targeting. Second, we are knocking down the expression using RNAi in flies and dsRNA in S2 cells. We have flies that express RNAi for SAF- A and SAF- B under control of an inducible UAS- GAL system.

525C Heterochromatin Comes Unhinged: Studies of HP1. Diane E. Cryderman, Karrie A. Hines, Andrew J. Petersen, Luka N. Zirbel, Beatrice Curio-Penny, Lori L. Wallrath. Biochemistry, University of Iowa, Iowa City, IA. Heterochromatin possesses the ability to spread along the chromosome and silence euchromatic genes. While the mechanism of spreading is unknown, gene silencing correlates with the association of Heterochromatin Protein 1 (HP1). HP1 is enriched within centric regions of the genome and has a conserved domain structure consisting of an amino terminal chromo domain (CD) that binds methylated lysine 9 of histone H3, a flexible hinge region possessing sites of possible phosphorylation, and a carboxyl chromo shadow domain (CSD) that homodimerizes. To determine the mechanism of HP1 spreading, we have targeted HP1 to sites upstream of euchromatic reporter genes. Targeting wild type HP1 nucleates the formation of silent chromatin that spreads bi-directionally from the target site; spreading is predominantly dependent on the histone methyltransferase SU(VAR)3-9. To determine the domains of HP1 required for silent chromatin spreading, domain truncations and amino acid substitutions that disrupt specific functions of HP1, including alterations within the hinge region were tested. Proper localization of HP1 requires both the CD and CSD. The hinge alone does not support silencing and spreading. Spreading requires the CD, CSD and a partner protein interaction domain. An initial screen of the deficiency kit has identified candidate genomic regions encoding potential partner proteins involved in silencing and spreading.

526A GENETIC INTERACTIONS BETWEEN RNA SILENCING COMPONENTS AND RNA POLYMERASE II. Harsh Kavi, James Birchler. Dept Biological Sci, Univ Missouri, Columbia, Columbia, MO. Heterochromatin is thought to be formed by transcription via RNA Pol II of aberrant/repeated regions such as those found in the centromeric regions that enter into the RNA silencing pathway and guide chromatin modification for gene silencing. Recent evidence in S. pombe indicates that mutations in the RNA Pol II second largest subunit affect the production of siRNA and the subsequent histone modifications associated with centromeric heterochromatin. It has also been postulated that the C-Terminal Domain of the largest subunit of RNA Pol II provides the scaffold for the assembly of RNAi silencing components. We examined this issue in Drosophila. Our results indicate genetic interactions between RNAi related components in Drosophila (dcr2, aub, ago2, hls and piwi) and the second largest subunit of RNA Pol II .The interaction between mutations in the RNAi silencing machinery and RNA pol II results in a strong suppression of PEV (Position Effect Variegation) indicating a possible effect on heterochromatin formation in Drosophila. The analysis of polytene chromosomes revealed a decrease in the H3-K9me2 modification at the chromocenter in the double heterozygous (hlsE616, hls125, dcr2 and RNA Pol II A5) mutants when compared with wild type. This study suggests a link between RNA Pol II and small RNA mediated heterochromatin assembly in higher metazoan cells. POSTERS: Chromatin and Gene Expression 267

527B Epigenetic blocking of an enhancer region controls irradiation-induced pro-apoptotic gene expression in Drosophila embryos. Nianwei Lin1, Yanping Zhang1, Gina Chan1,2, Rong Yuan1,2, Bing Yao2, Samuel Wu3, Pamela M. Carroll4. 1) Department of Molecular Genetics & Microbiology, Univ Florida, Gainesville, FL; 2) UF Shands Cancer Center, Univ Florida, Gainesville, FL; 3) Department of Statistics, College of Medicine,Univ Florida, Gainesville, FL; 4) Department of Applied Genomics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. It has been noticed that proliferating stem cells are highly sensitive to cytotoxic stimuli while their differentiated progenies can be much more resistant. How cells switch their sensitivity to exogenous stress is far from clear, but a similar transition was observed during the Drosophila embryogenesis and may shed light on the underlying mechanism. Drosophila embryos before developmental stages 11 are sensitive to γ-ray induced cell death, resulting from the up-regulation of two pro-apoptotic genes- reaper and hid. Whereas embryos become strongly resistant after stage 12, and none of the two genes could be induced by γ-ray. In this study we found that the sensitive-to-resistant transition is due to epigenetic silencing of a conserved 33kb region upstream of reaper locus, we called Irradiation Responsive Enhancer Region (IRER). The chromosome at IRER turns into a compact and repressive state, and becomes enriched with trimethylated H3K27 and H3K9 during the sensitive-to-resistant transition. Genetic mutations on histone modifying enzymes Hdac1 (rpd3), Su(var)3-9, Polycomb group protein Su(z)12 and Pc have effect on the sensitivity transition pattern. Although the silencing of IRER shares some common features with the canonic PcG-mediated silencing in homeotic genes, the two mechanisms differ in several aspects.

528C Studying the role of rDNA in gene expression. Silvana Paredes, Keith Maggert. Dept Biol, Texas A&M Univ, College Station, TX. Position Effect Variegation (PEV) can be observed as mosaic expression of a euchromatic gene when placed within or near heterochromatin. Mutations in genes involved in the formation of euchromatic environments cause increased repression of gene expression, known as enhancement of variegation. In contrast, mutations in genes responsible for heterochromatic environments cause increased gene expression, known as suppression of variegation. In Drosophila, the chromosomal inversion wm4 is used to study PEV, because of the variegating expression of the white+ gene. We use wm4 to study the effect of the rDNA genes on gene expression. The rDNA genes in Drosophila are localized in clusters on the X and Y chromosomes. Each cluster is organized in arrays of tandemly repeated units, each containing the 18S, 5.8S, 2S and 28S genes. The 28S gene contains a sequence that is recognized by the homing endonuclease I-CreI. We have developed a method to specifically reduce the number of rDNA cistrons from the Y chromosome-linked rDNA arrays using I-CreI. The damage is specific to the rDNA, enabling us to begin to probe the function of the repeated array of rDNA in genome organization and gene regulation. We have generated fly lines that have an extreme deletion of the Y-linked rDNA array. In these lines, we observed that the absence of rDNA acts as a strong suppressor of variegation or Su(var). Consistent with the behavior of the rDNA, during rDNA manification the suppressor effect is gradually lost as the Y chromosome is transmitted through several generations. Our genetic and molecular data show that the reversion of the suppressor phenotype starts when the number of rDNA copies has reached the minimal amount required for sufficient protein synthesis for survival. Thus, the increase in rDNA copy number decreases its suppressor effect. Our data suggests a role for the rDNA as a central regulator of heterochromatin in the nucleus.

529A Chromatin organization of the fourth chromosome of Drosophila melanogaster. Nicole Riddle, Wilson Leung, Kathryn Huisinga, Brent Brower-Toland, Sarah Elgin. Department of Biology, Washington University, St. Louis, MO. The fourth chromosome of D. melanogaster - often referred to as the “dot” chromosome due to its small size - is mainly heterochromatic in character, while maintaining one arm of approximately 1.2Mb that is amplified in polytene chromosomes, a characteristic of euchromatic domains. However, cytological studies show that the fourth chromosome is associated with HP1 and methylation of histone 3 at lysine 9, marks classically considered to be heterochromatic. Thus, the dot chromosome exhibits an interesting mixture of heterochromatic and euchromatic characteristics. Our laboratory has used various techniques including position effect variegation screens to probe the chromatin structure of chromosome 4. We find that heterochromatic and euchromatic domains are tightly interspersed on the fourth chromosome. 1360, a DNA transposon, correlates with heterochromatic domains in portions of the dot chromosome, and its contribution has been confirmed by genetic studies. However, the pattern of heterochromatic and euchromatic domains as a whole cannot be explained by the presence of any specific subset of repetitious sequences. We will present further evidence characterizing the fourth chromosome based on a variety of approaches, including genetic screens utilizing position effect variegation, cytology, chromatin immunoprecipitation, and gene expression studies. 268 POSTERS: Chromatin and Gene Expression

530B Studies on the function of the MSL complex. James Birchler, Harvey Fernandez, Xiaoping Sun, Lin Sun. Biological Sciences, University of Missouri, Columbia, MO. The MSL complex associates with the male X chromosome and causes an increase in H4Lys16 acetylation. Our laboratory has sampled X and autosomal gene expression in mutations that disrupt the MSL complex. Most X linked genes retain dosage compensation and many autosomal genes are increased in expression. Also, mutations that ectopically express MSL2 in females show no increase of expression of X linked genes but a reduction for many autosomal genes. These results led to the hypothesis that the MSL complex sequesters MOF to the X to prevent the otherwise upregulation of the autosomes that would result from X monosomy, but also counteracts the high level of acetylation to allow only the proper two fold up regulation of X linked genes. To examine this hypothesis further, a UAS-minimal promoter miniwhite reporter was recovered at several X and autosomal positions. The MOF acetylase was fused with the GAL4 DNA binding domain as was the mof1 point mutation and MSL2. Targeting of MOF to X and autosomal reporters in females causes a strong increase in expression. However, X and autosomal insertions in males are reduced in expression when targeted by GAL4-MOF. Using the GAL4-MOF1 construct eliminates the upregulation in females but not in males. Examination of autosomal insertions in males indicates that targeting of MOF to the reporter brings the other components of the MSL complex to the insertion site, suggesting that the repressive effect is not dependent on MOF activity but on other components. Using a GAL4-MSL2 construct does not have any effect on X or autosomal reporters in females, although immunolocalization and FISH indicate that the fusion GAL4-MSL2 protein is targeted to the reporters and the fusion protein will perform the normal MSL2 function of organizing the MSL complex on the X chromosomes. The results are consistent with the previous gene expression studies indicating a function of the MSL complex involved in overriding the effect of histone acetylation on the male X chromosome and that the MSL complex alone does not condition dosage compensation.

531C High-Resolution Mapping of histone modifications in Drosophila Stage 5 embryos. Sasha Langley, Gary Karpen. Dept MCB, Univ California Berkeley/LBNL, Berkeley, CA. The distributions of histone proteins, their variants, and post-translational modifications reflect the crucial roles they play in epigenetic regulation of gene expression and chromosome functions in eukaryotic organisms. Fine-scale mapping of the genomic locations of these elements of chromatin organization reveals a spectrum of complex features present during early embryonic development. We have examined the genome-wide distributions of histone modifications (H3K4me2, H3K4me3, H3K9me2, and H3K27me3) and the histone variant H2Av in stage 5 Drosophila embryos. As observed in other organisms, gene promoter regions contain two peaks of H3K4me2 and 3, and H2Av, flanking transcription start sites, and the enrichment for these marks is correlated with expression levels. We have identified a novel class of genes with ’plateaus’ of H2Av that span transcriptionally inactive loci lacking the ‘canonical’ mark of transcriptional activation, H3K4 methylation. We also demonstrate that stereotypic bivalent domains containing H3K4me3 and H3K27me3 associated with genes involved in developmental regulation and cell differentiation lack H2Av, regardless of transcriptional state. Finally, heterochromatic and 4th chromosome genes display H3K4 methylation and H2Av patterns similar to those in the euchromatin, despite significant differences in sequence, chromatin context, and gene structure. At the time of establishment during stage 5, the heterochromatic hallmark H3K9me2 is largely absent from coding regions of transcribed genes and is enriched in intergenic, transposon-rich regions of the pericentric heterochromatin.

532A Comparison of chromatin structure in wild type and BEAF (Boundary Element-Associated Factor) knockout larvae. Matthew Gilbert, Craig Hart. Dept Biological Sciences, Louisiana State Univ, Baton Rouge, LA. Insulators are DNA sequences that divide the genome into independently regulated domains. This is thought to be achieved through regulating enhancer-promoter communication and by preventing the spread of silencing heterochromatin into regions of active transcription. The mechanisms by which insulators and their associated binding proteins function are largely unknown. The two related insulator binding proteins BEAF-32A and BEAF-32B bind to the scs’ insulator and hundreds of other sites in Drosophila. Previous data from our lab has suggested that BEAF may function by affecting chromatin structure or dynamics. We have explored this hypothesis by digesting chromatin with DNase I and Micrococcal Nuclease to examine chromatin structure and probe for nuclease hypersensitive sites. Chromatin in nuclei of third instar larvae of wild type and a previously characterized BEAF knockout line was compared. Results from these and related experiments will be presented. POSTERS: Chromatin and Gene Expression 269

533B tRNA genes: a potential role as boundary elements in Drosophila melanogaster. Paola Guerrero, Keith Maggert. Department of Biology, Texas A&M University, College Station TX 77843. Paola Guerrero, Keith Maggert. Department of Biology, Texas A & M University, College Station TX 77843. Boundary elements are regulators of gene expression. They break enhancer-promoter communication when placed between the enhancer and promoter. Likewise, they prevent the spread of heterochromatin and the consequent silencing of transgenes. Hence, they may define the transition between repressive and active chromatin. Boundary elements have been identified in many organisms. Two tRNAs (tRNAAla and tRNAThr) have been found to act as boundaries in two yeast genera. At all centromeres in S. pombe, tRNA genes are at the transition between methylated Lysine 4 and Lysine 9 of histone H3 which mark active and repressed chromatin, respectively. We are currently investigating whether a subset of active tRNA genes have enhancer-blocking and/or heterochromatin- barrier activity in Drosophila, and how these activities are regulated. Our preliminary results indicate that single copies of tRNAAla, tRNAAsp, tRNAThr, tRNASer act as enhancer-blockers and/or short-range repressors. We are now working on distinguishing between these two mechanisms. We found that mutations of promoter elements and an intergenic control region (Box B), which is expected to eliminate transcription, result in an absence of repressor and/or boundary activity. Similarly, we are studying heterochromatin- barrier activity in “induced” and “natural” contexts using two tandem copies of a set of tRNA genes. We will evaluate euchromatic and heterochromatic histone modifications at both sites and within the boundary, and also whether transcription of the tRNA genes is required for their activity.

534C Genome-wide identification of BEAF binding sites in Drosophila. Nan Jiang1, Brenda Winbery2, Craig Hart1. 1) Dept Biological Sciences, Louisiana State Univ, Baton Rouge, LA; 2) Louisiana Scholar’s College, Northwestern State Univ, Natchitoches, LA. Insulators are DNA elements whose role in gene regulation suggests they could also play an architectural role in genome organization. Insulators have at least one of two activities. One activity is the ability of interposed insulators to block enhancer- promoter communication without inactivating either enhancers or promoters. The other activity is the ability to function as barriers that block the spreading of chromatin states. Insulator elements dispersed along the chromatin fiber are proposed to divide chromosomes into domains, with each domain having independent gene regulation. Insulators need to associate with specific proteins in order to function. One class of insulators in Drosophila is bound by the BEAF proteins (Boundary Element-Associated Factor, with two 32 kDa variants: BEAF-32A and BEAF-32B). While immunostaining of polytene chromosomes reveals that BEAF binds to hundreds of interbands and band/interband regions, very few BEAF binding sites have been identified. To better understand chromosomal domains and their gene regulation patterns, we are constructing a genomic map of BEAF binding sites. BEAF- associated chromatin fragments isolated from Drosophila embryos by chromatin immunoprecipitation (ChIP) are being analyzed in two complementary ways. One method is sequence tag analysis of genomic enrichment (STAGE), and the other is hybridization of ChIP DNA to genomic tiling microarrays. The resulting map of BEAF binding sites will allow us to better investigate the role of BEAF in gene regulation in a genomic context.

535A Does the Boundary Element-Associated Factor (BEAF) play a broad role in maintaining global patterns of gene expression? Swarnava Roy, Craig Hart. Dept Biological Sci, Louisiana State Univ, Baton Rouge, LA. Communication between an enhancer and promoter can be blocked by placing an insulator element between them. Based in part on this property, it is thought that insulators divide chromatin into functionally independent domains and thus should play a global role in gene regulation. We previously found evidence for such a global role. A genetic screen was performed with a dominant negative form of BEAF, based on a rough-eye phenotype. Most genetic interactions that were uncovered were with other insulator binding proteins, general transcription factors, and transcription factors involved in various aspects of head development. We hypothesize that these interactions are caused by general effects on gene regulation. If this is true, then transcription factor expression patterns, expression levels or accessibility to binding sites in chromatin should be altered in the absence of BEAF. We are testing this using flies with a knockout mutation in BEAF that we obtained by homologous recombination. Transcription factor expression patterns and levels are being examined in embryos by immunostaining and FISH, and binding patterns are being examined by immunostaining polytene chromosomes from third instar larval salivary glands. Results of these experiments will be presented. 270 POSTERS: Chromatin and Gene Expression

536B Gene expression analysis of Drosophila melanogaster after acute and short intermittent and constant hypoxia treatment. Priti Azad1, Gabriel Haddad1,2. 1) Departments of Pediatrics (Section of Respiratory Medicine) and Neuroscience, University of California-San Diego, La Jolla, CA 92093, USA; 2) The Rady Children’s Hospital, San Diego, CA 92123, USA. Constant and intermittent hypoxia can occur under normal as well as pathological conditions. Intermittent hypoxia (IH) is associated with sleep apnea, central hypoventilation syndrome and vascular occlusion associated with sickle cell anemia. Constant hypoxia (CH) is linked with pulmonary disease, congenital heart diseases or high altitude. Although a number of experiments have been done to elucidate oxygen sensing and signaling mechanisms, the molecular mechanisms that lead to injury or adaptation to constant and intermittent hypoxia are not fully established. Previous comparative studies between constant and intermittent hypoxia have demonstrated differences in the kinetics of protein kinases and ROS- related signaling mechanisms in both in-vivo and in-vitro experiments. We investigate here the changes in gene expression in D. melanogaster after acute intermittent and constant hypoxia treatment for 2 hours. Our microarray analysis has identified multiple gene families that are up- or down-regulated in response to acute constant and intermittent hypoxia. Some of these gene families include protein kinases, alkaline phosphatases and cuticle proteins. Interestingly, some genes that are upregulated during CH are down-regulated in IH suggesting the response to both treatments may differ and involve distinctive as well as inter-related pathways. These data will provide further clues about the mechanisms by which intermittent and constant hypoxia lead to cell injury and morbidity or adaptation and survival.

537C An investigation into the molecular function of the hybrid incompatibility gene, Lhr. Shamoni Maheshwari, Daniel A Barbash. Dept Molecular Biol & Genetics, Cornell Univ, Ithaca, NY. Reproductive isolation between closely related taxa is maintained via hybrid incompatibility (HI), the sterility and inviability of interspecies offspring. The developmental breakdown in hybrids is thought to be the result of interactions between incompatible alleles from the hybridizing species. Hybrid sons produced from a cross between D. melanogaster mothers and D. simulans fathers die as third instar larvae. We recently identified a candidate pair of interacting alleles, Hybrid male rescue (Hmr) in D. melanogaster and Lethal hybrid rescue (Lhr) in D. simulans. The Lhr gene has functionally diverged between the two species; only the Lhr ortholog from D. simulans participates in the lethal interaction that causes incompatibility in F1 hybrid sons. We want to address the molecular basis of this incompatibility by identifying differences between D. simulans Lhr and D. melanogaster Lhr in hybrids. These differences could be because the two orthologs are a) expressed at different stages of development b) expressed in different cells of the developing organism c) localized differently at the subnuclear level or bind to different regions of chromatin/DNA and/or d) interacting with a different set of protein partners thereby altering their function. In order to carry out this comparison, we have constructed transgenic lines which express epitope-tagged LHR under its native promoter. We have preliminary data which suggests that LHR has a restricted developmental profile, it is expressed highest in embryos and is absent in adults. Using immunoflourescence we are able to detect heterochromatic localization of LHR in early embryos. This is consistent with published results which show that LHR interacts with Heterochromatin Protein 1 (HP1) in a yeast two-hybrid assay, and localizes to heterochromatin when expressed in salivary glands.

538A Tissue-specific contributions of the Drosophila LEM domain protein dMAN1 in the nuclear lamina. Belinda Pinto1, Shameika Wilmington2, Lori Wallrath1,2, Pamela Geyer1,2. 1) Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA; 2) Department of Biochemistry, University of Iowa, Iowa City, IA. The integrity and organization of the nucleus depends upon the nuclear lamina, a protein network underlying the inner nuclear membrane. Lamina components include the LEM domain proteins, named for LAP2, emerin and MAN1. These proteins associate with Barrier-to-autointegration factor (BAF), a non-specific DNA binding protein, to establish bridges between the nuclear envelope and chromosomes. Although LEM domain proteins are globally expressed, loss of these proteins causes tissue-restricted human diseases, including cardiomyopathy, muscular dystrophy and bone density disorders. To gain insights into contributions that LEM domain proteins make to nuclear lamina function, we isolated mutations in the gene encoding dMAN1, the homologue of the vertebrate MAN1/LEM2 proteins. dMAN1 contains an N-terminal LEM domain, two transmembrane domains, a DNA-binding MSC domain and a C-terminal UHM. Mutations in dMAN1 reduce viability and cause age-enhanced, tissue-specific phenotypes, including wing patterning and positioning defects, male sterility, reduced female fertility, aberrant climbing and flightlessness; mutant phenotypes rescued by expression of the full length protein. To understand the tissue-specific functions of dMAN1, we are using the GAL4-UAS system to direct cell-type restricted expression of the wild type protein and determine effects on mutant phenotypes. Using the neuronal elav driver, mutant phenotypes of decreased locomotion, wing positioning and reduced female fertility were rescued, but not wing patterning defects. In contrast, use of the muscle specific mef2 driver failed to rescue any mutant phenotypes. These findings suggest that dMAN1 is required for proper neuronal function. Data from our studies will be valuable in understanding how globally expressed LEM domain proteins contribute to tissue-specific nuclear lamina functions and provide insights into the pathogenesis of human disease. POSTERS: Chromatin and Gene Expression 271

539B Epigenetic Regulation of Gene Expression by Drosophila Myb and E2F2-RBF via the Myb-MuvB/dREAM Complex. Hong Wen1, Laura Andrejka1, Jonathan Ashton1, Roger Karess2, Joseph S. Lipsick1. 1) Dept Pathology & Genetics, Stanford Univ, Stanford, CA; 2) CNRS, Centre de Génétique Moléculaire, France. The Drosophila Myb oncoprotein, the E2F2 transcriptional repressor, and the RBF and Mip130/LIN-9 tumor suppressor proteins reside in a conserved Myb-MuvB (MMB)/dREAM complex. We now show that Myb is required in vivo for the expression of Polo kinase and components of the spindle assembly checkpoint (SAC). Surprisingly, the highly conserved DNA-binding domain was not essential for assembly of Myb into MMB/dREAM, for transcriptional regulation in vivo, or for rescue of Myb null mutants to adult viability. E2F2, RBF, and Mip130/LIN-9 acted in opposition to Myb by repressing the expression of Polo and SAC genes in vivo. Remarkably, the absence of both Myb and Mip130, or of both Myb and E2F2, caused variegated expression in which high or low levels of Polo were stably inherited through successive cell divisions in imaginal wing discs. Restoration of Myb resulted in a uniformly high level of Polo expression similar to that seen in wild type tissue, whereas restoration of Mip130 or E2F2 extinguished Polo expression. Our results demonstrate epigenetic regulation of gene expression by Myb, Mip130/LIN-9, and E2F2-RBF in vivo, and also provide an explanation for the ability of Mip 130 null mutants to rescue the lethality of Myb null mutants in vivo.

540C Genetic analyses of bocksbeutel and otefin: genes encoding nuclear lamina LEM domain proteins. Shameika Wilmington1, Emma Hornick1, Belinda Pinto2, Lori Wallrath1,2, Pamela Geyer1,2. 1) Department of Biochemistry, University of Iowa, Iowa City, IA; 2) Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA. Proteins in the nuclear envelope and underlying lamina establish a network of interactions that provide mechanical stability to the nucleus and contribute to the regulation of nuclear trafficking, signal transduction and gene expression. One family of nuclear lamina proteins possesses the LEM (LAP2, emerin, MAN1) domain, an ~ 40 amino acid domain that associates with the DNA binding protein Barrier-to-autointegration factor (BAF). LEM domain proteins have redundant lamina functions that may involve anchoring chromatin to the nuclear periphery. LEM domain proteins are expressed in most cells, yet loss of these proteins leads to tissue- specific human diseases. Loss of emerin causes Emery-Dreifuss muscular dystrophy, while loss of MAN1 causes a bone density disorder. The Drosophila genome encodes five LEM homology proteins, Bocksbeutel, Otefin, dMAN1, CG3748 and CG8679. To understand the function of these proteins, genetic analyses of the genes encoding the putative emerin homologues, bocksbeutel (bocks) and otefin (ote), were undertaken. Two null alleles of bocks were generated by imprecise P excision. Flies homozygous for either allele are viable, fertile and show no visible adult phenotypes. An ote allele, oteB279, that carries a piggybac transposon inserted into the ote open reading frame, causes a loss of the C-terminal half of the protein. Flies homozygous for oteB279 are viable, female sterile and show no visible adult phenotypes. Interestingly, phenotypes caused by loss of bocks and ote are distinct from those caused by loss of dMAN1, where homozygous null flies show reduced viability and pleiotropic developmental defects. Evidence for shared function among the Drosophila LEM domain proteins was obtained by double mutant analyses; the dMAN1, ote double mutants die around the time of eclosion. Taken together, our data suggest that Drosophila LEM domain proteins make both distinct and over-lapping tissue-specific contributions to lamina function.

541A Defining the requirements for Polycomb group response elements (PREs) at the engrailed locus. Melissa Durant, Judy Kassis. NICHD, NIH, Bethesda, MD. The Polycomb group proteins (PcGs) play a vital role throughout development by maintaining precise gene expression patterns. It is known that PcG-mediated gene silencing is achieved through Polycomb Response Elements (PREs), however the mechanism for establishing this silencing is poorly understood. Currently, the requirements and composition of a working PRE are not fully understood. Recent work by Ringrose et al. (2006) attempted to narrow the definition of what protein binding sequences best define a PRE, resulting in an algorithm that aids in the prediction of PREs for any given DNA sequences. Here, I put these predictions to use in attempting to uncover PREs located within the engrailed (en) / invected (inv) domain. Previous evidence suggests many functional similarities between en and inv, and it is thought that the two genes may share regulatory elements, including PREs. Examination of DNA surrounding inv uncovered a number of potential PRE regions. We are testing the ability of these predicted PREs to maintain engrailed-like expression patterns of the lacZ reporter gene, as well as their ability to establish pairing-sensitive silencing of the mini-white reporter in pCaSpeR. Results of our on-going experiments will be presented. 272 POSTERS: Chromatin and Gene Expression

542B Functional analysis of the Polycomb repressive complex 2 (PRC2) histone methyltransferase. P Joshi1, N Jahren1, K Hines1, J Wang1, C Ketel1, D Cryderman2, E Miller1, E Carrington3, RS Jones3, LL Wallrath2, JA Simon1. 1) Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2) University of Iowa; 3) Southern Methodist University. Polycomb group (PcG) proteins form complexes that maintain repressed chromatin states. Fly PRC2 contains four core subunits, ESC, E(Z), SU(Z)12 and NURF55, and it methylates lysine 27 of histone H3. This modification marks PcG-repressed chromatin and helps recruit another PcG complex, PRC1. E(Z) is the catalytic subunit of the histone methyltransferase (HMTase), but it cannot work alone. ESC and SU(Z)12 are both key potentiators of E(Z) HMTase. In addition, several E(Z) domains besides the catalytic SET domain are required for HMTase. We wish to determine how these multiple inputs contribute to PRC2 HMTase function. We are testing recombinant PRC2 complexes bearing mutations that mimic loss-of-function alleles and/or that alter residues predicted to be critical for function. HMTase levels are measured by in vitro methyltransferase assay, with lysine-substituted histone mutants and mass spectrometric analysis of reaction products used to track lysine specificity and ability to produce mono-, di- and tri-methylated H3-K27. These studies define regions of the E(Z) SET domain, and surface loops of ESC, that are key for function. Since certain ESC mutations can disrupt HMTase without perturbing PRC2 binding to nucleosomes, ESC may potentiate by optimizing the E(Z) active site rather than by anchoring PRC2 to the nucleosome substrate. We also find that an ESC mutant lacking its 65 amino acid N-terminal tail, but retaining its β-propeller domain, can assemble into enzymatically active PRC2. Transformation rescue tests are underway to assess if the ESC tail, thought to harbor sites for ESC phosphorylation, is required for ESC function in vivo. We will also report progress on expressing LacI fusions to PRC2 subunits in order to engineer and study ectopic sites of H3-K27 methylation in vivo. Collectively, these studies will address requirements for and consequences of PRC2 histone methylation.

543C Recruitment of Drosophila Polycomb-group proteins by a novel Polycomblike containing complex. Urmi Savla, Judith Benes, Richard Jones. Dept Biological Sci, Southern Methodist Univ, Dallas, TX. Trithorax-group (trxG) and Polycomb-group (PcG) proteins respectively maintain the transcriptionally active and silent states, of Hox genes of the Antennapedia and bithorax gene complexes. To date three polycomb group proteins have been purified: PRC1, PRC2 and PhoRC. In embryos PCL is included in a subset of PRC2 complexes. PcG silencing requires the presence of a Polycomb Reponse Element (PRE), which contains binding sites for multiple proteins including PcG proteins PHO and PHO-LIKE (PHOL). Based on wing imaginal disc studies (in which Ubx is repressed) we have previously proposed a hierarchical binding pathway of PcG proteins to a PRE within the bxd regulatory region of the Hox gene Ubx. PHO and PHOL directly bind at the bxd PRE and recruit the PRC2 complex which trimethylates H3K27 facilitating recruitment of the PRC1 complex. We will describe a novel PCL- containing complex from larvae that is distinct from PRC1 and PRC2, but is required for chromosome binding by them and other PcG complexes.

544A Dissecting the functions of the CHD1 chromatin remodeling factor. Jennifer Armstrong, Ivy McDaniel, Kimia Raafat, Michael Dauer. Joint Science Dept, Claremont Colleges, Claremont, CA. The CHD1 chromatin remodeling factor is named for its N-terminal tandem chromodomains, a centrally located helicase/ATPase domain, and a C-terminal DNA-binding domain. CHD1 co-localizes with the elongating form of RNA polymerase II on third instar larval polytene chromosomes, suggesting that CHD1 may facilitate the passage of Pol II through chromatin during transcriptional elongation. To understand the function of the CHD1 chromatin remodeler, we generated null mutations in the chd1 gene. The chd1 gene is not essential, as homozygous chd1 mutant flies are viable. This is in contrast to the essential genes kismet, dMi-2, brahma, and ISWI, which encode related chromatin remodeling factors. Although not essential for viability, CHD1 is required for distinct developmental processes. chd1 mutants display notched wing margins, mutant males are sterile, and mutant females display greatly reduced fertility. Using the GAL4 driver system, we have characterized the consequences of overexpression of CHD1. Unexpectedly, overexpression of CHD1 results in females that retain their eggs. As a chromatin remodeling factor, CHD1 may affect higher order chromatin function. We have examined the consequences of both loss and gain of CHD1 function on polytene chromosomes. Loss of CHD1 does not obviously affect polytene chromosome structure, while gain of CHD1 function leads to decondensed regions of chromosomes, suggesting that the function of CHD1 may be to create open, transcriptionally accessible chromatin. POSTERS: Chromatin and Gene Expression 273

545B Blimp-1 Regulates Pupal Eye and Head Development. Gerald Call1, Joy Wu2, Chris Bui2, Stacy Chan2, Jingwen Tan2, Amrita Cheema2, Jiong Chen2, Utpal Banerjee2. 1) Dept of Pharmacology, Midwestern University, Glendale, AZ; 2) Molecular, Cellular and Developmental Biology, University of California, Los Angeles, Los Angeles, CA. B lymphocyte-induced maturation protein (Blimp-1) was recently identified in Drosophila melanogaster, but its function is still largely unknown. Blimp-1 contains a SET domain and a DNA-binding domain of five zinc fingers. Whether through direct or by recruiting other histone methyltransferases, Blimp-1 acts as site-directed chromatin remodeling factor. Using the ey-Flp/FRT system to induce Blimp-1 mutant clones in the eye in the presence of a Minute mutation leads to late pupal lethality. However, without the Minute mutation, Blimp-1 clones result in a unique raised glossy eye phenotype, reduced head size and bristle defects in adult flies. Staining with various developmental markers indicate that larval eye development is normal. However, staining in pupal eye discs reveal that the Blimp-1 mutation leads to cone cell patterning defects and loss or inappropriate development of bristle and tertiary pigment cells. The raised glossy adult eye may indicate overexpression of the lens protein, Crystallin (Cry). In situ hybridization for Crystallin demonstrated varying expression in Blimp-1 mutant pupal eye discs, suggesting that Crystallin expression may be regulated by Blimp-1. A Cry-LacZ stock is being used to further investigate the interaction between Blimp-1 and Crystallin. Preliminary results show that Crystallin expression is up-regulated in Blimp-1 mutant tissues compared with the neighboring wild-type tissue. Further studies will be performed to determine the role Blimp-1 of Blimp-1 in bristle development.

546C Functional cooperation between the Brahma chromatin remodeling complex and histone demethylase enzymes. Brenda J Curtis1, Daniel R Marenda2, Claudia B Zraly1,3, Andrew K Dingwall1,3,4. 1) Biochemistry Program, Loyola University Chicago, SSOM, Maywood, IL; 2) Dept Biological Sciences, Univ Sciences-Philadelphia; 3) Oncology Institute; 4) Dept Pathology. Eukaryotic organisms have evolved intricate means of organizing a seemingly overwhelming amount of genetic information by forming chromatin. In order for essential processes such as DNA replication, repair, recombination, and gene expression to occur, DNA must be accessible to regulatory proteins and transcriptional machinery. The regulation of chromatin status is two-fold: i) N- terminal histone tail modifications allow for recruitment of regulatory proteins; ii) energy-dependent multimeric complexes catalyze alterations in the physical contacts between DNA and histones. The most widely studied ATP-dependent chromatin remodeling complex is the evolutionarily conserved SWI/SNF complex. The Drosophila SWI/SNF counterpart is known as the Brahma (Brm) complex. Our research is focused on understanding the functional role of one of the key conserved core subunits, SNR1, that functions in part to restrict complex activity on important wing patterning genes during development. An antimorphic temperature- sensitive mutant allele (snr1E1) leads to target gene misregulation and formation of extra wing vein material that can be suppressed by mutations in the brm gene. We utilized an unbiased chromosomal deficiency screen to look for dominant enhancement or suppression of the snr1E1 wing patterning phenotype. We observed that large deficiencies that removed histone demethylase genes affected the mutant snr1E1 phenotype. To verify the screen results and test for a direct link between histone demethylating enzymes and the Brm complex, we next examined loss of function alleles of several histone demethylase genes for an enhancement or suppression effect. We found that Su(var)3-3/dLsd1 and certain members of the Jumonji family genetically interact with snr1E1, suggesting functional cooperation between chromatin remodeling complexes and demethylating enzymes in regulating gene expression. 274 POSTERS: Neurogenetics and Neural Development

547A The divergent TGF-β ligand Dawdle utilizes an activin pathway to influence neuronal function in Drosophila. Kavita Arora1, Jeremy Ellis1, Minh Nguyen1,2, Louise Parker1,3. 1) Dept Developmental & Cell Biol, Univ of California, Irvine, CA; 2) NIH/NIDCR Bethesda, MD; 3) Univ of California, Berkeley, CA. Axon guidance is regulated by intrinsic factors and extrinsic cues provided by other neurons, glia and target muscles. Dawdle (Daw), a divergent TGF-β superfamily ligand expressed in glia and mesoderm, is required for embryonic motoneuron pathfinding in Drosophila. In daw mutants, ISNb and SNa axons fail to extend completely and are unable to innervate their targets. We find that Daw initiates an activin signaling pathway via the receptors Punt and Baboon (Babo) and the signal-transducer Smad2. Furthermore, mutations in these signaling components display similar axon guidance defects. Cell-autonomous disruption of receptor signaling suggests that Babo is required in motoneurons rather than in muscles or glia. Ectopic ligand expression can rescue the daw phenotype, but has no deleterious effects. Our results indicate that Daw functions in a permissive manner to modulate or enable the growth cone response to other restricted guidance cues, and support a novel role for activin signaling in axon guidance.

548B Role of the Liprin family of proteins in axon targeting and synapse formation. Sergio Astigarraga, Kerstin Hofmeyer, Reza Farajian, Jessica Treisman. Developmental Genetics, Skirball Institute, New York University School of Medicine, New York, NY. In a genetic mosaic screen for genes required in photoreceptors for their normal projection pattern into the brain, our group identified null mutations in two genes that had similar effects on R7 axon targeting, Liprin-α and LAR. R7 axons mutant for either of these genes terminate prematurely in the R8 target layer in the medulla, instead of projecting deeper to reach their correct target layer. Liprin-α has also been shown to work together with LAR in regulating synapse size and shape at the larval neuromuscular junction (NMJ). LAR is a receptor protein tyrosine phosphatase, and Liprin-α binds to the intracellular domain of LAR. Liprin-α does not control LAR subcellular localization in Drosophila, as it does in C. elegans and in mammalian cells, suggesting that it has a signaling function downstream of LAR. In addition to Liprin-α, the Drosophila genome contains two more Liprin homologues. Liprin- β is closely related to vertebrate Liprin-β family members, while Liprin-γ has a closer uncharacterized vertebrate homologue. In situ hybridization to embryos shows that while the expression of Liprin-α and Liprin-γ is restricted to the nervous system, Liprin-β is ubiquitously expressed. In order to study the possible role of these Liprins during nervous system development we generated mutations in Liprin-β and Liprin-γ by imprecise excision of a P-element and X-ray mutagenesis, respectively. Mutant alleles that abolish the expression of Liprin-β and Liprin-γ do not affect viability or fertility. Although Liprin-β and Liprin-γ mutants show no obvious R7 targeting defects, double and triple mutant phenotypes suggest that Liprin-β and Liprin-γ may antagonize Liprin-α function. We have also found that Liprin-α physically interacts with both Liprin-β and Liprin-γ, but that in S2R+ cells it colocalizes primarily with Liprin-γ. We are currently testing the effects of Liprin-β and Liprin-γ on NMJ synapse formation.

549C Dissecting Dscam Localization and Signaling. Rachel Bortnick1, Hyoje Ryu1, Dan Dascenco1, Maria-Luise Erfurth1, Akhila Parthasarthy1, Michael Hughes2, Dietmar Schmucker1. 1) Department of Cancer Biology, Dana Farber Cancer Institute; Program in Neuroscience, Harvard Medical School, Boston, MA; 2) Department of Pharmacology, Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, PA. Drosophila Dscam is a single pass transmembrane protein of the immunoglobulin (Ig) superfamily that is alternatively spliced to up to 38, 0016 different transcripts and has important functions in the fly nervous and immune systems. A number of studies have suggested that each neuron expresses a unique set of Dscam isoforms. Each Dscam isoform is in turn capable of homophilic binding. Dscam has been shown to be required for precise axon targeting and branching in a variety of experimental systems, including Bolwig’s organ, olfactory neurons, the mushroom body, and mechanosensory neurons. In addition, recent evidence suggests that Dscam is required for dendritic self-avoidance and proper dendritic field development. The current model posits that the isoform specificity of Dscam enables sister-neurites to recognize and avoid each other and thereby elaborate appropriate dendritic fields. However, the nature of this repulsive signaling pathway is unclear and in vivo characterization of downstream effectors remains rudimentary. Moreover, the mechanism by which Dscam is localized to axons and dendrites is entirely unknown. The C-terminus of Dscam contains a putative PDZ-binding domain, and previous studies in our laboratory suggest an important role for the PDZ binding site in Dscam localization and/or signaling. We are currently conducting a genetic candidate screen examining whether any PDZ domain-containing or other signaling components are involved in the Dscam pathway. POSTERS: Neurogenetics and Neural Development 275

550A Transcriptional Regulation of Commissureless. Casey Jowdy, Mark Seeger. Dept. of Molecular Genetics and Center for Molecular Neurobiology, The Ohio State University, Columbus, OH. Commissureless (Comm) is required for proper commissural axon guidance and is a key regulator of the Slit-Robo signaling pathway. Comm functions by complexing with Robo receptors and preventing their cell surface accumulation. This post-translational regulation of Robo receptor distribution by Comm ensures that commissural growth cones will be unresponsive to Slit in the local environment, allowing commissural axons to the cross the CNS midline boundary. Tight temporal and spatial regulation of Comm transcription in commissural neurons appears critical for ensuring proper regulation of Robo. Not surprisingly, the Comm transcription pattern is quite dynamic in the developing embryonic CNS and different results suggest that the cis-regulatory regions for Comm are expansive and complex. We are pursuing two strategies to define and characterize the Comm cis-regulatory regions. First, we have created a set of molecularly defined deletions that remove varying regions of genomic DNA 5’ and 3’ of the Comm transcription unit. The effects of these deletions on Comm expression and commissure formation are being determined to define sequences that are necessary for proper Comm expression. Second, we are generating and testing genomic rescue constructs of Comm using BAC recombineering and the P[acman] transformation vector. These experiments will define sequences that are sufficient for normal Comm expression.

551B Vav, a Rho GTPase activator, controls axon guidance during Drosophila embryonic and larval development. Marianne BY Malartre, Maria D Martin Bermudo. Centro Andaluz de Biologia del Desarollo, Seville, Spain. The regulation of the actin cytoskeleton is a crucial event, which controls many biological processes such as cell division, adhesion and migration. This is mainly achieved by guanine exchange factors (GEFs), such as the vav proteins, that trigger the activation of the Rho GTPases. Despite their intense characterization in mammals, there is little information regarding the role of vav during development. Here we show that the unique Drosophila vav homolog is mostly expressed in the developing ventral midline, in both glial and neuronal cell populations, which prompted us to investigate a potential role for this gene in axon guidance. We have generated vav null mutant alleles and we show that mutant embryos display a midline crossing phenotype. Vav, like trio, is one of several GEFs, expressed in the CNS that can activate the rac GTPase. Strikingly we found that the vav;trio double mutants, exhibit a stronger phenotype than the single mutants, and which recapitulate the rac mutants defects. We propose that both GEFs control different aspects of rac-mediated CNS formation, according to their distinct expression patterns. We also show that vav mutants display defects in the targeting of photoreceptor axons in the larval optic lobe, suggesting that vav participate to a general rather than specific mechanism to control axon pathfinding. Moreover, rescue experiments demonstrate that vav is required within the photoreceptors to control their axon projections and that trio is not able to replace vav function in this system. Finally, unlike rac mutants, vav mutant eye discs exhibit ommatidia rotation defects that could also affect photoreceptor axons projection. Our results suggest that vav is able to regulate photoreceptor axon targeting through the control of both axon guidance via the activation of rac and ommatidia rotation via a rac-independent signalling pathway. Our model support the idea that vav, the only GEF that can be phosphorylated and contains several protein interaction domains, is a pleiotropic protein acting at the crossroads of multiple signalling pathways.

552C Analysis of the Drosophila crmp gene to determine the role of the CRMP protein in neurogenesis. Deanna Morris, John Rawls. Department of Biology, University of Kentucky, Lexington, KY. CRMP isoforms mediate growth cone dynamics and neuron polarity in cultured mammalian neurons through associations with a variety of signal transduction components and cytoskeletal elements. CRMP is a member of a protein family including the presumably ancestral DHP protein that carries out the second step in pyrimidine degradation. In Drosophila, CRMP and DHP proteins are produced by alternatively spliced transcripts of the crmp gene and are 91% identical, suggesting that the distinct functions of the two proteins are derived from differences in a small region of the protein. The two fly proteins appear to be expressed in non-overlapping sets of tisues, with CRMP found exclusively in neuronal tissues and DHP rather ubiquitously expressed in non-neuronal tissues. Loss-of-function mutantions of crmp that lack both proteins have been isolated; homozygous animals display DHP-null phenotypes but exhibit no overt developmental or neurological defects. Closer examination of crmp mutant embryos during neurogenesis is underway. Recently, defects in learning and memory have been detected in CRMP knockout mice, pointing toward new phenotypes to assess in Drosophila loss-of-function crmp mutants. Results of these experiments will be presented. 276 POSTERS: Neurogenetics and Neural Development

553A Dissecting the Frazzled cytoplasmic domain in-vivoand a genetic screen for novel regulators of axon guidance at the Drosophila midline. Mike P. O’Donnell, David S. Garbe, Greg J. Bashaw. Department of Neuroscience, University of Pennsylvania, Philadelphia, PA. Frazzled/DCC and Netrin constitute a phylogenetically conserved receptor-ligand pair that can induce outgrowth and attractive axon turning. Conserved cytoplasmic P-domains of DCC/Fra have been implicated as necessary mediators of the Netrin-induced signal. Studies in C. elegans have implicated the P1 and P2 domains in outgrowth, while conflicting vertebrate data has suggested that the P3 domain, but not P1 or P2, is required for Netrin-dependent outgrowth and turning. At least two models have been proposed to explain P3-mediated attraction, either through receptor self-association or through recruitment of focal adhesion kinase(FAK). We are examining the ability of Fra transgenic constructs to rescue midline axon crossing of a small subset of neurons, the EW neurons, in fra mutants. We have shown that the P3 domain is required for midline attraction in-vivo using this approach, whereas the P1 and P2 domains are dispensable. Interestingly, deletion of the P3 domain does not abrogate self-association, suggesting an alternative function for this domain in Drosophila. In vertebrates, Src family tyrosine kinases are important for Netrin- induced outgrowth and attraction in-vitro. As Drosophila Fra is phosphorylated in cell culture, the requirement for c-terminal phosphorylation of Frazzled in-vivo is being examined using this system. Additionally, expression of a truncated Frazzled receptor in which most of the cytoplasmic domain is deleted (FraΔC) causes defects in axon attraction, presumably by interfering with endogenous fra function. However, expressing this construct in a background lacking fra function causes more severe defects than a fra mutant, suggesting interference with additional attractive or repulsive pathways. Using this truncated receptor to create a sensitized genetic background, we are screening for novel mediators of axon guidance at the Drosophila midline. M. O’Donnell supported by Training Program in Cell and Molecular Biology T32 GM07229.

554B Helmsman is Expressed in Trachea and Retina: inactivation alters tracheal morphology and visually guided behavior. John Pollock1, James McKay2, Barbara Nightingale1. 1) Dept Biological Sci, Duquesne Univ, Pittsburgh, PA; 2) Department of Molecular Biology, University of Texas Southwestern Medical Center Dallas, TX. We have identified helmsman (hlm), which is expressed in the fruit fly photoreceptor cells during neural network development. Helmsman is also expressed in the elongating cells of the embryonic trachea. Both photoreceptor neurons and embryonic trachea cells elongate in precise, targeted growth for cell-to-cell specific recognition. Expression of anti-sense hlm interfering RNA during embryogenesis arrests elongation of the developing tracheal cells and blocks maturation. Expression of hlm interfering RNA during visual system formation results in reduced visual acuity and poor performance in optomotor response, indicative of abnormal neural network development. Helmsman is a unique cell surface protein with Complement-like protein interaction motifs. We have also cloned Helmsman from Lucilia cuprina (Australian blow fly), which is approximately 100 million years divergent from Drosophila, and find a remarkable 90% protein identity over the entire 558 amino acid protein. Analysis of the Helmsman sequence found in other species indicates a significant evolutionary pressure to maintain the Helmsman protein sequence. Our interpretation is that Helmsman is involved in cell maturation in both the elongating trachea and elongating photoreceptor cells. Cell adhesion and cell signaling, which are known to use immunoglobulin-like cell adhesion molecules, may use molecular systems analogous to Complement to create protein complexes to regulate growth.

555C Remodeling the larval motor system during metamorphosis: the fate of motor neuron subsets. Soumya Banerjee, Holly Rataiczak, Meredith Dorr, Badrinath Krishan, Joyce Fernandes. Zoology, Miami University, Oxford, OH. Holometabolous insects, such as fruit fly, Drosophila and moth, Manduca develop through distinct life stages, embryo, larva, pupae and adult. During the pupal phase, the larva is transformed into a morphologically and structurally distinct adult stage. This transformation is a key feature of holometabolous insects, known as metamorphosis. An intriguing feature of remodeling is that new neural circuits are established and consequently new behaviors are manifested. Adult motor neurons are thought to be persistent larval neurons and they connect with newly generated interneurons to establish an adult specific neural circuit. Thus, motor neurons once innervating larval muscles to regulate crawling and feeding movement may be respecified for adult specific function such as flight, walking, copulation, etc. During metamorphosis, locomotor control is shifted to the thorax, and this is accomplished by morphological changes in the CNS. The thoracic ganglion expands in size, whereas the abdominal ganglion is reduced. We are interested in examining how these changes are correlated with motor neuron remodeling in the CNS as well as in the periphery. In Drosophila, the identity of 34 motor neurons that innervate 30 skeletal muscles in each hemisegment is known, but their fate during metamorphosis is not understood. We are using neuronal drivers that label subsets of motor neurons to follow the fate of motor neurons during metamorphosis. One such label is dHb9, which innervates ventral and dorsal muscle targets in the larval stage. Our preliminary data suggests that the number of dHb9 expressing neurons is reduced during metamorphosis and that cell death is likely to be involved. POSTERS: Neurogenetics and Neural Development 277

556A CycE regulated cell fate specification in the embryonic CNS. Christian Berger1, Ramakrishnan Kannan2, LS Shashidhara2, Gerd M Technau1. 1) Inst Genetics, Johannes-Gutenberg Univ, Mainz, Germany; 2) Centre for Cellular and Molecular Biology, Hyderabad, India. The mechanisms leading to cell diversity in the central nervous system (CNS) represent a major unsolved problem. In the Drosophila embryo an array of 30 NBs per truncal hemisegment give rise to the CNS. NBs born at the same time, at the same position and expressing the same molecular markers acquire corresponding identities irrespectively of the segment they are located in. However, lineage analyses showed that five of these serially homologous NBs generate lineages that differ significantly in thorax and abdomen. We have examined the events by which segment-specific differences are generated within a neuroblast (NB) lineage in the embryonic CNS. NB6-4 generates both neuronal and glial cells in thoracic but only glial cells in abdominal segments. We have recently shown that this difference is dependent on the function of CycE. CycE is necessary and sufficient to generate neuronal cells in thoracic segments, where it is expressed exclusively in the neuronal part of the lineage. Loss of CycE leads to loss of neuronal cells in the thoracic lineage, whereas gain of CycE leads to de novo formation of neurons from the abdominal lineage. Intriguingly, all other regulators of cell cycle tested did not give any such phenotypes leading to the hypothesis that this new function of CycE in cell fate specification might be independent of its role in cell cycle progression. To further test this hypothesis, we are following different approaches: a) analyzing the function of CycE in the background of string mutation - NBs are born, but do not undergo divisions, b) generation of transgenic lines expressing various CycE-deletion constructs to test whether we can separate the function of the protein in regulating cell division and in cell fate specification. Furthermore, we are testing the impact CycE might have on the asymmetric localization machinery, which is used to generate two different cell types from a common precursor. Results from these approaches will be presented.

557B Development of the adult abdominal ganglion during metamorphosis: formation of the terminal nerve trunk. Meredith Dorr, Camilo Molina, Kathleen Broomall, Rakesh Rachamaduggu, Joyce Fernandes. Dept Zoology, Miami Univ, Oxford, OH. During its life cycle, Drosophila makes two motor systems: embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval motor system is restructured to give rise to the adult counterpart, a process that is integrated into the overall remodeling of the nervous system. One feature of the restructuring is that the thoracic ganglion expands and the abdominal ganglion reduces in size; this occurs to accommodate the shift of the control of locomotion to the thorax. The adult abdominal ganglion is reduced in size compared to its thoracic counterpart, and it does not function in locomotion. We have been studying the nature of the transition during metamorphosis and our goal is to understand how characteristics of the adult ganglion are established, such as the number of nerves, identity of persistent larval motor neurons, and new motor units. Our preliminary data indicate that the adult pattern of innervation involves a fusion of larval abdominal segmental nerves, specifically A4-A8, to form a terminal nerve trunk that then defasciculates in the peripheral bodywall. It is likely that glial remodeling is involved in this transition. We are also interested in examining how CNS restructuring relates to events in the periphery such as muscle development and NMJ formation.

558C Role Of ZFH-2 in Drosophila gene insulation and apoptosis. Ananya Guntur, Martha Lundell. Dept Biol, Univ Texas, San Antonio, San Antonio, TX. Zinc finger homeodomain-2 (ZFH-2) is a 330KD protein that encodes 16 zinc fingers and 3 homeodomain motifs. ZFH-2 is expressed in the embryonic CNS, wing and leg imaginal disc of 3rd instar larvae and in ovaries of adult fly. We are examining two different functions of ZFH-2 in Drosophila: 1) it acts as a component of gene insulation and 2) it can activate apoptosis. We are investigating the insulation function using a daughterless mutation dalyh which is a Springer retrotransposon insertion that disrupts wild-type da gene expression in the ovary by insulating enhancer and promoter interactions of the da gene. A mutation in zfh-2 suppressed this phenotype. We propose that ZFH-2 must bind to Springer element to promote insulation. Both molecular and genetic data support this model. We are investigating the apoptosis function of ZFH-2 using various mutant alleles of zfh-2 that rescue neurons that normally undergo apoptosis in the wild-type CNS. This has been observed in two different well characterized neuroblasts lineages, the 7-3 serotonin lineage and the 7-1 EVE+ lineage. TUNEL analysis on zfh-2 mutant brains show that there is marked decrease in the number of cells undergoing apoptosis when compared to wild-type. In addition immunohistochemistry for axonal markers 22C10 and FAS 11 suggests that there is an increase in the axonal density in the CNS, which we believe is due to an increase in the number of cells that fail to undergo apoptosis. We are currently examining how ZFH-2 may alter specific components of the apoptosis pathway. 278 POSTERS: Neurogenetics and Neural Development

559A Histone Deacetylase 1 (Rpd3) in Dendrite Targeting of Drosophila Olfactory Projection Neurons. Takahiro Chihara1,3,4, Joy S. Wu1,2,4, Liqun Luo1,2. 1) HHMI and Dept Biol; 2) Neuroscience program, Stanford University. Stanford, CA, USA; 3) Dept Gen, Grad Sch Pharmaceut, University of Tokyo, Tokyo, Japan; 4) These authors contributed equally to this work. The complex neural network in the brain results primarily from simple connections between axons and dendrites. Although axon targeting has been widely investigated, little is known about the mechanisms that regulate dendrite targeting. To reveal the molecular mechanisms for dendrite targeting, we utilize Drosophila olfactory projection neurons (PNs) whose dendrites precisely target one of the ~50 glomeruli in the antennal lobe. We performed a MARCM-based genetic mosaic screen with EMS mutants and identified several mutants affecting different aspects of dendrite morphogenesis, including dendrite targeting. Among these, one mutant has a missense mutation in the Rpd3 gene encoding Drosophila Histone Deacetylase 1 (HDAC1). Single-cell PN clones homozygous for Rpd3 show a mistargeting phenotype from the laterally located glomerulus DL1 to more medial glomeruli. Postmitotic expression of a UAS-Rpd3 transgene rescues the dendrite mistargeting phenotype of Rpd3 PN clones. Interestingly, mutations in the transcription factor Prospero yield similar dendrite mistargeting phenotypes, and overexpression of Prospero significantly suppresses the dendrite mistargeting phenotype of Rpd3 PN clones. These results suggest that regulation of a single transcription factor may account for a large fraction of the function of a ubiquitous chromatin remodeling factor in regulate neuronal connectivity of postmitotic neurons.

560B Protein stability of Glide/GCM regulated by F-box proteins Slimb and Archipelago during Drosophila glial development. Margaret Ho1, Hungwen Chen2, Angela Giangrande3, Cheng-Ting Chien1. 1) Dept Molecular Biol, Academia Sinica, Taipei, Taiwan; 2) Dept Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3) Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France. Gliogenesis in animal development is regulated spatiotemporally for precise control of proliferation, specification and differentiation. In Drosophila, the glial regulatory gene, glial cell deficient/glial cell missing (glide/gcm), serves as a binary switch between neuronal and glial cell fate during development, and promotes glial differentiation. Here we describe the ubiquitin-proteasome regulation of the GCM protein. GCM binds to Supernumerary limbs(Slimb) and Archipelago (Ago), two F-box proteins known to be adaptors of SCF E3 ligases. GCM is controlled by the 26S proteasome for its protein stability and inefficiently ubiquitinated when expressions of slimb and ago are silenced. In slimb and ago double mutants, glial cells improperly proliferate which can be phenocopied by increasing GCM expression during glial differentiation. Upregulated GCM protein levels also result in various defects such as incorrect lineage composition, target gene misexpression, disoriented glial migration, and shortening of the glial processes. We propose that downregulating GCM protein levels by Slimb and Ago ensures proper glial differentiation.

561C Degeneration of optic lamina caused by defective endocytic function in glial cells. Yuan-Ming Lee1,2, Y. Henry Sun1,2. 1) Inst Molecular Biology, Academia Sinica, Taipei, Taiwan; 2) Inst of Genomic Science, National Yang Ming University, Taipei, Taiwan. The visual system is composed of neurons and glial cells. In the optic lamina and medulla, there are also several distinct groups of glial cells. These are known to play a role in photoreceptor axonal projection and maintain the physiological function of the lamina monopolar neurons (L1-L5). We are interested in testing whether the glias play any function in the adult visual system. Since it was reported that the engulfing action of glia is involved in axon pruning (Awasaki and Ito, 2004), we blocked the endocytic pathway by expressing the temperature-sensitive dominant-negative dynamin (shibire) in glia and examined for the effect on the retina and the optic lobe in the brain. Shifting to non-permissive temperature caused the formation of hollow areas in the optic lamina. There is an increase of apoptosis, but the phenotype cannot be rescued by the coexpression of the anti-apoptotic p35. This result suggests that either the major defect is not due to increased apoptosis, or the apoptosis is caspase-independent. Our analyses also suggest that the hollows are formed cell-autonomously by the epithelial and marginal glia in the optic lamina. Therefore the endocytic function is required for the glia to survive. POSTERS: Neurogenetics and Neural Development 279

562A Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Christian M. von Hilchen, Ruth Beckervordersandforth, Christof Rickert, Benjamin Altenhein, Gerhard Technau. Institute of Genetics, Johannes Gutenberg University, Mainz, Germany. Peripheral glial cells (PGs) are required for the correct establishment of the peripheral nerves and the formation of the blood/nerve barrier. The embryonic PGs originate from sensory organ precursor cells and CNS neuroblasts, and they migrate over long distances to align along and finally ensheath the peripheral nerves. In order to gain more insights into the development of the PGs on the cellular level, we studied the spatial and temporal pattern, identity, migration, and origin of all PGs in truncal segments of wildtype embryos. We present a catalogue of markers, whose expression patterns indicate the establishment of individual cell identities and allow their discrimination. We uncovered the origin of each of the PGs by cell lineage analysis and linked them to identified central and peripheral neural stem cells. Using confocal 4D microscopy and GFP expression, we traced in vivo the migratory behaviour of the individual PGs during the course of embryonic PNS development. The order in which the individual PGs migrate into the periphery and their final positioning shows only minor variations. This detailed description of peripheral glia development and migration provides the basis for the examination of phenotypes on a single cell level under various mutant and experimental conditions.

563B Cultured Primary Mushroom Body Neurons Reveal a Notch-Deficient Phenotype. Randy Boyles1, Kate Shen1, Robert Kraft2, Linda L. Restifo2, Andrew Andres1. 1) School of Life Sciences, Univ Nevada - Las Vegas, Las Vegas, NV; 2) Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, Arizona 85721. Notch signaling, a pathway conserved through all metazoans, has been well characterized for its involvement in important cell fate decisions in the developing nervous system. Recent evidence suggests a role for Notch in post mitotic adult nervous systems specifically pertaining to synaptic plasticity and the preservation of neuronal ultru-structures. We, and others have previously shown that Notch is also necessary for long-term memory using a Pavlovian olfactory conditioning paradigm. We are studying the molecular pathway responsible for the effects of Notch signaling on the structure and function of specific neurons that can also be used to test long-term memory impairments. To that end we are using cultured primary neurons from third instar larvae that have components of the Notch pathway silenced in the mushroom bodies using transgenic RNAi strategies. We examined these neurons for anatomical anamolies such as defects in neurite outgrowth, changes in branchiness, and or changes in length of the primary extension. Presented are the results of that analysis.

564C Heterochronic microRNAs are required for appropriate cell division control and neuromuscular junction formation during Drosophila metamorphosis. Elizabeth E. Caygill, Laura A. Johnston. Department of Genetics & Development, College of Physicians & Surgeons, Columbia University, New York, NY. The let-7 microRNA belongs to a class of temporally expressed, non-coding regulatory RNAs which function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the evolution of complex body plans. The 21-nucleotide let-7 sequence is conserved among bilaterians, however, little is known about its requirement outside the nematode, or whether it universally controls the timing of developmental processes. We have mutated the let-7 locus in Drosophila, deleting let-7 and preventing the expression of the downstream microRNA miR-125. This mutation leads to specific defects during metamorphosis. We have focused on two distinct developmental processes. First, we demonstrate that mutants show abnormal timing of cell cycle exit in the wing, and that temporal mis-expression of let-7 leads to premature cell cycle arrest. Second, we find that mutants develop immature neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide, let-7 target, and examine its role in NMJ maturation. These results establish conservation of the heterochronic function of let-7, and identify the let-7 locus as a critical regulator of neuromuscular junction formation during metamorphosis. 280 POSTERS: Neurogenetics and Neural Development

565A The role of Crk-associate substrate (Cas) in neural development. Guang-Chao Chen1, Ai-Pei Chi2. 1) Inst Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2) Graduate Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan. Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein required for the regulation of actin cytoskeleton organization and cell migration in mammalian cells. However, the role of Cas during development is poorly understood. To investigate the function of Cas during development, we have identified the single Drosophila ortholog of mammalian Cas family, Dcas. Expressional analysis revealed that Dcas is highly expressed in the ventral nerve cord during embryogenesis and is enriched in the brain and visual system in third instar larvae. In addition, we found that reduction of Dcas expression by RNA interference (RNAi) resulted in abnormal CNS formation, photoreceptor (R cell) patterning, and bulged synapse boutons at the neuromuscular junction. Moreover, reduction of Dcas expression also resulted in aberrant bristle formation. These phenotypes are similar to those of kette mutants. Indeed, we found that Dcas genetically interacted with kette and Dcas colocalized with Kette in S2R+ cells. Kette is a regulator of WASP/WAVE complex in regulating actin cytoskeleton organization. We are currently investigating the role of Dcas in the Kette- mediated pathway.

566B Characterization of aaquetzalli (aqz), a gene required for development of the nervous system during Drosophila melanogaster embryogenesis. Miguel Mendoza-Ortiz, Juan Riesgo-Escovar. Dept Developmental Biol, Inst Neurobiologia, UNAM, Queretaro, Qro, Mexico. Many developmental processes that occur during embryogenesis require cell shape changes, and coordinate cytoskeletal reorganization. Previous studies have shown that aaquetzalli (aqz) is required for embryonic dorsal closure and head involution using germline clones of hypomorphic alleles. The name aaquetzalli derives from the fan-shaped appearance of mutant cuticles, as aaquetzalli is the nahuatl word for fan. In addition, aqz RNAi studies in embryos produce nervous system defects. We identified and characterized a new allelic series of aqz. Some of these alleles cause embryonic lethality with dorsal closure, segmentation, head involution, and nervous system defects. aqz expression occurs at all stages of the Drosophila melanogaster life cycle, and is dynamic. Embryonic phenotypes correlate well with the aqz expression pattern (transcript and protein).

567C Interstitial branching at glial boundaries determines the organization of the adult Drosophila brain. Shana R Spindler, Wayne Pereanu, David Nguyen, Volker Hartenstein. Molecular, Cell, Developmental Biology, University of California, Los Angeles, Los Angeles, CA. The unique organization of the Drosophila brain offers an ideal model to study neuronal branching dynamics. The Drosophila brain is fashioned so that a cortex of cell bodies surrounds an inner neuropile of highly branched axons and dendrites. This organization stems from the fact that most neurons in the Drosophila brain are unipolar, enabling all synapses to occur in a core location. Because dendrites in a unipolar neuron branch from the axon rather than emanate from the cell body, we can study branch point localization in the majority of Drosophila neurons. Setting a branchpoint at one place of the neurite versus another will significantly alter the way in which that neuron is connected to other neurons. The global mechanism by which neurons determine where dendritic processes should emerge on the axon is therefore key to understanding the initial development of a neuronal network. Using lineage-specific drivers and flip-out clones, we find that interstitial branching is a global mechanism by which Drosophila neurons in late larval and pupal stages form dendritic processes. In addition, we find that lineages of neurons derived from a single neuroblast all branch at the same point on the axon and establish this point as the bundled axons contact neuropile glia. These findings suggest that the majority of Drosophila neurons utilize interstitial branching, influenced by axon-glia contact, to localize dendritic branches to a specific domain of the axon. POSTERS: Neurogenetics and Neural Development 281

568A JAK/STAT signal regulates proneural wave progression in the optic lobe development. Tetsuo Yasugi, Daiki Umetsu, Satoshi Murakami, Makoto Sato, Tetsuya Tabata. Lab Morphogenesis, IMCB, Univ Tokyo, Tokyo, Japan. The Drosophila visual system is composed of the compound eye and the optic lobe in the brain. The latter contains three neural ganglia, namely, lamina, medulla, and lobula complex. These optic ganglia are derived from two neuroepithelial ectoderms, the outer optic anlage (OOA) and the inner optic anlage (IOA). The OOA generates the outer medulla and the lamina neurons, while the IOA generates the inner medulla, the lobula and the lobula plate neurons. The number of neuroepithelial cells (NE cells) of the OOA increases by repetitive symmetric cell divisions during larval stages. At early third instar, the NE cells located at the medial edge start to differentiate into medulla neuroblasts (NBs). Next, those NBs undergo asymmetric division and produce ganglion mother cells (GMCs), which divide again and become medulla neurons. Differentiation of NE cells to medulla NBs progresses in an medial to lateral direction. The precise control of the timing of the NB differentiation from NE cells is thought to be critical for ensuring correct amount of NBs and neurons. In this study, we present the evidence that the proneural proteins are responsible for the transition of NE cells to medulla NBs. At the border between NE cells and medulla NBs, expression of L(1)sc, one of the proneural proteins, always precedes the NB differentiation and hence we name this dynamic expression pattern of L(1)sc ‘‘proneural wave’’. We show that proneural genes including l(1)sc cooperatively regulate the medulla NB differentiation. We also found that the JAK/ STAT signal regulates the progression of proneural wave and hence the timing of medulla differentiation. We also the discuss the mechanism how topographic map (retinotopic map) is performed in the fly visual system.

569B Understanding the function of Sanpodo during asymmetric cell divisions in the Drosophila CNS. Burcu Babaoglan, Hemi Mistry, Kate O’Connor, Adam Schickedanz, Beth Wilson, James Skeath. Dept Genetics, Washington Univ. in St Louis, St Louis, MO. The Notch signaling pathway and Numb act in opposition to promote asymmetric cell divisions in flies and vertebrates. During these asymmetric divisions, the cell fate determinant Numb segregates exclusively into one daughter cell. In this cell, Numb blocks productive Notch signaling likely by inhibiting the localization of Sanpodo (Spdo) to the cell membrane and in so doing directs this cell to adopt the B fate. The absence of Numb in the other daughter cell, allows active Notch signaling in this cell, which in turn induces the A fate. The Notch signaling pathway regulates many developmental events. However, the Notch pathway only requires the function of spdo, which encodes a four-pass transmembrane protein, during asymmetric divisions. Here, we address the question: why does the Notch pathway require spdo function specifically during asymmetric divisions for productive signaling? We observe quantitative and qualitative changes in Notch levels and localization in asymmetric dividing cells in the Drosophila CNS; these changes coincide with decreased expression of cell adhesion molecules, such as E-cadherin and Echinoid in these cells. Our observations led to the proposal that Notch signaling is less efficient in asymmetrically dividing cells, due to decreased Notch levels and the less adhesive nature of these cells, and that proteins such as Spdo are required to increase Notch signaling above threshold levels. To test this model, we have been overexpressing wild-type forms of individual members of the Notch pathway in wild-type and spdo mutant embryos and assaying their effect on Notch/Numb dependent asymmetric divisions. To date, our work supports the model that spdo is not obligately required for Notch signal transduction, but rather that Spdo functions to potantiate signaling efficiency in the A cell above threshold levels. Moreover, our data also support the idea that the ability of Numb to block Notch signaling in the B cell depends, at least in part on spdo function.

570C extramacrochaetae (emc), a Notch target gene, is required for R7 and cone cell development. Abhishek Bhattacharya, Nicholas E. Baker. Dept Molecular Genetics, AECOM, Bronx, NY. Drosophila R7 photoreceptors are specified by synergistic activation of both Notch signaling and Sevenless mediated receptor tyrosine kinase (RTK) signaling pathways. In absence of Notch, presumptive R7 cells default to R1/6 photoreceptor fates. Without the Sevenless RTK, R7 cells acquire the Notch-dependent, non-neuronal cone cell fate. Prospero is identified the RTK target in R7 cells. We identified the extramacrochaetae (emc) gene is an effector of Notch during R7 and cone cell development. Drosophila emc gene encodes an Id-class Helix-Loop-Helix (HLH) protein that antagonizes bHLH transcription factor activity by forming non-functional heterodimers with them. We show using loss of function and ectopic expression studies that Notch signaling positively regulates emc transcription in eye imaginal disc. Study of emc null mutations reveals that Emc is autonomously required for R7 photoreceptor differentiation. Like Notch loss-of-function, emc mutant R7 cells instead acquire R1/6 photoreceptor fates. The emc gene is also required for the differentiation of supernumerary R7 cells by ectopic Notch activity. Like Notch, emc is also required for the development of cone cells. We also found that emc is required for multiple other aspects of retinal differentiation, including negatively regulating the morphogenetic furrow progression. We will present our understanding on how emc may function at the molecular level. 282 POSTERS: Neurogenetics and Neural Development

571A Analysis of the interactions between CK2, PP2A and E(spl) during neurogenesis. Anasua Bose, Ezgi Kunttas-Tatli, Bhaskar Kahali, Clifton Bishop, Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. Neural cell fate specification in the central and peripheral nervous system is regulated by Notch (N) signaling. N-mediated lateral inhibition is required to specify a precise number of neural precursors from a group of equipotent cells, the proneural cluster (PNC). Lateral inhibition is employed reiteratively during development of the eye and bristles, and involves the basic-helix-loop-helix (bHLH) repressors encoded by the E(spl)C. During specification of the R8 photoreceptors and bristle sensory organ precursors (SOP’s), E(spl) repressors antagonize proneural activators in the non SOP’s. Previous analysis demonstrated that a subset of E(spl), i.e., M5, M7 and M8 are phosphorylated by protein kinase CK2. Expression of a phosphomimetic variant of M8 elicited a dominant loss of R8’s. These results are supported by our findings that compromising CK2 levels/activity (CK2-RNAi) elicited ectopic R8’s and macrochaetes, indicating a requirement of CK2 for repression by E(spl). One question that remains open is whether phosphatase(s) regulate repression by E(spl). It has been shown that human CK2α forms a complex with the protein phosphatase 2A (PP2A) core- enzyme via a conserved HE165NRKL motif. Interestingly, this site is perturbed in Tik (H-E165D-NRKL), an allele of CK2α that encodes a dominant-negative variant. The studies we report here indicate interactions between CK2, Nspl and widerborst (wdb), a regulatory (beta’) subunit of PP2A. Nspl mutants exhibit a rough and reduced eye, and missing and split bristles. We find that reduced CK2 levels or increased PP2A activity, suppress the retinal defects of Nspl. In addition, we find that wdbKG02977 exacerbates the retinal defects when combined with Nspl. In Nspl, R8’s and SOP’s are rendered sensitive to inhibitory Notch signaling, presumably due to hyperactivity of M8. The possibility thus arises that increased PP2A dosage might lead to the accumulation of hypo-phosphorylated M8, a conformation that is not permissive for repression. These studies suggest that coordinated activities of PP2A and CK2 may regulate repression by M8.

572B Expression levels of the “pan-neural” factor Prospero controls neural vs support cell fate determination in the Drosophila eye. Mark A Charlton-Perkins, S. Leigh Whitaker, Tiffany Cook. Developmental Biology/ Pediatric Opthamology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH. Prospero (Pros) is a transcription factor recently shown to function as a molecular switch for stem cell proliferation and differentiation within larval sensory organ precursor (SOP) system. This occurs through the asymmetric inheritance of Pros into one of two daughter cells. Interestingly, during eye development, Pros is initially expressed equally in an equipotent precursor cell population, called the R7 equivalence group, one of which becomes a neuron (R7). R7 specification depends on activation of the tyrosine kinase receptor, Sevenless (Sev), in this cell, while the cells that do not receive Sev signal develop as lens-forming cone cells (CCs). Pros levels increase in the presumptive R7 cell specifically in response to Sev signaling, suggesting that its up-regulation is necessary for neural versus non-neural cell fate decisions. Here, using both loss- and gain-of-function experiments, we report that Pros is important for both R7 and CC development, and that levels influence which cell type forms. Moreover, we have characterized two additional transcription factors that genetically interact with Pros to affect CC number. Together, these data provide evidence that Prospero functions as a rheostat, rather than an on/off switch, for mediating cell fate decisions during retinogenesis. Future studies are aimed at testing this model in other sensory systems. Support from Research Preventing Blindness, the Ziegler Foundation for the Blind, and NIHNEI EY017907.

573C Genome wide dissection of eyeless function during retinal cell fate determination in Drosophila. Rui Chen1,2, Yumei Li1, Jianlan Peng1, Erin Haase1, Richard Gibbs1, Graeme Mardon1,2,3. 1) HGSC, Molec & Human Genetics, Baylor Col Medicine, Houston, TX; 2) Program in Developmental Biology, Baylor College of Medicine, Houston, TX; 3) Department of Pathology, Baylor College of Medicine, Houston, TX. Drosophila eye development is controlled by intricate genetic networks involving hundreds of genes. The earliest stage of this process, retinal cell fate determination, is regulated by eyeless (ey). A Pax6 homolog, ey functions as a master control gene that is both essential and sufficient for eye development. To better understand ey function during eye development, we conducted a genome- wide screen for its downstream targets using three independent methods. First, gene expression profiling has been performed in both gain and loss of function genetic backgrounds, which leads to the identification of several hundred candidate genes regulated by ey. In parallel, several computational approaches have been utilized to obtain a list of potential direct Ey protein binding elements. To complement the first two approaches, genome chromatin profiling experiments using DamID and ChIP-Seq are in progress to identify Ey binding sites in the fly genome. Many novel genes along with several genes that link ey with other genetic pathways, such as dpp and hh, have been identified. Further functional studies of these candidate genes will provide us with a comprehensive understanding of the mechanisms of retinal development. POSTERS: Neurogenetics and Neural Development 283

574A Microarray analysis of sensory neurogenesis in the embryo using FACS. Andrew Jarman, Sebastián Cachero, Petra zur Lage, Ian Simpson, Lina Ma, Fay Newton, Douglas Armstrong, Lynn Powell, Sadie Kemp. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom. Neurogenesis is triggered by the activity of a small group of bHLH transcription factors including atonal, scute, amos (proneural genes), and asense and cato. Whilst their role in sense organ precursor (SOP) selection is well known, how they activate subsequent events that eventually lead to neural differentiation is less well understood. In order to understand the gene regulatory network of early neurogenesis, we have conducted genome-wide gene expression analysis. Atonal-, Scute-, Amos-, and Cato-expressing SOP cells and their progeny have been purified from staged embryos by GFP tagging and FACS. Microarray analysis has revealed a large number of genes preferentially expressed during sensory neurogenesis. Cluster analysis reveals robust subsets of genes that may represent subnetworks within the overall neural gene network, including potential direct and indirect downstream targets of the proneural factors. This analysis is being combined with motif discovery to understand the regulatory relationships within the enriched gene dataset. Unexpectedly, a significant subset of terminal differentiation genes is expressed very early in neurogenesis, in some cases even at the SOP stage. This suggests that such genes may be required before overt terminal differentiation. It also suggests that some aspects of neural differentiation may be directly regulated by proneural factors, perhaps representing a vestige of the primordial function of such factors in animal neurogenesis.

575B Insights into the mechanism controlling stochastic spineless expression required for the color vision retinal mosaic. Robert Johnston, Claude Desplan. Dept Biol, New York Univ, New York, NY. Stochastic events play an important role in many biological processes including mating type selection in yeast, vulval development in worms, cone cell choice in the retina in humans, and fate commitment of hematopoeitic stem cells in mammals. In Drosophila, the R7 photoreceptor makes a stochastic fate choice that determines the formation of two distinct subtypes of ommatidia involved in color vision. Expression of specific rhodopsin pairs in R7 and R8 define ommatidia as either pale (p) or yellow (y) type. R7 cells make the p vs. y ommatidial choice and then impose fate onto R8. These ommatidial subtypes are distributed stochastically in the retina, similar to human cone photoreceptors. The Dioxin receptor transcription factor encoded by the gene spineless(ss) plays a critical role in this process. Ss is expressed in the y-R7 subtype. Loss of ss function results in all R7 and most R8 adopting the p fate whereas over-expression of Ss induces y-R7 fate. Stochastic expression of Ss in R7 cells is therefore necessary and sufficient to induce y-R7 cell fate. Though ss has been identified as a crucial factor controlling R7 subtype cell fate, the temporal dynamics of Ss expression and the mechanisms controlling this expression remain a mystery. Is Ss transiently expressed in a variable manner possibly controlled by transcriptional noise? What genes control Ss expression? To address these questions, we first used a Ss antibody to analyze the temporal aspects of Ss expression. We then quantitated levels of Ss expression in order to address if ss is controlled through variable, noise-based mechanisms or in a bistable (on/off) manner. Furthermore, we identified a region of DNA in the ss gene that is sufficient to drive reporter expression in R7 cells. Finally, we are using a RNAi-based knockdown screening approach aimed to identify genes required for subtype specification.

576C Analysis of a CK2 phosphorylation-specific variant of E(spl)M8 leads to a reinterpretation of the mechanism of E(spl)D. Bhaskar Kahali, Anasua Bose, Clifton Bishop, Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. E(spl) proteins mediate the inhibitory effects of N (lateral inhibition) by antagonizing the activities of the proneural activators Atonal (Ato) or Achaete Scute (ASC). E(spl) proteins mediate repression upon recruitment of the co-repressor Groucho (Gro). Analysis of the E(spl)D allele, encoding the truncated variant M8*, has impacted our understanding of the mechanism(s) by which E(spl) members antagonize Ato/ASC. M8* lacks the C-terminal domain and does not recruit Gro. Nevertheless, it displays exacerbated antagonism of Ato and abrogates the R8 fate in Nspl flies. The Nspl-dependency of M8* is thought to reflect lack of Gro-binding, and E(spl)D has thus been described as a Gro-independent hypermorph. In contrast, M8* behaves as an antimorph in N+ flies; the reason(s) for these disparate behaviors were unclear. Our finding that a CK2-phosphomimetic variant, M8S159D, elicits a severe reduced eye (akin to M8*) raised the possibility of a similar mechanism. However, the retinal defects of M8SD were Nspl-independent. To better define the relevance of these two variants, we neutralized the ability of M8SD to bind Gro (M8SD-GROm). We find that M8SD-GROm behaves as an antimorph in N+ flies, i.e., it elicits ectopic bristles. This phenotype is suppressed by increased dosage of M8 and enhanced by deficiencies that uncover the E(spl)C. In contrast to M8*, M8SD-GROm potently suppressed the retinal/ bristle defects of Nspl. This ability required its expression anterior to or within the morphogenetic furrow (MF). In contrast, M8- GROm did not elicit ectopic bristles in N+ flies or modulate the retinal/bristle defects of Nspl. Thus the ability to suppress the neural defects of Nspl requires a phosphomimetic replacement of the CK2-phosphoacceptor in conjunction with neutralized Gro-binding. To our knowledge, this is the first E(spl) variant that suppresses the neural defects of Nspl. Our analysis of this variant suggests that the absence of the CtD and its regulation by CK2 phosphorylation, rather than Gro-binding, might underlie the retinal defects of E(spl)D. 284 POSTERS: Neurogenetics and Neural Development

577A Anteroposterior control of stem cell identity: Specification of neuroblast 5-6 by overlapping action of Hox factors and Hox co-factors. Daniel Karlsson, Magnus Baumgardt, Stefan Thor. Dept. of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden. In the developing Drosophila ventral nerve cord, about 100 neurons express the LIM-HD gene apterous (ap). An easily distinguishable subclass of these, the Ap cluster neurons, consists of clusters of 4 cells located specifically in the lateral thoracic segments. In the Ap cluster, one cell, the Tv neuron, expresses the neuropeptide gene FMRFa, while another, the Tvb, expresses the Nplp1 neuropeptide gene. Studies from several labs have led to the elucidation of a highly cell-specific regulatory cascade acting to ensure proper Ap cluster specification, and to activate the cell-specific expression of FMRFa and Nplp1. Recent studies from our lab have determined that the origin of these Ap cluster cells is the neuroblast 5-6 (NB 5-6). With this information at hand, we are now asking the important question of which upstream regulatory events act to ensure that only the thoracic NB 5-6, and not the abdominal NB 5-6, generates these cells and activates the genetic cascade leading to the formation of the Ap cluster. We are finding that positive input from the Antp is important for activating the genetic cascade leading to Ap cluster specification in NB 5-6t. In the abdomen, Ubx and abdA act to block cell cycle progression of NB 5-6a, and also to block expression of certain Ap cluster determinants. Both the positive and negative action of these Hox genes requires the function of the Hox co-factors Hth and Exd, but Hth, Exd and Antp also play independent roles during NB 5-6t development. Interestingly, the generation of the Ap cluster requires input from Antp, and thus cannot be considered part of the thoracic “ground state”.

578B The C-terminal domain (CtD) of Enhancer of split M8, regulates repression in a CK2 phosphorylation-dependent manner. Jee-Eun Kim, Bhaskar Kahali, Umesh Karandikar, Clifton Bishop, Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. Lateral inhibition by Notch (N) is essential for the specification of binary cell fates. During neurogenesis, this process restricts neural fate to a single cell from a proneural cluster (PNC). The restriction requires antagonism of the proneural activators, Atonal (Ato) or Achaete-Scute (ASC), by the E(spl) repressors, and involves protein-protein interactions via the Orange domain of E(spl). Previous work conducted by us indicates that phosphorylation of E(spl)M8 by protein kinase CK2 displaces its CtD, which in the non-phosphorylated state restrains repression by blocking the Orange domain (termed ‘autoinhibition’). To further define the mechanism of ‘autoinhibition’, we have tested the isolated CtD’s from wild type M8 (M8-CtD) and from the CK2-phosphomimetic variant, M8S159D (M8SD-CtD), for their ability to interfere with endogenous M8. The former subdomain is predicted to inhibit (in trans), whereas the latter should not. Phosphorylation assays show that M8-CtD recapitulates the interactions of full length M8 with CK2. When expressed in vivo, M8-CtD elicits ectopic bristles akin to those upon loss of E(spl). We have also employed the Nspl allele, whose retinal defects reflect sensitivity of the R8 photoreceptors to inhibitory N signaling. In this case, it is expected that ectopic M8-CtD might ‘tune-down’ N activity and, perhaps, restore retinal patterning. Consistent with this, M8-CtD, but not M8SD-CtD, suppresses the retinal defects of Nspl. This ability requires expression of M8-CtD immediately anterior to the morphogenetic furrow (MF). Given the temporal dynamics of MF movement and the R8-specific defects in Nspl, this spatial requirement might allow for accumulation of the CtD fragment at levels sufficient to inhibit in ‘trans’ the activity of endogenous M8 in the MF. These effects also manifest at the level of the eye imaginal discs, in which Ato and Senseless levels are substantially restored. Together, these results suggest that interactions between the CtD and the Orange domain of M8 regulate repression in a phosphorylation-dependent manner.

579C Characterization of Echinoid and Friend of Echinoid Binding. Woongki Kim, Susan Spencer. Dept Biol, St Louis Univ, St Louis, MO. Echinoid (ed) and Friend of Echinoid (fred) are highly homologous cell adhesion proteins that regulate EGFR and Notch signaling cascades, crucial pathways for transmitting information between cells during development. We have previously shown that ed acts epistatically to fred during eye development and that Ed and Fred can bind both to themselves and to each other (homophilic and heterophilic binding). To measure the relative abilities of homophilic and heterophilic binding to promote cell adhesion, we have conducted aggregation assays using Ed- and Fred-expressing cultured cells. We have also used these assays to examine Ed and Fred binding to other transmembrane proteins. We will present the results of these experiments and discuss ways in which Ed- and Fred-mediated cell adhesion might be regulated. POSTERS: Neurogenetics and Neural Development 285

580A Potential Antagonistic Roles of CK2 and PP2A in Notch Signaling in Drosophila. Ezgi Kunttas, Anasua Bose, Clifton Bishop, Ashok Bidwai. Dept Biol, West Virginia Univ, Morgantown, WV. It is increasingly becoming apparent that dynamic control of phosphorylation and dephosphorylation underlies the proper spatial and temporal activities of many signaling circuits including Notch (N). Emerging evidence suggests that protein kinase CK2 mediates the effects of N during Drosophila eye and bristle development via phosphorylation of E(spl) repressors. Previously, it was shown in our laboratory that misexpression of a UAS-Tik construct encoding a dead catalytic subunit of CK2 (a CK2-DN) elicits neural defects in the eye and bristle, which are similar to those upon loss of E(spl). Specifically, UAS-Tik elicits ectopic and split bristles and a rough eye reflecting supernumerary SOP’s and R8’s respectively. These results suggest that impaired CK2 function compromises lateral inhibition, presumably due to hypo-phosphorylation of E(spl)M8. While these studies implicate a role for CK2 in the regulation of E(spl), the identity and/or involvement of a cognate phosphatase remain unknown. Protein phosphatase 2A (PP2A) is one of the candidates thought to be involved in N signaling. PP2A is a heterotrimeric enzyme that contains a structural (A), catalytic (C) and a regulatory (B) subunits. We have employed the Gal4-UAS system to assess the role of PP2A during eye and bristle development in wild type and/or Nspl flies. We find that increased dosage of C subunit (UAS-mts) suppresses the rough and reduced eye phenotypes of Nspl flies, a result that is also associated with decreased CK2 levels/activity. In addition, we find that UAS-mts elicits ectopic and missing bristles in N+ flies, both indicative of a loss of N signaling. Furthermore, UAS-mts elicits a rough eye phenotype characterized by aberrant ommatidial phasing and/or fusion and loss of the interommatidial bristles. These ‘genetic’ interactions will also be assesed at the protein level. Together, these results suggest that PP2A activity might attenuate, whereas CK2 augments, inhibitory N signaling. Given the previously described interactions between CK2 and E(spl)M8/M5/M7, PP2A might serve in an antagonistic manner to CK2.

581B Dbx represses Eve in the ventral nerve cord of Drosophila embryos. Haluk Lacin1, Yi Zhu1, Beth Wilson1, Heather Broihier2, James Skeath1. 1) Dept. Genetics, Washington Univ, St Louis, St Louis, MO; 2) Dept. of Neurosciences Case Western Reserve University, Cleveland OH. Homeobox transcription factors play important roles in determining cell fates in higher metazoans. Dbx belongs to the highly divergent H20 family of homeobox genes. In vertebrates, dbx promotes the development of a subset of interneurons, some of which coordinate left-right locomotor activity. Due to this role in verteberate CNS development and our interest in understanding the genetic regulatory hierarchy that controls cell fate specification, we are characterizing the role of dbx in the Drosophila CNS. To date our work has focused on a descriptive analysis of dbx expression, and a functional characterization of the role of dbx in neuronal cell fate specification in the CNS. We find that dbx is expressed primarily in interneurons located in the embryonic, larval, and adult CNS. Most Dbx+ neurons are located in the posterior of each hemisegment suggesting regulatory input from segment polarity genes. We have mapped most of these neurons to individual progenitor neuroblast lineages while also tracing their axonal projections. We find that dbx function is necessary and sufficient to repress Eve expression in specific neuroblast lineages. Interestingly, within these lineages Dbx+ interneurons and Eve+ motoneurons appear to have sibling relationship. Thus, our working model is that Dbx and Eve together with other proteins promote two sibling cells to acquire different fates. In the future we plan to assess the role of Dbx in controlling the differentiation of its expressing cells and to investigate the regulatory relationship between segment polarity genes and Dbx.

582C Functional studies of Optix in Drosophila. Yumei Li1,2, Kristi Hoffman2, Abanti Chattopadhyay2, Umesh Karandikar3, Keqing Wang1, Graeme Mardon2,3, Rui Chen1,2. 1) HGSC, Baylor College of Medicine, Houston, TX; 2) Dept Molecular & Human Gen, Baylor College of Medicine, Houston, TX; 3) Department of Pathology, Baylor College of Medicine, Houston, TX. Using a combinatorial approach of microarray analysis and phylogenetic shadowing, our lab had identified Optix as a direct downstream target of Eyeless during retinal development in Drosophila. The vertebrate homologs of Optix, Six3/6, are required for normal eye development. Mutation in the human SIX3 gene leads to holoprosencephaly and microphthalmia. Homozygous Six3 mutants in mouse have no eyes and severe cranio-facial defects. It is likely that Optix also plays important role in retinal determination in Drosophila. Optix encodes a homeodomain protein with a SIX protein-protein interaction domain. Like other known Retinal Determination genes, Optix is expressed prior to MF initiation and anterior to the MF during furrow progression in eye imaginal discs. Moreover, misexpression of Optix is sufficient to induce ectopic eye formation, suggesting that it functions near the top of the genetic hierarchy controlling retinal cell fate determination. However, the mechanisms of how Optix functions and interacts with the Retinal Determination network are not well understood. To elucidate it function, I have generated a molecular defined deletion using the P-element imprecise excision. This deletion loses the N-terminal part of Optix protein including SIX domain and homeodomain, and is likely to be a null. Flies carrying this deletion are homozygous lethal. Mutant phenotype and genetic interactions with genes in Retinal Determination network will be analyzed using this allele. Furthermore, an eye specific enhancer of Optix contains putative binding sites of Eyeless (Ey) and Sine Oculis (So), members of the RD network had been identified in our previous study. Using this enhancer as a tool, regulation of Optix by the RD network will be tested using both in vitro and in vivo assays. 286 POSTERS: Neurogenetics and Neural Development

583A The Notch ligands Delta and Serrate are necessary in the R1 and the R6 photoreceptors to prevent R7 fate via autonomous cis-inhibition of Notch activation. Adam Miller, Tory Herman. Institute of Molecular Biology, University of Oregon, Eugene, OR. The Notch (N) signaling pathway is utilized during development to specify different cell fates using two mechanisms: 1) lateral inhibition, wherein activated N divides a group of equivalent cells into two non-equivalent fates; 2) inductive N signaling where the signaling cells are already non-equivalent. During both processes, the N ligands Delta (Dl) and/or Serrate (Ser) activate the N receptor non-autonomously. However, Dl and Ser have also been shown to act autonomously to inhibit N activation (cis-inhibition) during wing margin specification, specifically when found at high levels (de Celis and Bray, 1997; Micchelli et al, 1997; Klein et al, 1997; Glittenberg et al, 2006). Each unit of the eye has eight photoreceptor neurons (R1-8), and inductive N signaling specifies R7 fate: when both R1 and R6 lack Dl the R7 precursor adopts the R1/6 fate while R1 and R6 adopt wild-type fates (Cooper and Bray, 2000; Tomlinson and Struhl, 2001). Surprisingly, we have found that the loss of both Dl and Ser from R1 or R6 causes them to adopt R7 fate. As mentioned above, loss of Dl alone has no effect on R1/6 fate, and likewise we found that loss of Ser alone has no effect, suggesting that either Dl or Ser is necessary for prevention of R7 fate in R1/6. We further found that R1/6s lacking Dl and Ser will adopt R7 fate only if R7 expresses N ligands: if both R1 and R7 lack Dl and Ser then R1 adopts wild-type fate. These results suggest that Dl and Ser inhibit N activation in R1/6 in cis. Since N and Dl/Ser are expressed in R1/6/7, how does cis-inhibition occur in R1/ 6 while not in R7? Our working model involves the timing of R neuron recruitment: Dl transcription is downstream of recruitment by EGF/Ras signaling (Tsuda et al, 2002); R1/6 are recruited before R7, express Dl first, and activate N in the neighboring R7 precursor, while concurrently building up high levels of Dl to allow cis-inhibition. R7 expresses Dl, but too late to either cis-inhibit N autonomously or trans-activate N in R1/6.

584B Identifying interactors of Sanpodo essential for Notch-mediated asymmetric divisions in Drosophila nervous system. Rajalaxmi Natarajan, James Skeath. Dept of Genetics, Washington University School of Medicine, St. Louis, MO. Asymmetric cell divisions produce daughter cells of different fates and are a basic mechanism that creates cell diversity during development. Notch/numb-mediated asymmetric divisions are one of the best-understood examples of asymmetric divisions in higher metazoans. Notch/Numb-mediated asymmetric divisions result from antagonistic interactions between the Notch signaling pathway and the membrane-associated protein Numb. While Delta, the ligand for the Notch receptor, signals to both daughter cells, Numb segregates exclusively into one daughter cell. The presence of Numb in one daughter cell somehow blocks productive Notch signaling in that cell, thereby promoting the B fate. The absence of Numb in the other daughter cell allows active Notch signaling to occur, which in turn, promotes the A fate. Previous studies from our lab have led to the model that Sanpodo, a novel four-pass transmembrane protein, functions at the cell membrane of the A cell to promote Notch signaling and that the presence of Numb in the B cell blocks Notch activation by keeping Sanpodo off of the cell membrane. To understand the molecular basis by which Sanpodo promotes Notch signaling during asymmetric divisions, we are using a genetic approach to identify genes that interact with sanpodo. A dominant modifier screen was conducted using >600 deficiency lines which together uncover >80% of the euchromatic Drosophila genome in two different sensitized genetic backgrounds- one optimized to identify enhancers of sanpodo and the other optimized to identify suppressors. Currents efforts to identify and characterize the functional relevance of genes(s) involved in the regulation of Notch/Numb-dependent asymmetric divisions will be described.

585C Examining Echinoid’s Role in the Endocytosis of EGFR, Notch, and Delta. Grant Simmons, Susan Spencer. Saint Louis University, Saint Louis, MO. Echinoid (Ed) is an Ig-superfamily cell adhesion molecule important for regulating EGFR and Notch signaling during neurogenesis in the developing retina in Drosophila (Spencer and Cagan, 2003; Rawlins et al., 2003). Mutations in ed result in the development of extra R8 photoreceptor cells in the developing eye. Ed acts similarly to DE-cadherin by mediating cell adhesion at adherens junctions and has been suggested to regulate endocytosis of cell surface proteins (Wei et al., 2005; Rawlins et al., 2003; Spencer and Cagan, 2003). However, it remains unclear how Ed regulates endocytosis of proteins such as EGFR, Notch, and Delta. To better understand the process, proteins that are known effectors of endocytosis of EGFR, Notch, and Delta are being examined for a genetic interaction with Ed. Results of these genetic interactions will be discussed. POSTERS: Neurogenetics and Neural Development 287

586A Changing sensory specificity in functional terminally differentiated photoreceptors by switching Rhodopsin expression. Simon Sprecher, Claude Desplan. Dept Biol, New York Univ, New York, NY. Functional specificity of sensory neurons is achieved by the restricted expression of one given sensory receptor gene per sensory neuron type. Once the choice is made to express a specific receptor, the cell has to maintain the expression of the correct receptor gene and exclude the expression of all other receptors. In Photoreceptor functional specificity is mediated through the expression of Rhodopsins. In addition to the photoreceptors of the compound eye, the fly also receives light dependent input from a small group of photoreceptors termed “eyelet”, which is required for entraining the circadian rhythm. The eyelet is derived from the photoreceptors of the larval eye, which is composed of about 12 photoreceptors, 4 expressing the blue-sensitive (Rh5), the remaining 8 are green- sensitive (Rh6). We find that, during metamorphosis, all eight green-sensitive (Rh6) larval photoreceptors die, while the four blue- sensitive (Rh5) switch fate to become green-sensitive (Rh6). We further show that Ecdysone receptor functions autonomously both for the death of Rh6-expressing larval photoreceptors and for the sensory switch of Rh5-photoreceptors. Surprisingly, we find that genetic program underlying sensory respecification requires novel mechanisms and does neither depend on the players required for Rh5/Rh6 regulation in the adult R8-cell, nor for the larval PR-subtypes. This is a previously unidentified example of sensory respecification of terminally differentiated neurons.

587B A Genetic Screen For Genes Controlling Tv Neuron Identity And FMRFamide Expression. Carina Ulvklo, Patrik Nilsson, Anna Angel, Fredrik Fransson, Stefan Thor. Molecular genetics, Clinical and Experimental Medicine, Linköping, Sweden. In the developing Drosophila ventral nerve cord, six neurons, the Tv neurons, express the neuropeptide gene FMRFamide. A number of regulatory genes have been identified that act to establish Tv neuron identity and/or to activate FMRFa expression. These include the COE gene collier/knot, the LIM-HD gene apterous, the zinc-finger gene squeeze, the bHLH gene dimmed, and the dachshund, nab and eyes absent transcriptional co-factors. FMRFa expression is furthermore critically dependent upon a TGFβ/ BMP retrograde signal provided by the target, the dorsal neurohemal organ. To identify novel genes acting at different levels of neuronal development, for instance stem cell competence and neuronal sub-type specification, we have conducted a large-scale forward genetic screen looking for genes that are critical for Tv neuron specification and differentiation. In addition, the identification of FMRFa expression as being dependent upon target-derived TGFβ/BMP signaling, fortuitously allows us to utilize its expression as a straightforward readout also of axon transport and retrograde signaling. We have generated transgenic flies where the FMRFa enhancer drives GFP expression, and have identified lines where the six Tv neurons are readily visible in the living late embryo and larvae. To verify the validity of the screen, several previously identified mutants have been crossed into this transgenic marker background and tested for effects upon GFP expression. We have completed our screen of the 2nd and 3rd chromosomes, 4,037 and 5,474 mutant chromosomes, respectively, and have identified 611 mutants that affect FMRFa-GFP expression. Mutations have been identified in several known FMRFa regulators, such as Antp, castor, collier, eya, gbb and mad, as well as in a large number of novel genes. Mutants with strong phenotypes have been analysed with a number of antibody markers, thereby placing them in different categories, such as lineage or TGFβ/BMP signaling effects. These results, and our efforts to map these mutants will be presented.

588C Senseless functions as a molecular switch for color photoreceptor differentiation in Drosohila. Baotong Xie, Mark Charlton- Perkins, Elizabeth McDonald, Brian Gebelein, Tiffany Cook. Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. The purpose of this study is to investigate the role of Drosophila transcription factor Senseless in late color photoreceptor (PR) differentiation. A major question in development is how specialized cell types arise from a common progenitor. In the adult Drosophila compound eye, color discrimination is achieved by UV, blue, and green-sensitive PRs. These PR subsets arise from neuronal precursors called R7 and R8 cells. Studies have demonstrated that R7-based UV-sensitive PRs require the repression of R8-based blue/green-sensitive PR characteristics to properly development. This repression is mediated by the transcription factor, Prospero (pros). Here, we show that Senseless (sens), a Drosophila ortholog to the Gfi1 zinc finger transcription factor, plays an opposite role to Pros by both negatively regulating R7-based features and positively enforcing R8-based features during terminal differentiation. In addition, we demonstrate that pros and sens function in conjunction with the pan-photoreceptor transcription factor Orthodenticle (otd) to oppositely regulate R7 and R8 photoreceptor rhodopsin gene expression in vitro. sens has previously been shown to be essential for neuronal specification in many developmental contexts. The data here indicate that sens is also necessary to selectively define specific neuronal subtypes during late ocular development. Interestingly, Pros has recently been shown to function as a tumor suppressor, whereas Gfi1 is a well-characterized oncogene. Thus, we propose that sens/pros antagonism is important for regulating many biological processes. 288 POSTERS: Neurogenetics and Neural Development

589A Drosophila dCtBP is required for adult peripheral nervous system development and sharpens a proneural transcriptional activity of Pnr. Inna Biryukova, Pascal Heitzler. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, BP 10142. The peripheral nervous system (PNS) is required for animals to detect and to relay environmental stimuli to central nervous sytem for information processing. Drosophila adult PNS displays regularly patterned external mechanosensory organs on the notum and wings. Two proneural genes achaete (ac) and scute (sc) are necessary for development of the sensory organs. Precise spatial and temporal pattern of the ac/sc expression is initiated and controlled by multiple enhancer elements driven by local combination of transcription factors that together form a prepattern. Currently, we have tried to understand the role of transcription co-repressor, dCtBP in the PNS development. dCtBP is required for embryonic segmentation and Hairy-mediated transcriptional repression. dCtBP genetically interacts with HDAC1-dSin3A and acts as context dependent activator and/or repressor. We found that during adult PNS development, dCtBP acts as a negative regulator of sensory organ emerging. Mitotic clonal analysis showed that cells lacking dCtBP exhibit ectopic sensory organs. However over-expression of dCtBP is characterized by dramatic loss of sensory organs. Observed mutant sensory organ phenotype is correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. We showed that dCtBP directly interacts with transcriptional activator Pnr and acts as a co-repressor of Pnr-mediated activation of the proneural genes. dCtBP mediates transcriptional repression in a HDAC- dependent or - independent manner. Towards indentifying a mechanism of the dCtBP-dependent regulation of the proneural gene expression, we are performing genetic and biochemical analysis of the dCtBP interacting genes, dSin3/HDAC1/Rpd3.

590B Understanding the function of twin of eyegone (toe) in eye development in Drosophila melanogaster. Abanti Chattopadhyay1, Abuduaini Abulimiti3, Keqing Wang3, Rui Chen1,2,3. 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) Program in Developmental Biology, Baylor Col Medicine, Houston, TX; 3) Human Genome Sequencing Center,Baylor Col Medicine, Houston, TX. Twin of eyegone (toe) is a paralog of the gene eyegone (eyg) in the genome of Drosophila melanogaster and both genes are located 30kb apart on chromosome 3L. Toe and eyg are structurally related to the PAX6 gene in mammals which is highly conserved in evolution from flies to human. Mutations in the Pax6 gene in humans lead to diseases such as aniridia (absence of iris), corneal opacity, cataract, glaucoma and long term retinal degeneration. Eyg promotes cell proliferation in the larval eye disc; however the function of toe is unclear. To this end, we have generated, using piggybac insertion lines, a deletion of the toe gene alone (ToeD40) and a double deletion of toe/eyg genes (eyg/toeD53). Flies homozygous for the Toe D40 deletion are eyeless whereas flies homozygous for the eyg/toeD53 deletion exhibit an eyeless and headless phenotype. Our preliminary results suggest that toe plays a redundant role with eyg since overexpression of toe can rescue the eyg/toeD53 mutant phenotype. Further functional analysis of toe during retinal development and its interaction with eyg will be reported.

591C Genetic interaction of dorsoventral patterning genes during embryonic development of the Drosophila brain. Janina Seibert, Dagmar Volland, Gerhard Technau, Rolf Urbach. Institute of Genetics, Johannes Gutenberg University, Mainz, Germany. The Drosophila embryonic brain originates from the procephalic neuroectoderm (pNE) which gives rise to about 100 neuroblasts (NBs) on either side. The brain can be subdivided into three major parts, from posterior to anterior: the trito- (Tc), deuto- (Dc), and protocerebrum (Pc), the latter forming the largest fraction of the brain. The expression and regulation of the columnar genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) which are known to be key players in dorsoventral patterning of the ventral nerve chord (VNC), reveals segment-specific differences among brain neuromeres and compared to the VNC. In the Tc, the columnar genes are expressed and regulated similar to the VNC, which is different from the Dc and Pc. These segment-specific differences and the fact that large parts of the pNE, including more than half of the brain NBs (mainly in the Pc) express neither columnar gene, suggest the existence of additional factors which control dorsoventral patterning of the early brain. Such factors might be the Drosophila Sox genes SoxN and Dichaete, as well as the transcription factor Nk6 and the Drosophila Epidermal growth factor receptor (Egfr), all of which are known to play important roles in the development of the VNC. We show that SoxN and Dichaete are expressed in an extensive and dynamic pattern throughout the pNE and developing brain. In the VNC, both genes are involved in the specification and formation of NBs, partly by interacting with ind and vnd. Therefore, we investigated in single and double mutant backgrounds wether and which interactions exist in the developing brain between the Sox genes (SoxN, Dichaete), Egfr and the columnar genes (vnd, ind, and msh). POSTERS: Neurogenetics and Neural Development 289

592A Over-expression of the Notch Intracellaur Domain in Neural Cells Potentiates Long-Term Survival of Drosophila in Hypoxia. DeeAnn W Visk1, Dan Zhou2, Gabriel Haddad2. 1) Biology, University of California, San Diego, La Jolla, CA; 2) School of Medicine, University of California, San Diego, La Jolla, CA. Hypoxia is a key factor in damage caused by stroke, heart attack, and lung disease. We generated a Drosophila strain that can tolerate extremely low, normally lethal, hypoxic conditions to elucidate strategies employed by organisms that can survive hypoxia long-term. Twenty-seven isogenic strains of Drosophila were crossed en masse and the F1 was subjected to gradually decreasing oxygen conditions for long term laboratory-selection of hypoxia tolerance. Gene expression profiling revealed significant up-regulation of the Notch signaling pathway in the hypoxia-selected flies during the third instar stage. Screening a variety of neural GAL4 drivers crossed to a UAS-Notch Intracellular Domain (NICD) fly lead to the discovery that this over-expression of the constitutively active Notch significantly enhanced hypoxia survival as determined in 5% oxygen. We believe that increased Notch signaling in these cells increases the pool of stem cells which may play a role in rescuing hypoxic damage in the brain. In summary, these results suggest that Notch plays an important role in long-term survival of hypoxia.

593B A mosaic screen for genes required for postembryonic neural proliferation. Jennifer Grant, Cedric Maurange, Louise Cheng, Julia Pendred, Alex Gould. Developmental Neurobiology, NIMR, London, London, United Kingdom. To identify regulators of postembryonic neural proliferation, we have been conducting a mosaic screen using the MARCM system. Screening of 4,600 EMS mutagenised chromosomes identified 177 lethal complementation groups on 3L and 3R that show altered spatiotemporal patterns of neural proliferation in the larval CNS. This includes a mix of mutations associated with smaller-than- normal and larger-than-normal neuroblast clones. We are currently characterising a subset of complementation groups that either shorten or lengthen the developmental time window of neuroblast divisions. We have mapped several of these lethal mutations to genes encoding tumour suppressors and chromatin remodelling proteins. These results identify several new neuroblast genes and shed some light on how neuroblast clone size is regulated by the balance between cell proliferation and apoptosis.

594C Characterization of the torp4a gene, a Drosophila homolog of human DYT1 (Torsin A) associated with early-onset dystonia. Noriko Wakabayashi-Ito1, Minoru Yamanishi2, Hideaki Moriyama3, James F. Gusella1, Naoto Ito1. 1) Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA; 2) School of Biological Science; 3) Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE. Dystonia is a neurological movement disorder characterized by involuntary muscle contraction that forces certain parts of the body into abnormal movements or postures. Dystonia can affect any part of the body including the arms and legs, trunk, neck, eyelids, face, or vocal cords. It is not usually fatal, nor does it affect intellect. Most cases of early-onset dystonia are associated with mutation of the DYT 1 (Torsin A) gene. In the Drosophila genome, one torsin-like gene, which is named torp4a, has been identified on the X chromosome. We have isolated seven null mutant lines of torp4a using the “ends-out” gene targeting method developed by Gong and Golic (2003). The mutations are semi-lethal and surviving adults are male sterile, and show thinner bristles, less pigmentation, and slow movement. Late 3rd instar larvae also show slow movement. All of these phenotypes are rescued by re- introducing the 1.9Kb genomic region containing torp4a. We established mutant lines balanced over an X chromosome balancer with an actin-GFP marker. We selected GFP negative larvae and examined at which stage mutant flies died. Most of the mutant flies died as late 3rd instar larvae or early in the pupal stage. In order to analyze the phenotypic difference between wild type and torp4a mutant larvae in more detail, we recorded larval motion with a video camcorder and counted the number of strides. In mutant larvae, the stride count (24.7± 16.3 per minute, n=29) was significantly reduced compared to wild type (53.3±12.0 per minute, n=33) (P<0.0001). We are currently quantifying the larval movement by a newly developed computer program “Prairie Dog”. This work will help us to understand the molecular function of torp4a gene in controlling locomotion in Drosophila and the mechanism of dystonia disease in human. 290 POSTERS: Neurogenetics and Neural Development

595A A role for Hox genes in regulating apoptosis during Drosophila embryonic central nervous system development. Ana Rogulja-Ortmann, Gerd M Technau. Institute of Genetics, University of Mainz, Mainz, Germany. Our research focuses on apoptosis as a patterning mechanism in the embryonic CNS. We previously identified two motoneurons in the embryonic CNS that undergo segment-specific apoptosis at the end of embryogenesis, the NB7-3 GW motoneuron (apoptotic in segments T3 to A8) and a NB2-4 motoneuron (apoptotic in T3 only), and we use these cells as models for our investigations into mechanisms regulating developmental cell death. We show that the homeotic genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate the fate of the GW motoneuron, with Ubx promoting GW apoptosis in segments T3 to A7, and Antp preventing it in segments T1 and T2. Our data indicate that these Hox genes are not merely responsible for the segment-specific identity of the NB7-3 lineage, but play an additional role in regulating apoptosis as a cell fate. In addition, we find that Ubx is required for segment- specific apoptosis of the NB2-4 motoneuron as well. Our current results suggest that, as in the case of the GW motoneuron, Ubx may not only specify the T3 identity of the NB2-4 lineage, but may be more directly involved in activating apoptosis of the NB2-4 motoneuron in this segment.

596B Characterization of Aedes aegypti rhodopsins in transgenic Drosophila. James H. England, Joseph Real, Xiaobang Hu, Zachary Lemmon, Aaron Lani, Jennifer Tung, Michelle A. Whaley, Joseph E. O’Tousa. Biological Sciences, University of Notre Dame, Notre Dame, IN. Analysis of the genome content of many different organisms allows comparative studies into the expression of genes in particular tissues and cell types. Members of the rhodopsin family of visual pigments are expressed in subsets of photoreceptor cells to tune the cells to colors of light. Analysis of this gene family in many insects has shown a wide scope of the type and number of opsin genes. The arrangement of photoreceptor cells and positioning of the rhabdomeric membranes within these cells also show significant differences. We aim to characterize the ten opsins found in Aedes aegypti, describe the cellular architecture of retina, and determine which opsins are expressed in each photoreceptor class. First, the spectral and structural properties of the full-length mosquito opsin genes are being analyzed in transgenic Drosophila. These flies carry the mosquito opsin gene under control of the Rh1 promoter in an Rh1 null background. Rh1 is the major rhodopsin gene expressed in R1-6 photoreceptor cells. ERG analysis of these transgenic flies establish that the Aedes rhodopsins tested in Drosophila, Lop1, Uvop8, and Sop9, are expressed and trigger a photoreceptor light response. Similar work on the other opsins is underway. Second, electron microscopy has determined the spatial organization of photoreceptors in the Aedes retina. Individual ommatidia consist of eight photoreceptors that elaborate rhabdomeric membranes only on the distal portion of the photoreceptor cell body. Nuclei are found in the proximal regions of all photoreceptors. The rhabdomeres are fused in a defined pattern and a cell body occupies the central area of the ommatidia. Third, to determine which opsins are expressed in each photoreceptor class, we are generating antibodies to the C-terminus of each Aedes opsin. The UVop8 antisera has shown that a single photoreceptor cell in each Aedes ommatidium expresses the UVop8 opsin. Similar work with other opsins is underway.

597C Gene-expression profiling of sensory organ precursor (SOP) cells from wing and leg imaginal discs. Mariano A Loza Coll, James W Posakony. Division of Biological Sciences, University of California at San Diego, La Jolla, CA. The body surface of adult flies is covered by sensory bristles of various kinds that detect mechanical and chemical environmental stimuli. Most of these simple organs arise via a stereotypical sequence of asymmetric mitoses starting with a single precursor cell, hence called the sensory organ precursor (SOP) cell. Over the last few decades, numerous studies have demonstrated the involvement of a limited number of genes and genetic pathways in the specification of SOP cells and their development into mature sensory organs. Despite these significant advances, we have still much to learn about the regulatory programs elicited in SOP cells and their progeny. We have taken the approach of attempting to catalogue the genes being expressed at each and all stages of bristle development. During sensory organ development, SOPs are first singled out from small groups of cells known as “proneural clusters” (PNCs). In previous work from our lab, we used a combination of microarray analysis and in situ hybridization to identify genes specifically expressed in the PNCs. We are now employing the same strategy to characterize gene expression in SOP cells. Wing and leg imaginal discs were dissected from white pre-pupae carrying a GFP reporter gene controlled by an SOP-specific enhancer (CG32150- eGFP). GFP-expressing SOP cells were isolated by trypsinization of the discs followed by fluorescence-activated cell sorting. RNA was extracted from the sorted cells, amplified and used to probe a genome-wide expression microarray. Here we present a preliminary analysis and discussion of our results. POSTERS: Neurogenetics and Neural Development 291

598A Retinophilin (CG10233) localizes to rhabdomeres of Drosophila photoreceptor cells. Kirk L Mecklenburg1, Benjamin Currie2, Joseph E. O’Tousa2. 1) Department of Biology, Indiana University South Bend, South Bend, IN, 46634; 2) Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556. CG10233 is expressed predominantly in Drosophila photoreceptors, and mammalian family members are known. We have initiated a genetic and molecular analysis of the gene. The protein consists of an N-terminal domain, four internal “Membrane Occupation and Recognition Nexus” (MORN) repeats, and a C-terminal domain. The MORN repeats are proposed to function in membrane associations, as they are found in junctophilin proteins responsible for junctions between the endoplasmic reticulum and plasma membrane. We determined the localization of the CG10233 protein by tagging with Red Fluorescent Protein (RFP). The CG10233- RFP fusion construct expressed specifically in photoreceptors R1-R6 is trafficked to the rhabdomere, co-localizing with a GFP- tagged Rh1 rhodopsin (Rh1-GFP). Initial work with anti-CG10233 antibodies to detect the native protein is consistent with these results. We constructed a series of CG10233-RFP deletions to define the role of specific CG10233 sequences necessary for subcellular localization. CG10233 protein deleted for the N-terminal domain of the protein is strongly detected in the rhabdomeres of young flies, but is excluded within 24 hours after eclosion. Deletions of the C-terminal domain and combinations of the MORN repeats are not localized to the rhabdomere at any age. Expression of the human CG10233 homolog fails to localize to the rhabdomere. These results suggest determinants within all domains are required for sustained rhabdomeric localization and at least one of these determinants is not conserved in the mammalian homolog. A major goal of our investigation is to determine the functional role of the of CG10233 gene product. We are carrying out two approaches and intend to report results from both. First, we utilized two piggyback transposon insertions flanking CG10233 to make a small deletion of the CG10233 region. In the second approach, we generated a CG10233 RNAi expressing strain through use of the pRISE-ftz vector.

599B Degradation of proneural proteins controls the timing of neural precursor division. Hai-Wei Pi1, Pao-Ju Chang1, Yun-Ling Hsiao1, An-Chi Tien2, Yi-Ju Li1. 1) Department of Life Science, Chang-Gung University, Tao-Yuan, Taiwan; 2) Program in Developmental Biology, Balor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. During development, cells divide in spatially and temporally regulated manners. In Drosophila, single sensory organ precursors (SOPs) divide a few rounds to generate daughter cells that constitute an external sensory organ. We find that phyllopod (phyl), a direct transcriptional target of proneural proteins Achaete (Ac) and Scute (Sc), regulates proneural protein degradation in SOPs prior to mitotic entry. In phyl mutants, proneural proteins accumulate in SOPs and G2-M transition is absent or delayed due to reduced mRNA levels of the mitotic inducer Cdc25. Phyl-induced proneural protein degradation requires the Sina E3 ubiquitin ligase and is mediated through the ubiquitin-proteasome pathway. Finally, the G2-M transition defect in phyl mutants is rescued by reducing the ac and sc gene dosage. Thus, our results suggest that auto-induction of a proneural protein degradation mechanism serves as the molecular clock to time the neural precursor division.

600C Feedback from Rhodopsin 6 protein is required to maintain pR8 identity through inhibition of Rh5 expression. Daniel Vasiliauskas1, Esteban O. Mazzoni2, Claude Desplan1. 1) Department of Biology, New York University, New York, NY; 2) Pathology Department, Columbia University, New York, NY. Generally, individual sensory neurons express single sensory receptors to avoid sensory confusion. This is known as a one neuron-one receptor rule. Neurons accomplish this by choosing to express one of a number of genes encoding alternative receptors, while repressing the rest. Studies of olfaction in mice raised an intriguing possibility that a feedback signal from the sensory receptor protein itself plays a role in the choice mechanism, possibly by directing repression of alternative genes. Photoreceptor cells (PR) in the adult Drosophila eye express one of five different rhodopsins, photon capturing G-coupled seven trans-membrane proteins and thus, follow the one neuron-one receptor rule. Each unit, omatidium, of the eye contains 2 inner PRs, R7 and R8, surrounded by 6 outer PRs which express Rh1. Excluding the dorsal rim area, 30% of the eye is populated by “pale” (p) omatidia which express Rh3 in R7 and Rh5 in R8, and 70% of the eye is populated by “yellow” (y) omatidia which express Rh4 in R7 and Rh6 in R8. p and y omatidia are randomly distributed throughout the eye. This pattern is set up through a stochastic decision in R7 cells, which then signal and determine R8 rhodopsin expression. By the end of pupation, robust, stable and exclusive rhodospin expression in the fly eye has been established. Here we ask whether feedback signals from rhodopsin proteins participate in regulating the choice of rhodopin gene expression. We find that in rh6 mutants, yR8 type is specified normally. However, in older adults yR8 cells start to express rh5, which normally is only expressed in pR8. Thus, Rh6 generates a signal which controls transcription. However, this signal does not participate in the initial rhodopsin choice, but rather acts to maintain yR8 identity by suppressing rh5 expression in the adult. 292 POSTERS: Neurogenetics and Neural Development

601A Identification of cofactors that participate in Orthodenticle-dependent development of the retinal mosaic. Michael Workman, Elizabeth McDonald, Tiffany Cook. Developmental Biology/Pediatric Opthalmology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. The Drosophila compound eye is composed of approximately 750 ommatidia, each containing six outer photoreceptor cells (R1- R6) and two inner photoreceptor cells (R7 and R8). Color vision is achieved in the inner photoreceptors which each express one of four different UV, blue, or green-sensitive rhodopsins (rh) in a paired manner: 30% express Rh3/Rh5 (pale subset) while the remaining

70% express Rh4/Rh6 (yellow subset). The K50 homeodomain transcription factor Orthodenticle (Otd) binds directly to the rh3 and rh5 promoters and is necessary for their activation in pale ommatida. However, Otd is expressed in all photoreceptors and its overexpression is not sufficient to change the highly conserved pale:yellow ratio. The purpose of our study is to identify factors that interact with Otd and participate in establishing a patterned array of rhodopsin expression in the developing retina. Using a modified yeast two-hybrid screen and a candidate gene approach, we have identified seven potential Otd-interacting factors including chromatin remodeling complexes and post-translational modifiers. We are currently testing direct Otd interactions and determining if these factors are necessary and sufficient for Otd-dependent activation of rhodopsins both in vivo and in vitro. Together, these studies indicate that Otd acts as a dual-regulator of rhodopsin expression through the differential use of cofactors and is necessary for generating the retinal mosaic required for color vision. Future studies are aimed at further examining the role of these factors in regulating other aspects of Otd-dependent functions during neurogenesis. Supported by Research Preventing Blindness and Ziegler Foundation for the Blind.

602B Drosophila EHBP-1 is Required for Notch Signaling during External Sensory Organ Development. Shinya Yamamoto1, Nikolaos Giagtzoglou2, Hillary K. Andrews1, Hao Wang3, Karen L. Schulze2, Hugo J. Bellen1,2,4. 1) Program in Developmental Biology; 2) Howard Hughes Medical Institute; 3) Summer Science for Seniors Program; 4) Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX. The notum of the fly is covered with hundreds of external sensory organs (ESOs) which are formed in a stereotypical pattern during pupariation. ESOs are comprised of four cells, all of which derive from a single sensory organ precursor (SOP) cell through asymmetric cell divisions. During the development of the SOP lineage, intercellular communication through Notch signaling regulates the acquisition of distinct cell fates. To identify new players in Notch signaling we performed an EMS mutagenesis screen based on an F1 Ubx-FLP FRT screen. We isolated four alleles of CG15609, the Drosophila homolog of EH-domain Binding Protein 1 (dEHBP- 1). Loss of dEHBP-1 in the notum leads to pIIa-to-pIIb transformation, resulting in an increase of neurons and sheath cells at the expense of shafts and sockets. Interestingly in dEHBP-1 mutant clusters, we observe that Delta fails to localize to the plasma membrane, yet intracellular Delta does not seem obviously affected. Furthermore, expression of Sanpodo is highly upregulated in mutant cells where it localizes to a vesicular compartment. These data indicate that dEHBP-1 regulates the trafficking of Delta and Sanpodo, thereby leading to a loss of Notch signaling phenotype. A thorough characterization of dEHBP-1 mutants will be presented.

603C The role of cato, an atonal-related gene, during Peripheral Nervous System development. Petra zur Lage, Andrew Jarman. SBMS, University of Edinburgh, Edinburgh, United Kingdom. In the Drosophila peripheral nervous system the proneural genes achaete, scute, atonal and amos are required for selection of sense organ precursors (SOPs). Two related genes, asense and cato, are expressed after SOP selection suggesting that they have roles in subsequent SOP development. cato (cousin of atonal) encodes a bHLH transcription factor related to atonal. In the embryo, Cato protein is initially expressed in Ato-dependent SOPs, which represent the chordotonal lineage, and the Amos-dependent SOPs. Later, expression is also switched on in some cells of the external sense organ lineage. At differentiation, expression persists particularly in multiple dendritic neurons. This expression pattern is recapitulated by GFP reporter constructs containing fragments upstream of the cato gene. We have generated two deletion mutations of the cato gene. Mutants exhibit a number of relatively subtle phenotypes, including chordotonal SOP selection and division defects. POSTERS: Neurogenetics and Neural Development 293

604A Fragile X protein functions in neural stem cells. Matthew A. Callan, Daniela C. Zarnescu. Molecular and Cellular Biology, University of Arizona, Tucson, AZ. Fragile X Syndrome (FraX) is the most common form of inherited mental retardation and affects approximately 1 in every 4000 males. The disease is caused by the loss of function for the FMR1 gene, which encodes an RNA-binding protein, FMRP. Currently, FMRP is thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs in neurons. Recently, loss of FMRP has been shown to cause an increased number of neurons at the expense of glia in neurospheres, a mammalian model for neural stem cells. Furthermore, loss of dFmr1 in developing ovaries causes premature differentiation of germ stem cells. Taken together, these data indicate that FMRP function may have an early developmental component, although the molecular mechanism remains to be elucidated. To uncover the mechanism by which FMRP functions early in neural development and in neural stem cells specifically, we are using larval brain neuroblasts as a model system. Preliminary data show that dFmr1 mutant brains exhibit cell cycle and differentiation defects. Currently, we are generating single neuroblast MARCM clones in the larval brain to determine the phenotypic consequences of dFmr1 loss of function early in neural development. Using a battery of cell cycle and differentiation markers we aim to determine the precise roles of FMRP in cell cycle and differentiation in this neural stem cell model and will report our progress.

605B The Tumor Suppressor Gene insensitive Encodes a Nuclear Protein That Controls Asymmetric Cell Division by Regulating Expression of lethal (2) giant larvae. Jamian D Reed, Nick Reeves, James Posakony. Division of Biological Sciences, Section of Cell and Developmental Biology, UC San Diego, La Jolla, CA. 92093. During asymmetric cell division cell fate determinants segregate to one side of a precursor cell. Unequal partitioning of determinants ensures that only one daughter cell will inherit specific protein functions establishing distinct cell fates. We have discovered an additional gene named insensitive (insv) that is required for asymmetric cell division in Drosophila sensory organs. Results: Loss of insv function in the sensory organ lineage leads to inappropriate Notch (N) signaling and the development of extra socket cells. Also, misexpression of insv in cells that receive an N signal converts these cells to the non-responder fate demonstrating along with the loss of function phenotype that the normal function of insv is to antagonize N signaling. Insv localizes to the nucleus and is expressed in all of the precursor cells generated by the sensory organ lineage. Like numb and l(2)gl, insv is also required for the proper internalization of Spdo. Since Insv is a nuclear protein we tested whether the insv phenotype is caused by affects on the expression of Numb or L(2)gl. In insv mutant sensory organs Numb is normally expressed and segregates to the pIIb cell but cortical L(2)gl expression is significantly reduced. Additionally, insv mutant larvae often develop imaginal disc tumors reminiscent of l(2)gl mutants. l(2)gl transript is undetectable in insv imaginal discs and re-supplying l(2)gl under heat shock control reverses the insv adult bristle phenotype demonstrating that insv regulates the expression of l(2)gl at the transcriptional level.

606C Falafel is a novel regulator of asymmetric segregation of the Miranda complex in Drosophila neuroblasts. Rita Sousa- Nunes1,2, William Chia1,2, W. Gregory Somers1,2. 1) MRC Centre for Developmental Neurobiology, King’s College London, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK; 2) Temasek Lifesciences Laboratory, 1 Research Link, National University of Singapore, 117604 Singapore. Drosophila CNS progenitors, neuroblasts, undergo asymmetric cell divisions and are an excellent model system to study this process and its consequence on cell fates. Each division produces yet another neuroblast and a smaller daughter, called ganglion mother cell. Neuroblasts are asymmetric throughout mitosis, exhibiting distinct protein complexes at opposing cortical domains. A number of proteins have been identified to play a critical role in establishing this asymmetry, but it is not fully understood how these molecules are correctly localized and in turn function to target other protein complexes. We performed a clonal screen on third-instar larval brains, designed to identify novel genes required for neuroblast asymmetric division. In this screen we identified falafel, which encodes for an evolutionarily conserved but poorly characterised protein. In dividing mutant neuroblasts the asymmetric Miranda/ Prospero/Staufen/Brat complex is displaced from the cortex to the cytoplasm whereas other asymmetrically localised complexes remain cortical. Falafel contains a Ran-binding domain (similar in structure to pleckstrin homology and EVH1 domains), a conserved domain of unknown function and armadillo/HEAT repeats. Functional analyses of homologous proteins in several species reveal their pleiotropy. Falafel or its homologues have been implicated in cellular and epithelial morphogenesis, cell-cycle progression, chemotaxis, ageing, stress-response and DNA repair. Antibodies generated to Falafel reveal it is a ubiquitous nuclear protein, which localises throughout the cell after nuclear envelope breakdown. Immuno-precipitation and yeast-two hybrid assays suggest that Falafel forms a complex with Miranda in vivo and that the interaction is direct. The mechanism by which Falafel regulates Miranda asymmetric localization will be discussed. 294 POSTERS: Neurogenetics and Neural Development

607A Co-regulators of programmed cell death in neuroblasts. Wei Tang1, Megumu Mabuchi1, Susan Pierre2, Reena Patel1, Barret Pfeiffer3, Kristin White1. 1) Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA; 2) Flybase, Harvard University, Cambridge MA; 3) Janelia Farm Research Campus, HHMI, Ashburn, VA. Correctly regulated apoptosis of large number of unwanted cells such as abnormal and dangerous cells is essential for normal development and homeostasis of the adult. The genes reaper, grim, hid and sickle, clustered in a 400 kb region of the third chromosome, regulate most developmental apoptosis in the fly. We have identified a genotype comprised of overlapping genomic deletions in this region that allows neuroblasts in the abdominal neuromeres, which normally die at the end of embryogenesis, to survive into pupal stages. We have found that neuroblast apoptosis is regulated by the combined functions of more than one of the cell death genes. Deletion of reaper alone does not allow abdominal neuroblast survival, while deletion of grim has a weak effect on neuroblast survival. Deletion of grim also prolongs the survival of neuroblasts that normally die at mid third instar. Knockdown of reaper and grim together by siRNA gives a stronger phenotype than deletion of grim alone, suggesting that the two genes coordinately regulate neuroblast apoptosis. We have characterized an enhancer region between reaper and grim that is likely to regulate the expression of one or more of these genes during neuroblast apoptosis. This analysis will eventually lead to a better understanding of how an individual cell is selected to die in the context of normal development.

608B Dfezl encodes a novel regulator of neural stem cell self-renewal in Drosophila. Mo Weng1,2, Shufen Situ2, Caitlin Gamble5, Cheng-Yu Lee1,2,3,4. 1) Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI; 2) Life Science Institute, University of Michigan, Ann Arbor, MI; 3) Molecular Medicine and Genetics, Unversity of Michigan, Ann Arbor; 4) Center for Stem Cell Biology, Unviersity of Michigan, Ann Arbor; 5) Department of Biology, Univeristy of Oregon, Eugene, OR. Precise regulation of stem cell identity and potential (self-renewal) is pivotal for generation of cellular diversity (differentiation) during normal development, maintenance of homeostasis, and tissue regeneration. Thus, how self-renewal is regulated is a central question in stem cell biology, developmental biology and cancer biology. Drosophila neural stem cells (neuroblasts) divide asymmetrically to self-renew a neuroblast and to generate a differentiating ganglion mother cell, and serve as an excellent model system for studies of self-renewal vs. differentiation. To better understand regulation of self-renewal, we conducted a genetic screen to identify mutations that resulted in either an increase or a decrease in brain neuroblasts. We discovered a novel mutation (l(2)5138) that showed a dramatic increase in neuroblasts at the expense of neurons. l(2)5138 was mapped to the cytological location 22B5- 8, and sequence analysis identifies a single nucleotide substitution resulting in mis-sense mutation at a gene CG31670. CG31670 encodes a novel zinc-finger transcription factor that shows strong homology in amino acid and in organ expression pattern to mammalian Fez and Fezl (forebrain embryonic zinc-finger and zinc-finger-like) proteins, and is renamed dfezl. In dfezl mutant brain, differentiation occurs normally while excessive neuroblasts are produced. Dfezl doesn’t affect localization pattern of polarity proteins and thus functions more downstream as a transcription factor. Currently, we are conducting detailed characterization of the dfezl mutants and analysis of the dFezl protein.

609C RhoGAP100F is Required for R7 Photoreceptor Axon Targeting. Scott Holbrook, Tory Herman. Institute of Molecular Biology, University of Oregon, Eugene OR 97403. We are analyzing the layer-specificity and retinotopy of R7 photoreceptor axons because they are particularly amenable to genetic manipulation: we can analyze the development of individual homozygous mutant R7s in an otherwise heterozygous animal. This has allowed us to dissect the roles even of genes required more generally for synaptogenesis or other aspects of development. Loss of the cell adhesion protein N-cadherin, the receptor tyrosine kinase LAR, or the scaffolding protein Liprin-alpha causes R7 axons to retract to the incorrect target layer. We have recently identified a new mutation that causes this phenotype as the first known loss- of-function allele of RhoGAP100F. RNAi of RhoGAP100F was previously shown to cause no phenotype. The C. elegans RhoGAP100F homolog SYD-1 promotes synapse formation by positively regulating Liprin-alpha. However, SYD-1 lacks a conserved Arginine domain required for GAP activity in other RhoGAPs, suggesting that its function may differ from that of RhoGAP100F and its vertebrate homologs.We are currently investigating whether despite this sequence difference RhoGAP100F and SYD-1 play conserved roles in synapse formation, and we are determining RhoGAP100F’s place in the molecular pathway that includes N-cadherin, LAR, and liprin-alpha. POSTERS: Neurogenetics and Neural Development 295

610A The role of Rac GTPase activity in synaptic structural and functional plasticity at the Drosophila neuromuscular junction. Maude Warren-Paquin, Kazuya Tsurudome, Pejmun Haghighi. Department of Physiology, McGill University, Montreal, Canada. The actin cytoskeleton plays an essential role in the establishment of normal neuronal morphology. Small GTPases of the Rho family, which include Rho, Rac, and Cdc42, are key regulators of the actin cytoskeleton. When activated, they bind various downstream effectors to transduce signals into changes in cytoskeletal dynamics. Although small GTPases are known to be important for normal neuronal development, their role in the regulation of synaptic growth is not well understood. We are presently investigating the action of small GTPases at the Drosophila NMJ. Our preliminary results show that affecting levels of Rac, but not of Cdc42 or Rho, leads to structural and functional synaptic defects. The Drosophila genome contains three highly redundant genes collectively referred to as the Rac genes: Rac1, Rac2 and Mtl. We began our study by determining whether a graded genetic loss of Rac causes synaptic phenotypes. Our preliminary results suggest that the removal of four or five genetic copies of Rac causes a decrease in the number of synaptic boutons and alters synaptic function. Flies lacking all Rac genes are embryonic lethal, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we are using the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single-cell motor neuron clones devoid of all genetic copies of Rac. These experiments will allow us to examine single motor neuron NMJs that have developed in the complete absence of Rac activity. In addition, using a reverse genetic approach, we are currently conducting genetic interaction experiments to identify molecules that interact with Rac to regulate synaptic growth at the NMJ. 296 POSTERS: Neurophysiology and Behavior

611B Involvement of the circadian clock in xenobiotic responses of Drosophila melanogaster. Shawn M Butcher1, Henry Priest2, Louisa Hooven1, Todd Mockler2, Jaga Giebultowicz1. 1) Zoology, Oregon State University, Corvallis, OR; 2) Botany and Plant Pathology, Oregon State University, Corvallis, OR. Circadian clocks control daily rhythms in behavior, physiology and gene expression in many species. Genetic disruption of clock components has been observed to cause arrhythmicity and alteration of the expression of various genes. It has been shown in previous genome-wide microarray studies that genes related to the detoxification pathway such as various cytochrome p450s and GSTs may be expressed in a daily rhythm. Here we utilize physiological and molecular approaches to focus on two p450s, cyp6a2 and cyp6g1, which have been implicated in pesticide metabolism. Our data demonstrate that wild-type flies exposed to a fixed dose of pesticide across varying time points exhibit daily rhythms in their susceptibility. Our goal is to investigate whether the circadian clock governs these daily variations in Drosophila melanogaster response to pesticides. We utilize mutants in specific clock genes to analyze how loss and recovery of clock function, overexpression of clock genes, and loss of function of multiple genes affects both daily susceptibility and general pesticide sensitivity in flies. Our results suggest that specific mutations in clock genes significantly alter pesticide susceptibility. To further pursue the mechanism linking the core clock mechanism with circadian expression of detoxification genes, computational methods are being applied to identify statistically overrepresented DNA motifs in the regulatory sequences of Drosophila protein coding genes. The results of this computer-assisted search will provide us with 3-12mer motifs which may play a role in circadian expression patterns. These results, combined with currently known E-box and Parre elements may help elucidate how circadian clocks play a role in xenobiotic processes.

612C What’s Lov got to do with it? A role for Jim Lovell in Drosophila courtship. Sonia Bjorum, Kate Beckingham. Dept Biochem & Cell Biol, Rice Univ, Houston, TX. Using an eight choice-point vertical maze, we isolated a collection of Drosophila P {GawB} mutants with defects in gravitaxis. Eighteen genes have thus been identified as having roles in gravitaxis, including several novel genes with unexplored functions. One of these genes has proved to be related to the gene fruitless which specifies male courtship behavior. Like fruitless, this gene CG16778, Jim Lovell (Lov), has BTB-POZ and heliz-turn-helix domains, suggesting roles in ubiquitin-mediated degradation and transcription. Imprecise excision has been used to generate additional mutants of Lov in order to understand more fully the roles of the gene in Drosophila. Molecular characterization of the excisions is underway. One of the Lov mutants, Lov66, appears to show defects in both fertility and courtship. Initial immunolocalization shows a tentative role for Lov in spermatogenesis. mRNA expression of individual transcripts for Lov is being examined with in situ hybridizations.

613A A logjam in the neural circuits controlling oviposition behavior. Ginger Carney, Kara Boltz, Stephanie Grady. Dept of Biology, Texas A&M Univ, College Station, TX. logjam (loj) is one of the few genes known to affect female post-mating behaviors. Loj is expressed in a variety of tissues, particularly in the adult central nervous system (CNS) and developing eggs. Our earlier studies showed that loj mutant females produce mature eggs but are unable to oviposit. Neural expression of loj is sufficient to rescue this defect. loj encodes a p24 protein with predicted function in intracellular trafficking. p24 proteins are present in all eukaryotes, and at least one p24 protein has required functions in animal development. However, the specific molecular functions of these molecules are unclear. Mutations in two of the nine fly p24 genes, eclair (eca) and baiser (bai), also prevent oviposition. We hypothesize that p24-expressing cells define an oviposition neural circuit and predict that Loj is required in neurosecretory cells to traffic a molecule required to transmit the egg-laying signal. We are testing this hypothesis by expressing loj in peptidergic neurons in a loj mutant genetic background. Our studies indicate that Loj, Eca and Bai may be present in non-overlapping populations of neurons. Therefore, each p24 protein may function in distinct subpopulations of cells that together comprise the egg-laying neural circuit. Alternatively, a few cells that co- express Loj, Eca and Bai may constitute the oviposition circuit. To distinguish between these possibilities, we need a better understanding of the specific cellular locations of the p24 proteins in the CNS. We are currently performing co-immunolocalization studies to determine if Loj, Eca and Bai are in the same or different cells. Once completed, our studies will define a neural circuit controlling a complex behavior, the 1st step toward elucidating how changes in neuronal function alter complex behavioral outputs. POSTERS: Neurophysiology and Behavior 297

614B Fitting it all together: How the courtship- and mating-responsive fit gene affects male reproductive behaviors. Lisa L. Ellis, Ginger E. Carney. Dept Biol, Texas A&M Univ, College Station, TX. Environmental stimuli, nervous system function, and genetic and epigenetic interactions regulate behavioral outputs. In order to better understand how behaviors are genetically regulated, we are studying the stereotypical courtship behavior of Drosophila melanogaster. The sex-determination pathway regulates reproductive behaviors in males and females; however, only a few targets of this pathway are known. Social experience also influences behavior, but how such experiences affect gene expression and nervous system function remains unclear. To address this problem, we utilized microarray technology to determine how gene expression in the Drosophila nervous system is affected by courtship behavior. From a comparison of expression profiles from heads of males that courted a female, mated with a female, or were mock-exposed we identified a group of 31 courtship-responsive and 61 mating-responsive candidate genes. Of particular interest is a target of the sex-determination pathway, female-specific independent of transformer (fit). In situ hybridization confirms that fit is expressed in the fat body surrounding the brain and abdomen in both males and females. fit is up regulated in males in response to courtship as well as mating. We are utilizing RNAi and null alleles to determine fit’s role in sex determination and reproduction as well as to understand how the fat body affects neural physiology and behavior.

615C Takeout family members play a role in courtship in non-neuronal tissues. Valbona Hoxha1, Hyeeun Kim1, Paul E. Hardin2, Gregg Roman1, Brigitte Dauwalder1. 1) Department of Biology and Biochemistry, University of Houston, Houston, TX; 2) Department of Biology, Texas A&M University, College Station, TX. The takeout (to) gene encodes the prototypical member of a large family of lipophil binding proteins in Drosophila that are similar to juvenile hormone binding proteins from other insects. takeout is specifically expressed in the male head fat body that surrounds the brain. We have recently shown that Takeout protein is present in the hemolymph and acts as a secreted factor in male courtship. A mutation in takeout reduces male courtship behavior and interacts genetically with fruitless, a main regulator of male mating behavior. We are analyzing whether other takeout family members are playing a similar role. All of the genes in the takeout family for which we were able to detect a transcript are preferentially expressed in males, indicating that takeout related genes constitute a family of male specifically expressed genes. Mutants exist for two of the genes and we have found that they exhibit significantly reduced courtship. Thus, several of the genes in the gene family are involved in controlling efficient courtship. Like takeout, the two other family members affecting courtship appear to be expressed outside of the nervous system. In contrast to takeout, however, they are not expressed in fat body, but in glial cells. We are currently examining their role in these cells.

616A Non-transitive sex and violence between genotypes in Drosophila melanogaster. Sergey V Nuzhdin, Larry Cabral, Brad Foley. Molecular Computation Biology, University of Southern California, Los Angeles, CA. In a panel of Drosophila melanogaster lines, we found unpredictable genotype×genotype interactions in the outcomes of aggressive trials between pairs of males, and variable patterns of mating success in those same dyads with females from different lines. Past studies in D. melanogaster show weak, populationlevel preference of females for territorial males, but we demonstrate that the outcomes of territorial interactions between males can be intransitive among different combinations of genotypes. This suggests a way in which directional sexual selection might maintain variation in aggressive behaviours. Additionally, female choices among particular male genotypes are not necessarily consistent across contexts. Instead, females express genetic differences in their patterns of mate choice that are a result of complex interactions between the genotypes of the males being competed, and also their relative territorial status. Selection may result in a mix of behaviours in contexts where the genotype of the interactant matters, and thus in a population there may be no optimum behavioural phenotype. We have shown that the effects on mating success of genetic interactions between individuals can be potentially as evolutionarily important as population mean preferences. 298 POSTERS: Neurophysiology and Behavior

617B Social decision-making in Drosophila melanogaster. Julia Saltz. Population Biology Graduate Group, UC Davis, Davis, CA. Social interactions play a fundamental role in the evolutionary process of virtually all organisms, shaping selective forces in taxa as diverse as primates (e.g., Silk et al., 2007 Science 317(5843):1347-1351) and amoebas (Gilbert et al., 2007 PNAS 104(21):8913- 8917). Since different social environments generate different fitness outcomes (Wolf et al., 1998 TREE 13(2):64-69), mobile organisms should experience selection to make adaptive choices, preferentially associating with a subset of conspecifics while spurning others. Analogous to linkage disequilibrium, such patterns of non-random social associations dictate the spatial distribution of genotypes on a micro-habitat scale. To detect genetic associations between animals and their population-level outcomes, I exploit the tools available in D. melanogaster in conjunction with a novel, whole-room behavioral assay. I control for ecology by providing flies with four identical food patches, thus dissociating social group choice from habitat choice on a relevant spatial scale. By capturing fly aggregations in situ and genotyping both adults and progeny, I demonstrate a genetic basis for variation in aggregation preference and its relevance to mate choice.

618C An ecdysone signal lost: How is loss of EcR influencing adult behavior? Christoph C Schwedes, Ginger E Carney. Biology, Texas A&M University , College Station, TX. Ecdysone signaling through the three isoforms of the ecdysone receptor (EcR) in Drosophila melanogaster has been studied extensively during development and metamorphosis. However, the influence of ecdysone signaling through EcR receptors in modulating adult reproductive behavior is less clear. EcR mutant adults have altered reproductive behaviors such as decreased female egg laying and increased levels of male-male courtship yet the role of each EcR isoform in modifying these behaviors is still unknown. Our immunostaining of mature adults revealed broadly expressed EcR-A and EcR-B1 isoforms in male and female nervous tissue, gonads, fat body, and gut. Understanding the role of specific isoforms and which EcR expressing tissues are important for regulation of reproductive behaviors will be determined by behavioral analyses of isoform-specific EcR mutants. EcR regulated secretion of signaling molecules from the fat body could alter nervous system function and/or reproductive tissue activity. Modification of EcR expressing neurons in the brain may influence integration of courtship stimuli or reproductive status. To first identify which isoforms of EcR impact reproductive behaviors, we are screening for increases in male-male courtship, interruptions in courtship conditioning, and suppression of egg-laying in EcR mutants using isoform-specific deletion alleles. To qualify tissue- specific effects of lacking influential EcR isoforms, we will assay these same behaviors in flies with tissue-specific expression of EcR RNAi constructs for EcR-A and EcR-B1. Our goal is to understand at the isoform and tissue level how EcR mediated hormone signaling influences adult reproductive behavior in D. melanogaster.

619A Neuronal Mechanisms of anesthetic tolerance. Yazan M Al-Hasan, Harish Krishnan, Alfredo Ghezzi, Yan Wang, Nigel Atkinson. Neurobiology, Neuroscience, Austin, TX. Our previous experiments have demonstrated a role for the BK channel (slo) in tolerance and resistance to benzyl alcohol (BA). These experiments are mostly published in Ghezzi et al. Here we use RNAi to knock down slowpoke mRNA expression post developmentally, and found that slo expression during development is not sufficient for the proper development of tolerance. We have induced a slo cDNA using various Gal4 drivers and identified the mushroom bodies to be important sites for BA resistance. We have also identified other biochemical pathways involved in the proper development of tolerance. These pathways include the vesicle recycling and fusion pathways and the Histone Deactylases. POSTERS: Neurophysiology and Behavior 299

620B An Epistatic Analysis between Catecholamines up and α-synuclein in Drosophila. Faiza Ferdousy, Hakeem Lawal, Carrie Williams, Janis O’Donnell. Department of Biological Sciences, Univ Alabama, Tuscaloosa, AL 35401. The neurotransmitter dopamine, the predominant form of catecholamines in the central nervous system, plays an important role in many cellular and signaling processes. Dopamine synthesis is initiated by the action of tyrosine hydroxylase (encoded by pale in Drosophila) the first and rate limiting enzyme in this pathway. Misregulation of dopamine homeostasis has been associated with many neurodegenerative diseases such as Parkinson’s disease. Previously, our lab has shown that the Drosophila gene Catecholamines up, predicted to be a seven transmembrane domain protein, acts as a post-translational negative regulator of tyrosine hydroxylase, the first and rate-limiting enzyme in the dopamine biosynthesis pathway and GTP cyclohydrolase, the first and rate-limiting enzyme in the synthesis of tetrahydrobiopterin. As such, Catsup plays a crucial role in dopamine homeostasis and the neurodegerative diseases associated with dopamine homeostasis. Our lab has shown that mutations in Catsup protect dopaminergic neurons against oxidative stress induced by paraquat in a Drosophila model of Parkinson’s disease. Here we study the role of Catsup in a Drosophila model of Parkinson’s disease involving exogenously expressed human α-synuclein, a protein involved in the etiology of Parkinson’s disease. We present evidence of an epistatic interaction between Catsup and α-synuclein. We also show that a mutation in Catsup is able to rescue the reduced dopamine levels and tyrosine hydroxylase activity characteristic of exogenous expression of human α-synuclein. Taken together our data supports a novel neuro-protective role for Catsup against oxidative stress induced by α-synuclein.

621C Site-directed mutagenesis of the Drosophila NMDA-R1 using homologous recombination. Moon Draper, Mariah Dedrick, Adron Harris, Nigel Atkinson. Dept Neurobiology C0920, Univ Texas, Austin, TX. Ongoing research has identified specific residues in transmembrane domains of the NMDA receptor that are believed to be targeted by alcohol. Site-directed mutatgenesis of the mouse R1 subunit at specifc amino acids has shown a loss of inhibitory effects due to alcohol and other anaesthetics in oocytes. However, in vivo models are not currently available. The Drosophila homolog is highly conserved in these TM segments compared to the mammalian genes. Using a multiple recombinase schema, single residues in the fly NMDA R1 gene have been mutated from phenylalanine to alanine. cDNAs containing this mutation in the fly gene are also tested in oocytes along with behavioural assays of lines with a P-element disruption in the NMDA gene.

622A α δ straightjacket, a Drosophila calcium channel 2 subunit, is required for the proper localization of synaptic voltage gated α 1 2,3 2,3,5 2 1,2,3,4 calcium channel 1 subunits. Cindy Ly , Chi-Kuang Yao , Patrik Verstreken , Tomoko Ohyama , Hugo Bellen . 1) Department of Neuroscience, Baylor College of Medicine, Houston, TX; 2) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3) Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX; 4) Program in Developmental Biology, Baylor College of Medicine, Houston, TX; 5) VIB Department of Developmental Genetics, K.U.Leuven Center for Human Genetics, Leuven, Belgium. In a screen designed to identify genes involved in synaptic function we isolated lethal mutations in Drosophila straightjacket (stj), α δ an 2 subunit of the voltage gated calcium channel (VGCC). stj mutant photoreceptors develop normal synaptic connections but display reduced ‘on-off’ transients in electroretinogram (ERG) recordings which indicates a failure to evoke postsynaptic responses, thereby suggesting a defect in synaptic transmission. stj is expressed in neurons but excluded from glia. The mutants exhibit seizure-like activity when we record endogenous neural activity indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions (NMJs) exhibit ~4-fold reduction in synaptic release compared to controls. In addition we find impaired calcium handling at stj synapses suggested by a rightward shift in the calcium dependence of synaptic release and α increased paired pulse facilitation. These defects in stj mutants likely stem from a mislocalization of Cacophony (Cac), the N-type 1 VGCC subunit. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, α δ α indicating a role for the 2 calcium channel subunit in mediating the proper localization of an N-type 1 subunit at synapses. 300 POSTERS: Neurophysiology and Behavior

623B The development of a semi-automated phototaxis assay to identify genes responsible for ethanol tolerance. Kapil V Ramachandran, Rosie B Ramazani, Nigel S Atkinson. Section of Neurobiology, University of Texas at Austin, Austin, TX. According to NIDA statistics in 2007, the consequences of drug abuse costs Americans over half a trillion dollars each year. Our lab studies the effect of ethanol on the Drosophila model system. We have developed an assay to measure ethanol tolerance. We define tolerance as ethanol-induced resistance to the effects of ethanol. The assay is based on phototaxis, the movement towards a light source. The flies are sedated and an automated camera system takes pictures at defined intervals. The images are then processed by programs as described in Ramazani et al. and then the number of recovered flies are counted. We have identified a deficiency mutant, df-7589, that does not acquire tolerance. The deficiency deletes a portion of the 3L chromosome, encompassing nine genes. After measuring gene expression of these nine genes, we identified candidate genes that may be responsible for the lack of tolerance phenotype. Using gene knockdown and P-element induced mutations specific to these genes, we are currently studying the effects of these candidate genes on ethanol tolerance.

624C Elucidating the role of the evolutionarily conserved elements in the promoter region of the BK-type Ca2+-activated K+ channel, slowpoke, in the induction of tolerance to alcohol. Maureen R. Scholl, Xiaolei Li, Nigel S. Atkinson. Section of Neurobiology, University of Texas at Austin, Austin, TX. The BK-type Ca2+-activated K+ channel gene, slowpoke (slo), encodes a large conductance K+ channel that is highly conserved in both sequence and function in both mammals and invertebrates. These channels play an important role in regulating neural firing patterns and the efficacy of synaptic transmission by integrating calcium, electrical and metabolic signals. Previous research in Drosophila has shown slo to be involved in drug-induced tolerance. As expression of the slo channel is increased, Drosophila acquires tolerance. A knock out mutation of slo prevents acquisition of tolerance to alcohol and other organic solvents, whereas an artificial induction of slo causes tolerance in naïve flies. Therefore, increased expression of the slo channel is required to acquire tolerance. In order to determine how tolerance is acquired, it is necessary to study the machinery of how the drug influences expression of slo. Previous studies have suggested that the effects of alcohol cause epigenetic changes and thus influence the binding affinity of transcription factors to DNA due to histone modification. One example is the transcription factor CREB, which recruits the acetyltransferase CBP to cause histone acetylation. The transcription control region of the slo gene contains several evolutionarily conserved elements, suggesting an important role in the regulation of gene expression. One of these conserved elements, region 6B, was observed to be a highly acetylated region compared to controls at 6 hours and 24 hours after drug treatment. I propose to examine the role of the 6B promoter region of slo, in the induction of tolerance to alcohol, in Drosophila melanogaster, in order to improve the general understanding of alcohol addiction. Determining the steps that are necessary to the induction of tolerance may lead to the discovery of drug targets to treat alcohol addiction.

625A JAK/STAT signaling is required for long term memory in Drosophila. Tijana Copf, Thomas Preat. Genes et Dynamique des Systemes de Memoire, CNRS-ESPCI, Paris, France. Drosophila can form diverse types of memory and, importantly, the underlying molecular and cellular mechanisms appear to be evolutionarily conserved. While gene expression is clearly required in the formation of long term memory (LTM), only few transcription factors and signaling pathways have been implicated in the process. Here we show, using the associative olfactory paradigm, that JAK/STAT signaling is selectively required for LTM formation. The pathway is active in the adult mushroom bodies, the olfactory memory center, as visualized by STAT activity GFP reporter. Down-regulation of any of the pathway components (the secreted ligand upd, the membrane receptor dome, the kinase hop or the transcription factor stat92E) by transgenic RNAi, over-expression of negative regulators or dominant negative alleles of the pathway in the adult mushroom bodies all result in severe loss of LTM, while short term memory and senses are unaffected. Our results identify JAK/STAT signaling as a key pathway for LTM formation in Drosophila, and for the first time implicate this pathway in brain function or behavioral output in any model organism. We are currently confirming the brain expressions of theses genes. POSTERS: Neurophysiology and Behavior 301

626B Impact of reduced numbers of Kenyon cell classes on odor memory in Drosophila. Brian Dunkelberger, Christine Serway, Nicole Nolan, J Steven de Belle. University of Nevada, Las Vegas, School of Life Sciences, Las Vegas, NV. Mushroom bodies (MBs) are paired neuronal assemblies that have been implicated as sensory integration and olfactory memory centers in the Drosophila brain. Genes that influence MB development were initially identified in mutant screens for brain defects in the early 1980s . Most remain poorly characterized in terms of their genetics and influences on anatomy and behavior. Here we describe the nature of MB reduction in three MB mutants: mushroom body miniature B (mbmB), small mushroom bodies (smu), and mushroom bodies reduced (mbr). Histological preparations viewed with fluorescence microscopy and planimetric measurements of the calyx verify the severe MB reductions previously reported in these mutants. Mutant alleles were then combined with GAL4 enhancer elements to target expression of green fluorescent protein in either the nuclei or the cytoplasm of MB neurons. Whole mounts of adult brains from these flies were viewed under a confocal laser scanning microscope, revealing reductions in cell number and aberrant patterns of axonal architecture in these mutants. Based on our analysis, we believe that each gene affects the development of lobe subsets in a different manner. Anatomical studies during development were used to refine when disruption occurs in each mutant. Classical conditioning of odor memory decay curves were generated for mbmB and smu to look at the dynamics between lobe disruption, Kenyon cell number and memory impairment.

627C Distinctive Neuronal Networks and Biochemical Pathways in Appetitive and Aversive Memory in Drosophila Larvae. Ken Honjo, Asami Tanaka, Katsuo Furukubo-Tokunaga. Graduate School of Life and Enviromental Sciences, University of Tsukuba, Tsukuba, Japan. Abilities of finding foods and avoiding noxious substances are essential for animals to survive. Associative learning enables animals to predict the occurrences of foods or toxic substances, and increases their adaptability to various environmental conditions. In classical conditioning, animals learn association of originally neutral stimuli (conditioned stimuli, CS) with unconditioned stimuli (US), which elicit reflexive responses. Whereas associative strength between CS and US is thought to determine learning efficacy, little is known about the neuronal processes that underlie differential CS-US association in the brain. We have established a novel associative learning paradigm for Drosophila larvae, which have a simpler brain than the adult fly. We found that appetitive training with sucrose induces six times longer memory in larvae than aversive training with quinine. Whereas both conditionings produce comparable short-term memory depending on cAMP signaling, only appetitive training induces medium-term memory, which depends on amn and CREB function. Neurocircuitry analyses with UAS-shits1 suggest that memory is stored before the presynaptic termini of mushroom body neurons in either training. However, neural output of octopaminergic and dopaminergic neurons, which exhibit distinct innervation patterns on the mushroom bodies and antennal lobes, is required for appetitive and aversive memory formation, respectively. These results suggest that genetically programmed US pathways involved in memory acquisition may determine different memory spans in different conditionings activating additional molecular components in the brain.

628A A critical component of the nuclear pore complex is implicated in learning and mushroom body development in Drosophila; mushroom body miniature B is Pendulin, the Drosphila Importin alpha 2. Christine N. Serway1, Nicole W.C. Nolan1,2, Stephanie Freer1,3, J. Steven de Belle1,4. 1) School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV; 2) Creighton University Medical School, Omaha, NE; 3) The University of North Carolina at Chapel Hill, Chapel Hill, NC; 4) The National Science Foundation, Arlington, VA. Drosophila melanogaster has long been used as a model organism to study the molecular and genetic mechanisms underlying the development of neuronal circuitry in the brain. This circuitry is responsible for a generating a wide range of complex behaviors. In particular, the mushroom bodies (MBs), paired neuropil structures in the insect brain, are believed to be centers of sensory integration and association, with a predominant role in olfactory classical conditioning. Without them, flies are unable to learn in an olfactory-based Pavlovian paradigm. We have shown that a mutation in the gene mushroom body miniature B (mbmB) displays a significant reduction in MB calyx volume as well as a learning defect, yet they have no gross defects in sensory acuity (odor avoidance and shock reactivity), and walking tests. Sterility in homozygous mbmB females (described previously) and reduced MB volume were found to be tightly linked. Using sterility as a convenient recessive screening phenotype, we mapped mbmB by complementation against multiple types of genetic reagents representing 95% of the second chromosome to the cytological region 30F4-31A2. After sequencing genes in this region, we discovered that mbmB produces a premature stop codon in the gene Pendulin (Pen a.k.a. Importin alpha2), which is a central component of the nuclear pore complex (NPC). We are currently working to rescue the anatomical and behavioral phenotypes associated with mbmB as well as characterizing PEN protein levels and expression pattern in the brains of mbmB mutants. Our molecular characterization of mbmB implicates trafficking across the nuclear membrane in learning and MB development. 302 POSTERS: Neurophysiology and Behavior

629B Second-Order Learning in Drosophila melanogaster. Christopher Tabone, J. Steven de Belle. School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV. Training Drosophila melanogaster utilizing a Pavlovian training protocol provides the means by which researchers can elucidate the mechanisms underlying the behavior of learning and memory. Here we present an analysis of second-order learning using a modified version of the Pavlovian olfactory paradigm. We demonstrate that flies display an ability to form second-order memories in which a previously conditioned stimulus (CS1) can serve as the unconditioned stimulus (US) for a novel conditioned stimulus (CS2) in a second episode of training. Similar results have previously been reported by other researchers utilizing the Drosophila flight simulator. We also demonstrate that flies are capable of elemental learning wherein they can distinguish single odors from complex mixtures during this second-order training paradigm. The study of complex behavior and second-order learning in Drosophila may help to characterize the neuronal circuits and cellular functions underlying the association of conditioned and unconditioned stimuli during memory formation.

630C Induction of the Stress Response Mitigates Heat Disruption of Mushroom Body Development and Gene Expression in Drosophila melanogaster. Xia Wang1, Lisa L. Strobel2, J. Steven de Belle1, Stephen P. Roberts1. 1) School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV; 2) Lehrstuhl für Genetik und Neurobiologie, Universität Würzburg, Am Hubland, Würzburg, Germany. Environmental stress (nutritive, chemical, electromagnetic and thermal) has been shown to disrupt central nerve system development in every model system studied to date. In Drosophila, a brief daily heat shock (HS, 39.5 °C for 35 min) throughout larval and pupal development reduces odor learning by disrupting mushroom body development. In many organisms, a heat pretreatment induces stress response genes that alleviate thermal injuries caused by heat shock. When a heat pretreatment (PT, 36 °C for 1h) is given before each daily HS treatment (PT+HS), MB development is partially restored in male flies, but not in females. Furthermore, microarray analysis in adult male flies revealed that 24 genes show a greater than 1.5 fold change in response to the PH+HS treatment, while 226 genes show greater than 1.5 fold changes due to the HS treatment alone. These HS-affected genes encode proteins involved in diverse biological processes including proteolysis and electron transport. Further behavior tests of PT+HS treated flies will be performed to examine whether heat pretreatment can protect odor learning and memory from impairment caused by heat shock.

631A Creating “Alzheimer” Drosophila via ΦC31-mediated RCME. Anastasia Yemelyanova, Christopher J. Jones. Department of Biological Sciences, Moravian College, Bethlehem, PA. Familial Alzheimer’s disease (FAD) has been linked to mutations in the gene coding for presenilin. Excessive cleavage by presenilin may be responsible for the accumulation of amyloid plaques in the central nervous system of Alzheimer patients. The morphological effects of various “Alzheimer” mutations have been examined in Drosophila melanogaster and have been shown to correlate with FAD age of onset (Seidner et al. 2006). We are interested in examining behavioral effects of these mutations in flies. Preliminary studies using presenilin lines provided by Dr. Mark Fortini (NCI) were inconclusive, probably due to the fact that the transgenes were inserted randomly into the Drosophila genome. By means of a new transformation technique via ΦC31-integrase-mediated cassette exchange (Bateman et al. 2006) we now can assure identical location and expressivity of presenilin transgenes. Currently, we are working to subclone the wild-type presenilin gene into a targeting vector to deliver the insert to a specific site within the fly genome. The resulting construct will then be mutated by site-directed mutagenesis and used in transformation. Behavioral and morphological studies will be performed once the “Alzheimer” presenilin fly stocks are established. POSTERS: Neurophysiology and Behavior 303

632B PKA activity differentially regulates multiple processes required for wing expansion in bursicon-secreting neurons. Fengqiu Diao, Nathan C. Peabody, Benjamin H. White. Laboratory of Molecular Biology, NIMH/NIH, Bethesda, MD. We have previously reported that a dominant-negative Protein Kinase A regulatory subunit, PKAinh, blocks bursicon secretion into the hemolymph and inhibits wing expansion when expressed in Crustacean Cardioactive Peptide-expressing neurons (NCCAP) (Luan et al. 2006, J. Neurosci. 26: 573-584). Because bursicon-secreting neurons (Nburs) are a subset of NCCAP in the pharate adult, we asked whether blocking PKA activity solely within Nburs might inhibit wing expansion. This is, in fact, the case. Selective expression inh of Gal80 in Nburs rescues the wing expansion deficit caused by PKA expression in NCCAP, and, more importantly, flies expressing inh UAS-PKA in Nburs under the control of a bursicon-Gal4 driver, fail to expand their wings.

Nburs consists of two populations of neurons, one located in the abdominal ganglion (BAG), the other located in the subesophageal ganglion (BSEG). The BAG, but not the BSEG, are included in the expression pattern of the c929-Gal4 enhancer-trap line, and we find that flies expressing UAS-PKAinh (two copies) under the control of c929-Gal4 also fail to expand their wings. While these data demonstrate that PKA activity is required in the BAG for wing expansion, it remains unclear whether PKA activity also is needed in the inh BSEG. Behavioral examination of flies expressing UAS-PKA under the control of the bursicon-Gal4 and c929-Gal4 drivers suggests a requirement for PKA activity in the BSEG. Wing expansion is supported by two principal behaviors, abdominal contraction and air swallowing. Flies expressing UAS-PKAinh inh in only the BAG execute both behaviors normally. In contrast, flies expressing UAS-PKA throughout Nburs contract their abdomens normally, but fail to accumulate air in their gut, suggesting a deficit in either air swallowing or air retention. Our results thus indicate inh that PKA acts within the BAG to block a non-behavioral process necessary for wing expansion (perhaps cuticle plasticization), and within the BSEG to interfere with normal air swallowing.

633C Neprilysin 4: An essential endopeptidase is expressed in eve-positive pericardial cells and in the CNS of Drosophila melanogaster. Mareike Panz, Eva Haβ-Cordes, Achim Paululat, Heiko Meyer. University of Osnabrueck, Department of Biology, Section of Zoology. Proteins belonging to the family of neprilysins are typically membrane bound endopeptidases (M13) responsible for the termination and/or activation of peptide signalling events on cell surfaces. Mammalian neprilysins are known to be involved in the metabolism of various regulatory peptides especially in the nervous, immune, cardiovascular and inflammatory systems. Although there is still much to learn about their participation in various diseases they are potential therapeutic targets. Here we report on the identification of neprilysin 4 (Nep4) from Drosophila melanogaster. Reporter lines as well as in situ hybridisation combined with immunolocalisation showed an expression in eve-positive pericardial cells of stage 11 to stage 15 as well as in the central nervous system of stage 13 to stage 17 embryos. Additionally, in 3rd instar larvae, Nep4 transcripts were detected in the brain hemispheres and in the ventral ganglion. The presence of two predicted isoforms could be validated by northern and western blot analysis. Corresponding mRNAs / expressed products of at least one isoform were found in every developmental stage indicating protein activities required throughout the whole life cycle of Drosophila. In addition, western blot analysis could confirm the prediction of one membrane bound and one soluble isoform. This isoform specific solubility has never been reported for any neprilysin before. NEP4 deficient mutants are severely slowed in their development and show lethality in the mid pupal stage, an observation which demonstrates an essential function for Nep4 in Drosophila melanogaster.

634A Genetic analysis of the proprotein convertase amontillado (amon): amon is required for growth control and glucose homeostasis. Jeanne Rhea1, Lowell Rayburn1, Christian Wegener2, Michael Bender1. 1) Dept Genetics, Univ Georgia, Athens, GA; 2) Department of Biology, Phillips University, Marburg, Germany. Proprotein convertases (PCs) proteolytically cleave and activate an array of peptide hormones that regulate growth, development, and physiology. In mammals, two endocrine hormones, insulin and glucagon, are key regulators of glucose homeostasis that are processed and activated by PC1 and PC2. Given the high degree of conservation of the insulin signaling pathway, we hypothesize that amon, the Drosophila homolog of mammalian PC2, regulates hemolymph sugar levels in Drosophila by proteolytically activating drosophila insulin like peptides (dilps) and adipokinetic hormone(akh) - a peptide hormone that is the functional equivalent of mammalian glucagon. We have previously presented data supporting amon’s activation of the dilps to regulate larval growth. Here, we focus on the role of amon in the activation of akh. Measurements of first instar larvae of amon mutants reveal a growth defect in these animals when compared to wild-type. amon null mutants also have significantly reduced hemolymph sugar levels. In addition, mature akh levels are greatly reduced in amon mutants as shown by mass spectrometric peptide profiling techniques. To dissect cell-specific requirements for amon, we are reducing amon function via cell-type specific expression of amon-RNAi transgenes. Reduction of amon in the AKH producing cells of the corpora cardiaca results in animals with reduced sugar levels. This phenotype resembles that seen in animals in which the AKH cells have been ablated. Together, our data supports the hypothesis that akh is a substrate of amon. In a complementary approach, we have restored amon expression in the AKH cells using cell-type specific expression of a uas-amonconstruct. Surprisingly, expression of amon in the AKH cells of amon mutant larvae using an AKH-GAL4 driver partially rescues the growth defect seen in amon mutants, suggesting that amon activity in the AKH cells of the CC is sufficient to promote larval growth. 304 POSTERS: Neurophysiology and Behavior

635B

The 5-HT7Dro Serotonin Receptor: Expression in the CNS and Function. Jaime Becnel, Oralee Johnson, Charles D. Nichols. Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center, New Orleans, LA. Serotonin (5-HT) is a neurotransmitter that influences a variety of behaviors that include circadian rhythms, sleep, appetite, aggression, locomotion, perception and sexual behavior. The many effects of serotonin are mediated through G-protein coupled receptors, which initiate multiple effector pathways. Disregulation of serotonin signaling in humans has been implicated in neuropsychiatric disorders including depression, anxiety disorders, anorexia and schizophrenia. Drosophila expresses orthologs of four mammalian 5-HT receptor subtypes: 5-HT1ADro and 5-HT1BDro, 5-HT2Dro, and 5-HT7Dro. The 5-HT7Dro receptor is orthologous to the mammalian 5-HT7 receptor and activates adenylate cyclase through Gαs. We have mapped 5-HT7Dro receptor expression, and begun an investigation of its role in behaviors. We generated a 5-HT7-GAL4 strain and used it in combination with a UAS-mCD8-

GFP strain to map the circuitry of the 5-HT7Dro receptor. In 3rd instar larvae, 5-HT7Dro expression is observed in discreet populations of neurons in the hemispheres as well as in neurons of the ventral ganglia. 5-HT7Dro expression does not co-localize with anti-5-HT immunoreactivity, indicating that 5-HT7Dro is postsynaptic. In adults, 5-HT7Dro is highly expressed in R-field neurons that innervate the ellipsoid body, a region of the brain believed to be involved in higher order behaviors. Expression is also seen in the antennal lobes, as well as in discreet clusters of neurons between the central brain and visual lobes. Staining with anti-PDF antibodies revealed that cells expressing 5-HT7Dro cluster with the PDF-expressing sLNvs and lLNvs in the brain, suggesting a possible role in modulating circadian rhythms. Pharmacological analysis using the 5-HT1A/7 receptor agonist 8-OH-DPAT in conjunction with the 5-

HT1A receptor antagonist WAY100635 demonstrated an abatement of circadian activity in Drosophila. These results suggest that 5-

HT7Dro may modulate circadian activity in the fly. Further studies to determine the role of 5-HT7Dro in additional behaviors are ongoing.

636C Fly Tracker as a novel system for analyzing movement behaviors in Drosophila melanogaster. David J. Moore, Young-Cho Kim, Jeong-Hye Min, Kyung-An Han. The Huck Institutes Neuroscience Graduate Program, The Pennsylvania State University, University Park, PA. Drosophila melanogaster respond to a variety of stimuli including mechanical and olfactory disturbances as well as addictive drugs ethanol and cocaine through changes in their locomotor activity. Thus, the analysis of stimulus-induced locomotor behaviors is a useful method for identifying the underlying mechanisms and associated behavioral plasticity. Currently, measuring instantaneous parameters of Drosophila locomotor activity is accomplished by video analysis methods. Here, we developed Fly Tracker as a novel, non-commercial locomotor tracking system. Our system provides an accuracy and flexibility that is not feasible with commercial systems. Commercial systems such as Digital Image Analysis System (DIAS) define objects based upon light intensity thresholds and thus the tracking includes dead flies and non-fly objects in the recorded image whose light intensity is below the threshold. This error is avoided with Fly Tracker in which a background image is defined as objects that remain immobile for certain duration of time. We also improved accuracy by tracking two colliding flies as separate objects rather than one, unlike commercial systems. Furthermore, Fly Tracker is an adaptable system that can be modulated to analyze complex movement behaviors such as circling and jumping behavior. We tested the effectiveness of Fly Tracker by analyzing ethanol-induced behaviors in the Drosophila mutants with elevated or reduced levels of dopamine throughout development. The hypodopamine mutants show a decreased response to ethanol in their locomotor activities whereas the hyperdopamine mutants exhibit the opposite effect. These findings suggest a role for the dopaminergic system in the ethanol-induced locomotor behavior. Additionally, we show that Fly Tracker is a novel locomotor tracking system that can be implemented in the analysis of movement behaviors associated with a variety of external stimuli and drugs such as ethanol, cocaine and other psychostimulants.

637A Mutations of the Drosophila Vesicular Monoamine Transporter decrease larval locomotion and the adult response to cocaine. Anne Simon1, Richard Daniels2, Rafael Romero-Calderon1, Grygoruk Anna1, Wu Mark3, Amita Sehgal3, Larry Ackerson1, Nigel Maidment1, Aaron DiAntonio2, David E. Krantz1. 1) Semel Institute for Neurosciences and Human Behavior, UCLA, Los Angeles, CA; 2) Dept of Mol. Biol. and Pharma., Washington University, School of Medicine, St. Louis, Mo; 3) Howards Hugues Medical Institute, Dept of Neurosc., School of Medicine, University of Pennsylvania, Philadelphia, PA. Aminergic signaling pathways regulate a variety of complex behaviors in both the fly and mammals but the mechanisms remain obscure. The Drosophila vesicular monoamine transporter (dVMAT) is responsible for the storage of dopamine, serotonin and octopamine in synaptic vesicles, and dVMAT mutants provide a platform to explore the function of multiple aminergic circuits. dVMAT mutant larvae show reduced spontaneous movements, but locomote similarly to the wild type in response to light touch. Similarly, suction electrode recordings from segmental nerve roots show reduced baseline activity, but respond when the cuticle is stimulated. These data suggest that exocytic release of amines regulates locomotion at a central site, and further experiments will help define the relevant aminergic pathways. Under conditions of low population density, dVMAT mutants pupate, eclose and appear grossly similar to wild type as adults, despite dramatically reduced quantities of serotonin and dopamine. The dVMAT homozygote females are sterile, and males show reduced fertility. Despite a reduced life-span, adults are viable for up to 2 months, allowing a phenotypic analysis of behavior. Adults are mildly impaired in baseline activity and geotaxis, but show an increase in free-range locomotion and a surprising increased response to light in fast phototaxis tests. Interestingly, dVMAT mutants show a blunted response to cocaine. We suggest that the dVMAT mutants may be used as a genetic model to study the adaptive changes that occur in response to decreased amine release, as well as the mechanisms by which amines govern synaptic transmission and behavior. POSTERS: Neurophysiology and Behavior 305

638B Zinc and Dopamine: A functional relationship? O’Neil Wright, Janis O’Donnell. Dept Biological Sci, Univ Alabama, Tuscaloosa, AL. The misregulation of dopamine (DA) is an integral part of many neurological disorders such as Parkinson’s Disease, Schizophrenia and Alzheimers Disease. One of the foci of our lab is understanding the relationship between genetic factors involve in regulating DA biosynthesis and known neurological disorders associated with this process. Catecholamines up (Catsup) appears to be a key gene in the regulation of DA homeostasis. Catsup, a seven transmembrane domain protein, serves as a negative regulator of tyrosine hydroxylase (TH), the rate limiting step in DA synthesis; and GTP cyclohydrolase (GTPCH), the rate limiting enzyme in tetrahydrobiopterin (BH4) synthesis, a regulatory cofactor required for the production of TH. Analysis of Catsup protein sequence has shown that it has a domain similar to known zinc transporters in mammals, and another domain with extensive histidine repeats, suggesting that the protein has the ability to bind zinc. Previous research has shown that exposure of adult wild-type Drosophila to excess zinc induces tremors, paralysis and other neurological symptoms. Moreover, analysis of neurons in wild-type adults in which GFP is expressed using TH-GAL4 and Ddc-GAL4 drivers has shown that zinc induces neuronal toxicity and that subsets of dopaminergic neurons appear particularly sensitive. Though these findings are interesting, the molecular role of zinc in Catsup function, or whether zinc has a direct role in DA regulation, remains elusive. We present the results of a structure-function analysis of the histidine repeat region and the phenotypic effects of a 57 bp deletion of a portion of these repeats, on Catsup protein levels, DA regulation, zinc toxicity, and behavior in adult flies.

639C The effect of individual Acps on sperm competition and uterine muscle contraction. Frank Avila. Dept. of Molecular Biology and Genetics, Cornell University, Ithaca, NY. During mating in Drosophila, both sperm and accessory gland proteins (“Acps”) are transferred to the female reproductive tract in the male ejaculate. The receipt of Acps results in numerous physiological and behavioral changes within the mated female, including being necessary for the proper storage of sperm. Sperm storage in females allows for extended progeny production after a single mating, and for competition between ejaculates of different males. Additionally, during and after mating, the lower reproductive tract of Drosophila females undergoes a stereotypical series of conformational changes that are thought to facilitate sperm storage. Acps, but not sperm, trigger the muscle contractions of the female reproductive tract. Mutational and/or RNAi knockdown studies have identified five Acps necessary for some aspects of sperm storage: Acp36DE, CG1656, CG1652, CG9997, and CG17575. Acp36DE is necessary for sperm entry into storage; the other four Acps regulate the release of sperm from the sperm storage organs. These Acps were examined for their physiological roles in sperm storage and sperm competition. Transgenic RNAi lines were used knock down male levels of these Acps, individually—with the loss of each Acp examined for its effect(s), in their mates, on reproductive tract muscle contraction, sperm movement within the female reproductive tract, and sperm storage parameters.

640A A Cluster of Autonomic Neurons regulates peristolsis Drosophila larval midgut. Dennis LaJeunesse, Brooke Johnson. Department of Biology, University of North Carolina, Greensboro, NC. The Drosophila larval midgut consists of a two thin layers of visceral muscle that push food through an endothelial tube. The mechanisms that propagate peristaltic muscle action and the digestive process in the larval midgut are unknown. Neurons innervate the proventriculus and adjoining anterior portion of the midgut, however the remainder of the midgut is devoid of neuronal input. This organization suggests that peristalsis is maintained and regulated from within the midgut itself. We have identified a novel component of the Drosophila autonomic nervous system that we call the Superior Cupric Autonomic Nervous System or SCANS. The SCANS region is located at the juncture between the distal portion of the anterior midgut and the copper cell/acidic region of the gut. The SCANS is characterized by three structures: (1) a muscular valve; (2) a cluster of 7-9 Lettuce Head Cells, which express nervous system markers including Choline acetyltransferase and Dopa decarboxylase; and (3) two sets of specialized longitudinal muscles that project from the tips of the dorsal gastric cecae, which connect the SCANS to the proventriculus. Each Lettuce Head Cell contacts a longitudinal visceral muscle, extends through the intestinal wall and ultimately projects a lamilopodial head into the lumen of the gut. Using the UAS ricin/Gal4/Gal80ts system we have ablated the Lettuce Head Cells from the SCANS region and observed abnormal valve closure and muscle contractions in the midgut. Our experiments demonstrate that the SCANS and the Lettuce Head Cells are required for the regulation of peristalsis and food movement within the gut. Furthermore, the function and structure of the Lettuce Head Cells suggests that they may be the Drosophila analogues to the interstitial cells of Cajal, which function as pacemakers in vertebrate intestines. 306 POSTERS: Neurophysiology and Behavior

641B Dissecting patterns of aggressive behavior established by fruitless. Steven Nilsen1, Dylan Nelson2. 1) Biology, Oxford College of Emory University, Oxford, GA; 2) Neurobiology, Harvard Medical School, Boston, MA. Can neural circuits responsible for different aspects of sexually dimorphic behavior be genetically separated? A screen for mutants that variably reverse sexually dimorphic behavior would be ideal, but intractable, approach to this question. For a large screen, detailed behavioral assays must be compromised; pleiotropy of mutations often alters complex behavior; thus, screens can select supernumerary mutants. To avoid these issues, we perform a low throughput screen by interfering male-specific fruitless transcripts in specific neurons. Selected GAL4 enhancer trap lines, from the flytrap collection, were crossed with females carrying multiple copies of UAS-fruIR transgene, at 25 °C. Progeny were raised and tested (from embryos or N1 larva) at 29.5°C, the temperature where this RNAi works most efficiently. Many sexually dimorphic behaviors were scored using a standard aggression assay (Chen, Lee and Bowens et al., 2002, PNAS 99(8): 5664-8). A few lines were selected for retesting using different controls (including females) and a larger sample size, thus revealing one GAL4 line of interest. Using a UAS-GAL4 “amplifier” transgene, we correctly predicted that increasing GAL4 levels would augment an abnormal phenotype, which falsifies the possibility that genetic background or transgene position caused the phenotype. Many sexually dimorphic patterns of Drosophila melanogaster aggression still require elaboration. One example is the likelihood of both animals attacking with a thrust pattern during the same social encounter, a reciprocal thrust: in males, this is rare, but much more common in females. During reciprocal thrusts both animals are attacking, just as during boxing and/or tussling. Together we group these patterns as forms of a form of intense retaliatory behavior that appears to require significant energy output by both animals. We identify a few sexually dimorphic neurons (in the SOG and superior protocerebrum), from one GAL4 pattern, that contribute to the frequency of retaliation.

642C Drosophila CG16801/fdx modulates eclosion, wing expansion behaviors, and possibly fertility. Steven Robinow, Qing Chang, Laura Wong, Elizabeth Nguyen, Nelson Lazaga, Carl Sung. Dept Zoology, Univ Hawaii, Honolulu, HI. We have been characterizing CG16801, the ortholog of the vertebrate photoreceptor specific nuclear receptor gene, in an effort to develop a more comprehensive understanding of the role nuclear receptors play in neural development and function. CG16801 cDNA was cloned by RT-PCR. Northern analysis demonstrates that CG16801 generates two major transcripts of 3.3 kb and 2.2 kb. While the 3.3 kb transcript is the major species during early embryonic development, the 2.2 kb transcript becomes dominant during the mid and late stages of embryogenesis and continues to be the dominant transcript during post-embryonic stages. In situ hybridization demonstrates that CG16801 transcripts are localized within the central nervous system. Expression is observed in discrete clusters of cells including the neurons of the mushroom body. To investigate the function of CG16801, a mutant allele was generated by homologous recombination. Animals lacking CG16801 displayed one of three phenotypes. 20% of the mutant/deficiency animals failed to eclose. 25% of the mutant animals eclosed but failed to expand their wings. The remaining 55% of the mutant animals eclosed and expanded their wings. However, all of these animals were sterile, independent of gender. We have rescued the eclosion and wing expansion phenotypes but have been unable as yet to rescue the sterility observed in mutant/deficiency animals. Based on the phenotypes that are rescued by a fdx transgene, we propose to rename this locus as failure to deploy or expand (fdx). We are undertaking efforts to identify independent alleles of fdx and utilize an RNAi transgene to independently test whether the sterile phenotype maps to the fdx locus. We are testing the hypotheses that fdx modulates the neuroendocrine control of eclosion and wing expansion, and that fdx is required for the proper development or function of the neurons that innervate the testes and ovaries.

643A Development of Assays Used to Study Tolerance to Drugs of Abuse in Drosophila. Nyssa A. Sherazee, Jascha B. Pohl, Kevin Bieri, Tanzeen Yusuff, Nigel S. Atkinson. Department of Neurobiology, University of Texas at Austin. Drug abuse is a continuous problem costing our society over $200 billion a year. Drosophila provide a good model system for studying many drugs of abuse due to similarities in behavior to humans and the wide variety of genetic tools available. Our lab has been studying tolerance, defined as a reduced response to the effect of a drug due to previous exposure, which is an important component of addiction. However, little is understood about the neuronal mechanism behind this behavior. We are developing assays to investigate the effects of a variety of abused drugs. For instance, we have designed an assay to investigate the sedative effects of ethanol. We have found that Drosophila gain tolerance to the knockdown effects of ethanol after previously being sedated, thus demonstrating tolerance. We are isolating mutants that show abnormal tolerance to determine important genetic components. Interestingly, flies with a loss of function mutation for the period gene, involved in circadian rhythmicity, do not gain tolerance. We are currently developing assays to study other drugs of abuse. POSTERS: Neurophysiology and Behavior 307

644B Genetic mapping of the bas mutation in Drosophila. Simon Tabchi, Nathaniel Tussey, Christopher J. Jones. Department of Biological Sciences, Moravian College, Bethlehem, PA. In Drosophila, the bang sensitive mutations cause paralysis of the fly upon mechanical shock. One specific recessive mutation from this group is in fact called “bang-sensitive” (bas). Ganetzky and Wu (1982) mapped this mutation to a region between the g (garnet) and sd (scalloped) mutation markers, corresponding to the 12F region of the genome. We are attempting to identify the bas gene, initially through deletion mapping. We have used both extant deletions and deletions created using Exelixis P-element lines to narrow down the location of bas to the 12F1-12F4 region on the X chromosome. Mapping of bas is ongoing in an attempt to narrow the list of candidate genes further.

645C Mapping the bss mutation in Drosophila. Nathaniel Tussey, Simon Tabchi, Christopher J. Jones. Department of Biological Sciences, Moravian College, Bethlehem, PA. The bang senseless mutation, bss, is one of a number of bang-sensitive mutations in Drosophila; flies carrying one of these mutations become paralyzed upon mechanical shock. Previous recombination mapping has limited bss to the 14B2 - 14B9 region of the X chromosome between the markers sd (scalloped) and f (forked) (Ganetzky and Wu 1982). Although Ganetzky and Wu found bss to be semidominant, in our hands both existing bss alleles are dominant, precluding traditional deletion mapping. Previously described recessive temperature-sensitive paralysis (Grigliatti et al. 1973) as well as differences in the duration of paralysis between bss homozygotes, heterozygotes, and deficiency heterozygotes (Rutherford, 1995) were retested but not confirmed. We are currently outcrossing bss into several genetic backgrounds in preparation for repeating these tests. We are also constructing multiply-marked chromosomes for recombinant mapping.

646A Pain sensitization induced by tissue damage in Drosophila larvae. Daniel T Babcock1, Christian Landry2, Michael J Galko1. 1) Department of Biochemistry & Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX; 2) ProDev Engineering, Houston, TX. Pain sensation is a crucial defense mechanism that allows detection of and rapid response to potentially damaging stimuli. In vertebrates, alterations in pain sensation often occur as a result of inflammation or tissue damage. An increase in pain sensitivity may result in an exaggerated response to noxious stimuli (hyperalgesia) or a sensation of pain when presented with normally innocuous stimuli (allodynia). Here we establish a novel assay for studying alterations in pain sensation resulting from tissue damage. Recent studies demonstrated that Drosophila larvae exhibit a stereotypical “rolling” withdrawal response when presented with painful stimuli. We constructed a thermal probe to stimulate larvae in individual body segments at precise temperatures. We determined the pain threshold using our assay to be approximately 39oC, the lowest temperature at which we saw any response in our w1118 control strain. At 44oC nearly all of the larvae respond after an average stimulation of 9.3 seconds, and when temperatures approach 48oC the response is very rapid. To induce tissue damage we irradiated larvae with a sub-lethal dose of ultraviolet (UV) radiation that causes morphological deterioration of the epidermis and activation of the apoptotic protease caspase-3. Nearby sensory neurons are grossly intact in this assay but the irradiated larvae show clear alterations in pain sensation. 24 hours after UV irradiation, the response to noxious temperatures (44oC) is nearly twice as fast as in control larvae (hyperalgesia) and the pain threshold itself is lowered by several degrees (allodynia). Our results introduce a new genetically tractable model of pain sensitization that we will use to identify the cellular and molecular mechanisms by which tissue damage leads to alterations in pain sensitivity. 308 POSTERS: Neurophysiology and Behavior

647B aguesic is a gustatory-related DEG/ENaC ion channel. Yehuda Ben-Shahar1, Michael Welsh1,2. 1) HHMI; 2) Internal Medicine, University of Iowa College of Medicine, Iowa City, IA. Members of the Degenerin/ENaC family of ion channels can be found in genomes of all animals. Several members of the DEG/ ENaC family have been implicated in various sensory modalities such as mechanosensation, nociception, and salt taste. Here we describe aguesic (agu), a new member of the Deg/ENaC family in flies. agu expression was abolished in the poxn mutant background, suggesting it is enriched in chemosensory structures. To further establish the role of agu in chemosensory functions, we generated transgenic flies that express a GFP reporter driven by the putative agu promoter. Multiple independent transgenic lines showed strong GFP expression in gustatory hairs in the adult appendages, suggesting a possible gustatory role for agu. In addition, a GFP- tagged agu localized to sensory cilia in taste neurons, suggesting the protein may be involved in gustatory chemosensory transduction. Preliminary analysis of mutations in agu suggested that this ion channel can modulate sensory sensitivity to some gustatory stimuli. In conclusion, we used various approaches to show that agu is a chemosensory-specific channel, contributing to gustatory behaviors in flies. Further studies should establish whether agu is acting as an independent sensory transducer, or whether it is a modulatory component of other sensory molecules such as members of the gustatory GPCR family.

648C john glenn (jog), a gene implicated in gravitaxis, may be involved in axon outgrowth and function downstream of Drosophila APP (APPL). Cassidy B. Johnson1, Vanaja Konduri1, Sven Huelsmann2, Nick Brown2, Kathleen M. Beckingham1. 1) Dept. Biochemistry & Cell Biol, Rice Univ, Houston, TX; 2) Gurdon Institute and Dept. of Physiology, Development and Neuroscience, University of Cambridge, UK. In a previous screen of a collection of P{Gal4} transposon-insertion mutants, our lab identified the gene john glenn (jog; previously CG11940) as playing a role in gravity based behavior. Sequence analysis indicates that Jog encodes the single Drosophila member of the MRL family of proteins, which have roles in cell adhesion and motility. The single C. elegans MRL protein Mig-10 affects axonal growth via the Slit and Netrin pathways. A mammalian homolog of Jog is also known as β-amyloid precursor protein-binding, family B, member 1- interacting proteins (APBB1-IP) based on its interaction with components of the signaling pathway downstream of the Alzheimer’s related membrane protein APP. jog encodes two protein isoforms, the longer one containing a short additional domain. Our gravitaxic mutant is predicted to affect this longer isoform. Additional mutations that delete genomic DNA encoding one or both isoforms have been prepared. We have produced an antibody for the domain specific to the large isoform and are preparing one for the common region. Experiments to investigate possible roles of jog in axon guidance and in signaling downstream of the Drosophila APP protein are in progress.

649A Characterization of Drosophila bitter taste receptor. Seok Jun Moon, Craig Montell. Department of Biological Chemistry The Johns Hopkins University, Baltimore, MD. The ability to distinguish nutritious food from noxious substances is critical senses for the survival of all animals. The bitter taste associated with many toxic chemicals prevents animals from eating noxious substances, even when they are mixed with nutritious foods. Thus, the ability to detect bitter chemicals is important to help animals avoid ingesting harmful tastants because failing to do so could cause lethality. Many naturally occurring by-products from plant are bitter and toxic. However, they are structurally not related. Nevertheless, animals are able to detect these diverse chemicals using a restricted number of taste receptors. In Drosophila, there are 68 gustatory receptors (Grs) which are subdivided into two groups based on whether they are co-expressed with either Gr66a or Gr5a. Gr66a and Gr5a expressing gustatory receptor neurons (GRNs) are representing bitter and sweet sensing GRNs, respectively. Because of the diversity of bitter chemicals, indeed most of Grs are expressed in a subset of Gr66a positive GRNs and predicted as bitter receptors. However, little is known about the function of individual bitter receptor candidate including Gr66a. Recently, our lab showed that Gr66a is required to sense caffeine in vivo. The flies missing Gr66a showed greatly reduced avoidance to caffeine in a behavior assay. Caffeine-induced neuronal impulses are abolished in the Gr66a mutant flies, but other bitter and sweet responses were not altered. Growing body of evidence suggests that Drosophila Grs can function as heteromultimers. We have found that ectopic expression of Gr66a was insufficient to give rise to a caffeine response, raising the possibility that it may also heteromultimerize with another receptor. We are currently examining other Grs, to identify the putative co-receptor. POSTERS: Neurophysiology and Behavior 309

650B Atypical soluble guanylyl cyclases in Drosophila are expressed in taste chemosensilla and are involved in taste preference behaviors. Anke Vermehren, Judith Stewart, Wendy Timmermans, David B Morton. Integrative Biosciences, OHSU, Portland, OR. The Drosophila genome contains five genes that code for soluble guanylyl cyclase (sGC) subunits. Three of these genes, Gyc- 89Da, Gyc-89Db, and Gyc-88E, code for the atypical sGC subunits which have been identified as likely molecular oxygen sensors in invertebrates, primarily regulated by oxygen. Using promoter-GAL4 fly lines to drive the expression of the red fluorescent protein dsRED, Gyc-89Da and Gyc-89Db have been localized to the CNS and peripheral sensory neurons. Taste sensilla are hair-like structures that house 2-4 gustatory and one mechanosensory neurons, and the Gyc-89Da and Gyc-89Db subunits have been found to innervate the short, intermediate and long taste sensilla on the labellum of the adult fly, as well as some tarsal and wing chemosensilla. To determine the role played by the atypical sGC subunits in feeding preference behaviors we used Gyc-89Da and Gyc-89Db mutants that contained a transposon inserted within each gene. Using a standard larval preference test, we showed that wild type larvae prefer 50 mM sucrose to water, and avoid 10 mM caffeine. Tests performed with Gyc-89Da and Gyc-89Db mutants showed that, while the Gyc-89Da mutant larvae also preferred sucrose, the Gyc-89Db mutants did not, and while the Gyc-89Db mutants avoided 10 mM caffeine, the Gyc-89Da did not. Adult flies show similar responses to sucrose (preference) and caffeine (avoidance), and in contrast with our larval preference results, wild type and Gyc-89Da and Gyc-89Db mutants preferred 10 and 100 mM sucrose to water. On the other hand, while wild type flies avoided 1 mM caffeine, both Gyc-89Da and Gyc-89Db mutants did not. All fly lines avoided 10 mM caffeine. All these results are supported by assays in which cGMP was reduced in Gyc-89Da and Gyc- 89Db neurons by expressing bovine phosphodiesterase 5 (cGMP specific) in these cells using the GAL4 system. Support contributed by: NS29740.

651C Bitter taste in Drosophila. Linnea A. Weiss, Anupama Dahanukar, Jae Young Kwon, John R. Carlson. Molec., Cell. & Dev. Biology, Yale University, New Haven, CT. We examined the molecular and cellular basis of bitter taste. Taste stimuli are detected via neurons in chemosensory sensilla. ~30 labellar sensilla have been classified (L,M,S,I,P) by morphology and location. A family of 60 gustatory receptor (Gr) genes encoding 68 receptors has been identified. Gr66a is expressed in bitter-sensitive neurons and 8 receptors have been shown to be expressed in Gr66a+ neurons. We have generated flies with promoter-GAL4 drivers for 61 of the 68 Gr proteins. At least 25 Gr-GAL4 drivers are expressed in the Gr66a+ neurons of S,I and/or P sensilla. None of these 25 is expressed in L or M sensilla. S sensilla express the most drivers and fall into two classes: S1,2,3,7,10, which express 19-21 drivers, and S4,6,9, which express 9-10 drivers. I and P sensilla express 5-6 drivers and divide into two classes (I1P1-5 and I3P6,7) based on the complementary expression of two receptors. Thus, each class of sensilla expresses a unique combination of Gr genes. Our expression data predicted that flies possess many bitter receptors and there are functional differences among sensillar classes. We analyzed the electrophysiological responses of each labellar sensillum to structurally diverse bitter compounds. None induced action potentials in L or M sensilla in agreement with their lack of expression of the 25 putative bitter receptors. Robust responses were observed in the remaining sensilla. S sensilla are broadly tuned, but fall into two groups with distinct responses.

These two groups correspond exactly to S1,2,3,7,10 and S4,6,9 as identified in our expression analysis. I and P sensilla are more finely tuned and divide into two classes that respond to complementary subsets of bitter compounds. These groups correspond to I1P1-5 and I3P6,7 as identified in our expression analysis. We demonstrate functional differences between and within classes of sensilla and find a correlation between receptor expression and breadth of response. Our data support roles for these 25 Gr proteins in bitter taste and provide a receptor and physiological map of the fly labellum.

652A Phosphatidyl inositol 4,5 bisphosphate signaling determines synaptic morphology of the Drosophila neuromuscular junction. Ron L.P. Habets1,2, Thang M. Khuong1,2, Patrik Verstreken1,2. 1) Laboratory of Neuronal Communication, K.U.Leuven, Center for Human Genetics, Belgium; 2) VIB, Department of Molecular and Developmental Genetics, Belgium. Correct formation and maintenance of synaptic architecture is crucial for proper neuronal communication. One of the regulators of cellular signaling is phosphatidyl inositol 4,5 bisphosphate (PIP2). To determine the effect of this lipid on the morphology of the neuromuscular junction we manipulated PIP2 levels genetically and assessed PIP2 abundance by determining the accumulation of a GFP-fused phospholipase-Cδ1 PH domain (PHplcδ1-GFP), a peptide known to bind PIP2. PIP2 levels were increased to about 150% using loss of Synaptojanin function, an inositol phosphatase located at the synapse. Conversely, PIP2 levels were significantly decreased in tweek mutants that we identified in a screen of a collection of EMS mutated flies for reduced synaptic PHplcδ1-GFP. Interestingly, tweek mutants show an increase in synapse length and bouton number and a corresponding decrease in the number of active zones per synapse area, in contrast to synaptojanin mutants. Furthermore, preliminary analyses with a dominant negative probe that masks the available synaptic PIP2 shows very similar defects when compared to tweek mutants, suggesting that PIP2 acts to restrain synaptic growth. We are currently exploring the role of PIP2 as a critical regulator of cytoskeletal organization at the synapse. 310 POSTERS: Evolution and Quantitative Genetics

653B Variability of the Dras1 gene in D. virilis sibling species. Anna Chekunova1, Vyacheslav Sergienko1, Helen Zelentsova2, Larisa Gauze1, George Bakhtojarov1, Alex Kulikov1, Vladimir Mitrofanov1. 1) Dept Genetics, Koltsov Institute of Developmental Biology RAS, Moscow, Russian Federation; 2) Dept of Molecular Mechanisms of Biological Adaptation, Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Moscow, Russian Federation. Drosophila ras1 gene product is 75 % homologous to mammalian HA-ras gene product. First 80 amino acids of human N-Ras protein are identical to the ras of Drosophila. The analysis of 27 alleles of all three ras genes in D. melanogaster showed very low level of protein polymorphism. Ras proteins are a part of the large group of GTP-binding proteins. The ras1 gene product plays an important role in signal transduction and ras gene is expressed continuously throughout development. Thus, ras1 can serve as good control for determination of molecular variability in Drosophila, because exclusively nonsense mutations are allowed to be preserved in this locus. At the same time it is necessary to have information on localization of the locus to the particular region of the chromosome in order to better understanding of mechanisms of expression regulation of the locus. The gene Dras1 vir (Dras1 virilis) is localized to the second chromosome of D.virilis section 25 (Gubenko map). We used as a probe a conservative fragment of the Dras1 vir gene (300 b.p.), which was amplified by PCR with TACGATCCCACCATCGAGGA and TTGTTGCCCACGAGCACCAT forward and reverse primers accordingly. We created restriction maps of the ras1 vir gene for both D. virilis and D. lummei. We studied polymorphism or 11 species of the D.virilis group using the same PCR generated probe of the conservative region of the Dras1 vir gene. DNA was cut with PvuII, EcoRI, HindIII and TaqI endonucleases. PvuII polymorphism is very interesting so far as the PvuII fragment lengths fluctuated from 6.5 to 5.0 kb and the presence of only one band suggests absence of the additional restriction sites within the gene. Using Southern blot hybridization we showed that all species have there unique restriction pattern. The study was supported by the RFBR grant #05-04-49450.

654C Testing the functional consequences of adaptive protein evolution at the bag of marbles gene for Drosophila germline stem cell differentiation. Heather A. Flores, Charles F. Aquadro, Daniel A. Barbash. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY. Drosophila germline stem cell (GSC) differentiation is controlled at multiple levels. GSCs are present in a niche environment, and extrinsic signals from the niche in addition to intrinsic signals act to maintain the stem cells. The bag of marbles (bam) gene is both necessary and sufficient for GSC differentiation, acting as a switch to move from the stem cell state to a differentiated daughter cell in oogenesis. Given the intricate control exhibited in this pathway, one might expect that the genes involved would be under extreme functional constraint. However, recent work by our groups and others has shown that many, but not all, of these genes are experiencing rapid, adaptive protein evolution in Drosophila melanogaster and the closely related species, D. simulans. We are focusing on the bag of marbles gene to understand the functional consequences of this rapid protein evolution. We are using interspecies complementation to test whether the bam ortholog from D. simulans can fully complement a bam null mutation in D. melanogaster. Additionally, we are using cytological methods to determine the level of conservation of oogenesis in species of the melanogaster subgroup. By combining direct functional analyses with our statistical inferences of adaptive evolution, we aim to identify the function(s) of bam that are altered by selection and to identify the selective forces acting to diversify the genes that regulate germline stem cells.

655A Evolution of Hox gene expression and function and the effect on limb specification in arthropods. Cheryl Hsia1, Adam Paré1, Matthew Ronshaugen2, William McGinnis1. 1) Division of Biology, University of California, San Diego, La Jolla, CA; 2) Faculty of Life Sciences, University of Manchester, Manchester, UK. Hox genes are evolutionarily conserved transcription factors that pattern the body along the anterior-posterior axis in metazoans and have been implicated in the evolution of diverse body plans. Most hexapod insects, for example, have limbs in the thoracic but not in the abdominal pre-genital segments, while most of their crustacean ancestors have limbs throughout their pre-genital trunk segments. In order to gain a deeper understanding of the molecular events contributing to the limb number differences between the insect Drosophila melanogaster, and the crustacean Artemia franciscana, the expression patterns of abd-A transcript and protein in Artemia were determined and the function of Artemia ABD-A protein tested in Drosophila embryos. There was no detectable ABD- A protein expression in the pre-genital trunk of Artemia, including the limb primoridia, prior to and during trunk development when the limb-promoting gene, Distal-less, is activated. This finding in Artemia is the first example of the silencing of a functional Hox gene throughout early developmental stages. The early ablation of segmental identity function apparently allows the development of limbs on Artemia trunk segments and may represent a Hox gene in evolutionary transition, temporarily silenced during trunk formation, and capable of evolving novel patterning functions. POSTERS: Evolution and Quantitative Genetics 311

656B Distinct modifications of EGF receptor signaling in homoplastic evolution of eggshell morphology in genus Drosophila. Tatsuo Kagesawa, Yukio Nakamura, Minori Nishikawa, Kenji Matsuno. Dept. Biol. Sci. / Tec., Tokyo Univ. Sci., Japan. Homoplasy is a phenomenon that organisms independently acquired similar traits in different phylogenetic group. However, it is largely unknown how similar morphological traits were manifested during the evolutionary changes of developmental mechanisms. In genus Drosophila, their eggshells have the various numbers of the dorsal appendages (DAs). DAs function to supply the developing embryos with oxygen. In this genus, all species of the subgenus Sophophora, including Drosophila melanogaster (D. melanogaster), have eggshells with two DAs, while most species in the subgenus Drosophila, including Drosophila virilis (D. virilis), have those with four DAs. Four-DA-characteristic is an ancestral trait from which two-DA-characteristic was derived. Phylogenetic analyses suggest that Drosophila melanica (D. melanica), which belongs to subgenus Drosophila, acquired two-DA-characteristic independently of the species in subgenus Sophophora. Patterning of the DAs is tightly regulated by epidermal growth factor receptor (EGFR) signaling in D. melanogaster. A single DA is formed from each cluster of follicle cells where EGFR signaling is activated. Here, we compared the patterns of EGFR signaling activation in the species of subgenera Drosophila and Sophophora. Our analysis demonstrated that distinct patterns of EGFR signaling activation in each subgenus were consistent with their phylogenetic relationship. In spite of their common two-DA- characteristics, the EGFR signaling activation pattern in D. melanica was significantly diverged from that of subgenus Sophophora species. Our results suggested that homoplastic two-DA-characteristic was derived by different modifications of a developmental system, which were independently occurred at least twice in genus Drosophila.

657C Transkingdom sex: viral mediated cytoplasmic incompatibility in the Wolbachia/Drosophila symbiosis system. Timothy Karr, Ben Heath. Dept Biol & Biochemistry, Univ Bath, Bath, United Kingdom. Wolbachia is a pervasive endosymbiont associated with a wide variety of arthropods and filarial nematode hosts. As reproductive parasites, Wolbachia manipulate host reproduction by a variety of mechanisms including cytoplasmic incompatibility (CI), male- killing, parthenogenesis and feminization. CI represents a unique form of paternal-effect rendering sperm incapable of completing karyogamy following fertilization and is similar to defects observed in ms(3)K81, a paternal effect mutation in D. melanogaster. Despite intense scrutiny, the cellular basis of CI remains unknown. Recently prophage DNA sequences have been recognized in Wolbachia genomes. Consistent with this observation, viral particles have been observed in ovaries and testes. Here we describe the life cycle of the Wolbachia “B” (WoB) virus using PCR assays specific for Wolbachia and (pro)phage DNA. Remarkably, we show using PCR and electron microscopy that intact WoB virions are, (i) produced following a (presumptive) lytic cycle and viral assembly, (ii) incorporated into developing sperm during spermatogenesis, (iii) transferred to the female reproductive tract following copulation and (iv) delivered to the egg during fertilization. EM and previous genome analyses places WoB virion as a member of viral particles similar to the podoviridae (T7-like) family of viruses. Tetracycline treated males devoid of detectable Wolbachia are nonetheless virus positive and such males express incompatibility in the form of egg lethality strongly implicating WoB as the causative agent of CI. Evidence of viral infection of resident Wolbachia in infected eggs supports a “Transkingdom sex” hypothesis that explains the presence and maintenance of active prophages in an obligate intracellular proteobacterium. Transfer and inheritance of active eubacterial-derived virions within a eukaryotic sperm cell raises important evolutionary and population genetic issues. Maintenance of a lysogenic virus in Wolbachia may also provide a mechanism of horizontal gene transfer often seen in Wolbachia lineages.

658A Various evolutionary rates in the Drosophila virilis species group. Alex Kulikov, Oleg Lazebny, Vladimir Mitrofanov. Dept Genetics, Koltsov Institute of Developmental Biology, RAS, Moscow, Russian Federation. Polymorphism of sequences of five genes in 11 sibling species of the D. virilis species group was investigated. The following tests to estimate the irregularity of evolution rates in different lineages were conducted: Takezaki’s test to compare branch lengths, Tajima’s test of neutrality, and a joined test of Gillespie and Bulmer to estimate substitution rate differences in different lineages. The effect of lineages on accumulation of substitutions was shown as well as differences in evolutionary rates among coding and non- coding regions. Study was supported by Russian Foundation for Basic Research grant 05-04-49450 and by the Research Program of Russian Academy of Sciences “Dynamic of Gene Pools”. 312 POSTERS: Evolution and Quantitative Genetics

659B Interspecies variability and genetic analysis of phallus hairiness in sibling species of the Drosophila virilis species group. Alex Kulikov, Nick Gornostaev, Oleg Lazebny, Vladimir Mitrofanov. Dept Genetics, Koltsov Institute of Developmental Biology, RAS, Moscow, Russian Federation. Copulatory organ has arisen as a separate morphological structure in Apterygots for the first time in evolution of animals. The 9- 11th abdomen segments are supposed to take part in its formation in these species. It is known that rate of evolution of traits related to the structure and functioning of animal reproductive system of closely-related species is many times higher than that of other characters. Sibling Drosophila species are the most convenient living model for studying genetic basis of evolution of male mating organ. Microchaetes, being a mechanical sensory organ, occupy a special place among numerous characters of male mating organ. In insects, the whole body including abdomen is covered by chaetae of proneural origin. One may expect that evolutionary transformations of abdomen to internal genitals should lead to microchaetes reduction. Nevertheless, phallus hairiness is a well known phenomenon in Leucophenga, Scaptodrosophila and others. In this study genetic determination of phallus downiness was studied in sibling species of the D. virilis species group. Microchaetes thickly cover the top of the middle part of aedeagus in species of the D. montana phylad occupying up to a quarter of its length. In species of the D. lummei phylad only males of D. lummei have microchaetes on the phallus top, while other species are characterized by bald-headed aedeagi. Strain of D. virilis with chromosomes marked with several recessive mutations and a wild type strain of D. lummei were used for genetic analysis. 475 phallus samples were prepared and analyzed. Leading role in microchaetes formation on the phallus top of the second and sixth chromosomes has been showed. The current study was supported by Russian Foundation for Basic Research grant 05-04-49450 and by the Research Program of Russian Academy of Sciences “Dynamic of Gene Pools”.

660C Evolution of head development and axis specification in higher flies. Steffen Lemke, Matteen Rafiqi, Urs Schmidt-Ott. Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL. In Drosophila, localized transcripts of the class 3 Hox gene bicoid are required to establish the anterior-posterior axis of the embryo and to determine the position of the embryonic head. The evolution of this patterning mechanism in higher flies (Cyclorrhapha) correlates with the loss of extraembryonic cell fate in the anterior blastoderm. We have identified a cyclorrhaphan fly (Episyrphus baltetus, Syrphidae), in which the extraembryonic anlage extends to the anterior pole, possibly reflecting an evolutionary reversion to the ancestral state. Episyrphus may lack a bicoid orthologue (79 kb of the Episyrphus Hox3 locus were sequenced, and degenerate PCRs with multiple primer combinations were negative), and it does not contain a functionally conserved bicoid-responsive cis- regulatory element upstream of the proximal hunchback promoter, although this trait is conserved in a wide range of cyclorrhaphan flies. We have identified Episyrphus genes with a potential role in head determination or anterior-posterior axis specification, including homologues of nanos, orthodenticle, giant, hunchback, caudal, and torso. We will report conserved and diverged expression patterns and patterning functions of these genes in Episyrphus.

661A Genetic analysis of between-species differences in embryonic pattern formation. Susan E. Lott1, Michael Z. Ludwig2, Martin Kreitman1,2. 1) Committee on Genetics, Univ Chicago, Chicago, IL; 2) Department of Ecology and Evolution, Univ Chicago, Chicago, IL. Egg size is an adaptive trait in Drosophila, varying clinally within species and differing between species. Because the A-P axis in Drosophila is established by diffusion of maternally supplied morphogens, egg length can be viewed as an external challenge to mechanisms establishing precision in the spatial localization of segmentation landmarks. In previous work, we showed that the spatial expression of the gap genes Kruppel and giant and the pair-rule gene even-skipped scales with embryo length in F3 embryos produced by crosses between D. melanogaster lines differing by ~25% in embryo length. Here we report the analysis of similar crosses between D. simulans and D. sechellia, which also differ ~25% in embryo length, but unlike the D. melanogaster strains, also differed in scaled gene expression. F1 females produced by a cross between D. simulans mothers and D. sechellia fathers were twice backcrossed to each of the parental species to create two F3 embryo classes. The F3’s produced by backcrossing to D. sechellia produced intermediate-sized embryos with stripe scaling that resembled D. sechellia. In contrast, the F3’s produced by backcrossing to D. simulans were similar length to D. simulans, but exhibited scaled patterns roughly intermediate between those of the parental species. Within each F3, the pattern scales to embryo length within a line, but this scaling is different between the lines. There are additionally no easily discernable phenotypic classes of embryo length or pattern within an F3, so either these traits have many loci of small effect, are buffered within each line, or are the product of differential fitness of some or many stages in the cross. We also examined other aspects of development that may be informative as to how expression patterns are localized, such as cell numbers and density. POSTERS: Evolution and Quantitative Genetics 313

662B Components of protein complexes linked to cognition are conserved and co-expressed in the Drosophila brain. Bilal R Malik1, Andrew J Pocklington1, Alex Bayes2, Richard Emes3, Seth GN Grant2, J Douglas Armstrong1. 1) ANC, School of Informatics, University of Edinburgh; 2) Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK; 3) Institute for Science and Technology in Medicine, School of Medicine, Keele University, Staffordshire ST5 5BG, United Kingdom. Synapses are instrumental in connecting neurons and generating behaviour, yet little is known about their molecular evolution. We examined synapse molecular evolution from single cell eukaryotes to humans using comparative genomic and proteomic methods, focusing on the postsynaptic density and MAGUK associated signalling complexes (MASCs) underlying learning and memory. At the gene level, striking changes in signalling complexity were observed with expansion in key synapse components, notably receptors, adhesion/cytoskeletal and scaffold proteins, at the metazoan and chordate boundaries leading to novel functionality. The earliest orthologues of synaptic components found in unicellular organisms regulate protein synthesis and structural plasticity. Direct proteomic comparison of Drosophila and mouse MASCs was performed using a peptide affinity purification technique based on the C-terminal Dlg binding domain of NMDAR2. The complement of proteins identified in the two species revealed common core of proteins present in both, with an expansion in signalling complexity in the vertebrate complex.

663C Cell number and distribution in cycle 14 Drosophila embryos selected for large and small egg size. Cecelia Miles, Martin Kreitman, Michael Ludwig. Dept Ecology & Evolution, University of Chicago, Chicago, IL. Early development of Drosophila melanogaster embryos proceeds through 13 nearly-synchronous cycles of DNA replication and nuclear division in a common syncitium. After mitotic cycle 13, cellularization occurs at the periphery of the embryo and subsequent mitotic cycles are no longer synchronous. The assumption has been that cell number at cleavage cycle 14 is constant regardless of embryo size. Yet, there is considerable genetic variation for embryo size within D. melanogaster strains and this variation is very likely adaptive. In order to examine the relationship between cell size, number, and distribution and embryo size we used replicate lines of D. melanogaster artificially selected for large and small egg volumes. These lines exhibit a mean increase of approximately 20% in large-egg lines relative to small-egg lines. Embryos were identified as cycle 14 (stage 5) after in situ staining for expression of giant (gt) with DAPI used as the nuclear stain after the techniques of Lott et al. (2007). We then used confocal microscopy to obtain images to test the assumption of constant cell number at the cellular blastoderm stage and compare cell size and distribution across embryos. We used the same technique to compare these results to lab strains of D. melanogaster showing genetic variation for egg size.

664A Comparative genomics reveals that the TGF-beta and Wnt signaling pathways have distinct patterns of developmental evolution. Stuart Newfeld1, Charlotte Konikoff1, Michael Stinchfield1, Sudhir Kumar1, Robert Wisotzkey2. 1) Sch Life Sci, Arizona State Univ, Tempe, AZ; 2) Dept. Biol, California State Univ East Bay, Hayward, CA. Intercellular signaling by TGF-beta and Wnt family members regulates numerous developmental decisions in many organisms. In the TGF-beta pathway, serine-threonine kinase receptors and Smad signal transducers are required for proper responses. In the Wnt pathway, Frizzled receptors and Disheveled signal transducers are often required for the modulation of Wnt target genes. We have taken a comparative genomics approach to understanding the evolutionary history of developmental signaling pathways. We were interested in determining if the pattern of evolution displayed by the TGF-beta pathway was typical of other signaling pathways. Our Wnt pathway analysis included all Wnt ligands, Frizzled receptors and Disheveled proteins in mouse, fly and nematode. Our results show that the Wnt pathway has a very distinct tempo and mode of evolution from the TGF-beta pathway. While both pathways are found only in multicellular animals there are numerous differences in their evolutionary trajectories. For example, in contrast to the TGF-beta family there are Wnt family members in the fly and nematode that have no homologs in mice. In contrast to TGF-beta receptors, the extracellular and intracellular domains of Frizzled family members have different evolutionary relationships. In contrast to the three functionally distinct classes of Smad signal transducers, Disheveled proteins have remained essentially unchanged throughout their history. These findings provide new insight into molecular mechanisms governing the evolution of developmental signaling pathways and the diversification of the metazoan body plan. 314 POSTERS: Evolution and Quantitative Genetics

665B How different are the mitochondria during spermatogenesis? Chitra Chandrasekaran1, Esther Betran2. 1) Biology, Texas Wesleyan University, Fort Worth, TX; 2) Biology, University of Texas Arlington, Arlington, TX. We recently studied gene duplicates generated trough retroposition and found that our retrogene set includes 15 retrogenes with similarity to known mitochondrial genes: CG17856, CG6255, CG4706, CG9582, tomboy40, Hsp60B, CG9920, EfTuM, CG14508, CG5718, CG11913, CG10748, CG10749, CG18418 and CG7514. A large fraction of the retrogenes in these pairs (87%) is expressed in testis and some of them are known to have testis-specific functions. In testes, mitochondria are known to change shape (i.e. condense) and change function during spermatogenesis. In humans, while spermatogonia can utilize aerobic pathways (i.e. glucose) for energy production, spermatocytes have limited access to glucose and rely on lactate and pyruvate from Sertoli cells. All these changes are known to be accompanied by changes in gene expression. Some of these changes may be accomplished through gene duplication followed by evolution of a male-specific pattern of expression for one of the paralogs. Our results suggest that retroposition could be a major mechanism underlying the genetic innovation necessary for this physiological transition in Drosophila. We are studying how different mitochondria are during spermatogenesis and to what extend retroposition and other duplication mechanisms contribute to this physiological change.

666C Mining Odorant Receptor Genes from 12 Drosophila and Other Insect genomes. Seong-il Eyun1, Stephen O. Opiyo1, Pooja K. Strope1, Etsuko N. Moriyama1,2. 1) School of Biological Sciences; 2) Plant Science Initiative, University of Nebraska-Lincoln, Lincoln, NE 68588, USA. Olfaction is a crucial sensory modality for most animals mediating behavioral responses to, e.g., food, mates, and predators. This odorant recognition capacity relies on a set of multigene families and its major player is the odorant receptor (OR) proteins, a member family of the G-protein-coupled receptor (GPCR) superfamily. While the number of ORs in Drosophila melanogaster is much smaller compared to those in other animals, the average level of sequence divergence among Drosophila melanogaster ORs is approximately three times higher than that among human ORs. To elucidate the mechanisms of molecular evolution of ORs in different species, we conducted thorough mining of odorant receptor (Or) genes from 12 Drosophila and 5 other insect genomes. In addition to BLAST and PSI-BLAST, we applied more sensitive protein mining methods including both alignment-based (e.g., profile hidden Markov models) and alignment-free (e.g., support vector machines). We identified 740 Or candidates in total from 12 Drosophila genomes (60 Or candidates per genome on average). The number of Or candidates identified from insects varied from 31 to 225 depending on the species. There are frequent gene loss and gain events including species-specific expansion of some OR groups. Although many of the GPCR families are characterized with gene duplication and divergence, Ors represent the case of extreme. Birth and death process appears to be the prominent part of odorant receptor evolution.

667A Measuring spontaneous mutation rate at locus dumpy in Drosophila melanogaster. Olga G Grushko1, Christopher Duchesneau1, Amber Carmon2, Ross J. MacIntyre2, Alexey S. Kondrashov1. 1) Life Sciences Institute, University of Michigan, Ann Arbor, MI; 2) Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. Data on spontaneous mutation rates in multicellular eukaryotes are still insufficient. For D. melanogaster current estimates of the per nucleotide mutation rate vary from 10-9 to 10-8 (Keightley, Eyre-Walker, 2000; Haag-Liautard et al, 2007). Here we report preliminary results from a single-generation phenotype-based study of spontaneous mutations at locus dumpy (dp). All loss-of-function dp mutations have good fitness and clear-cut phenotypes affecting wing shape and/or dorsal thorax cuticle and are unable to fly when present as a compound heterozygote with dpov1. In order to detect new recessive mutations we cross sibship of wild type heterozygous parents dp-A/dp-B with homozygous strain ed dpov1 cl/ed dpov1 cl, raise the offspring in the absence of selection and then perform large-scale screening for new mutants dp+A/dpov1 or dp +B/dpov1 using the original flight-ability-testing devise. Using sibships of parents allows us to distinguish between clusters of new mutations and pre-existing heterozygosity. About 1% of offspring refuse to fly for a variety of reasons and has to be screened for dp phenotypes. Among ~140000 flies screened to date 11 individuals with mutant phenotypes, both full-bodied and mosaics, transmitted their mutant alleles to the next generation. Thus our estimate of the overall per locus mutation rate at dumpy is 0.8x10-4. So far no sequencing has been performed but PCR assay for dumpy locus (> 100 kb) has already been designed (Carmon et al, 2005). Since we generate mutant allels in an isogenic progenitor chromosomes (dp-A and dp-B), amplicones produced from genomic DNA isolated from new mutant/wild type heterozygotes (dp+A/dp-A or dp+B/dp -B) can be cleaved by Surveyor nuclease at the site of mismatches and analyzed by dHPLC. Then identified amplicone will be a subject for direct sequencing. Our goal is to recover at least 30 new dumpy mutants which will be sufficient for a good estimate of spontaneous mutation rate. POSTERS: Evolution and Quantitative Genetics 315

668B Effect of X-linkage on rates of evolution of sex-biased genes in Drosophila. Tatiana Gurbich, Doris Bachtrog. Division of Biological Sciences, University of California San Diego, La Jolla, CA. A large fraction of genes in higher eukaryotes are expressed differentially in males and females, suggesting that they have different fitness effects in the two sexes. Sex-biased genes show unusually high rates of interspecies divergence, and previous studies revealed that sex-biased genes commonly experience adaptive evolution. However, the effect of genomic location on strength and mode of selection acting upon these genes is yet to be studied. Evolutionary theory predicts that genes that have opposing fitness effects in males and females show different rates of evolution if X-linked. Here we test whether X-linkage influences rates of evolution of sex-biased genes by comparing sex-biased genes on the neo-X chromosome of D. miranda with their autosomal orthologs in D. pseudoobscura. The neo-X chromosome in D. miranda formed recently as a result of an autosome (element C) becoming fused to the Y chromosome while no such fusion happened in D. pseudoobscura. Thus, this unique situation allows us to compare rates of evolution in orthologous sex-biased genes when they are autosomal (D. pseudoobscura) and when they are X- linked (D. miranda). We present polymorphism and divergence data for about 100 sex-biased genes located on the neo-X chromosome of D. miranda, and compare their evolution to a similar number of non-biased genes.

669C Molecular Evolution of Glutathione S-Transferases in the Genus, Drosophila. W.Y Low1, H.L Ng2, C. J Morton2, M.W Parker2, P Batterham1, C Robin1. 1) Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Vic 3010, Australia; 2) Biota Structural Biology Laboratory and the ACRF Rational Drug Discovery Facility, St. Vincent’s Institute of Medical Research, Melbourne, Vic 3065, Australia. As classical phase II detoxification enzymes, Glutathione S-Transferases (GSTs) have been implicated in insecticide resistance and may have evolved in response to toxins in the niche-defining feeding substrates of Drosophila species. We have annotated the GST genes of the twelve Drosophila species with recently sequenced genomes and analyzed their molecular evolution. Gene copy number variation is mainly attributable to unequal crossing over events in the large delta and epsilon clusters. Within these gene clusters there are also GST genes with slowly diverging orthologs. This implies that they have their own unique functions or have spatial/temporal expression patterns that impose significant selective constraints. Searches for positively selected sites within the GSTs identified G171K in GSTD1, a protein that has previously been shown to be capable of metabolizing the insecticide DDT. We find that the same radical substitution (G171K) in the substrate-binding domain has occurred at least three times in the Drosophila radiation. Homology modelling places site 171 distant from the active site but adjacent to an alternative DDT-binding site. Preliminary testing of kinetics of Dmel GSTD1, Dmel GSTD1G171K, and Dsim GSTD1 for DDTase activities indicates that they are all DDTases. However, sequencing of alleles from contemporary and historical populations suggests that these activities pre-date the use of DDT. We propose that the parallel evolution observed at this site may be an adaptive response to some other environmental toxin.

670A Radiation sensitivity and progeny production in Drosophila melanogaster under selective pressure. Lucy McNamara, John Jenkins, Nicole Boyle, Valerie Vassor. Biology Department, Swarthmore College, Swarthmore, PA. Populations under selective pressure adopt many strategies in order to survive and reproduce. In this study, we investigated the possibility that populations of Drosophila melanogaster had adopted either of two adaptive strategies under the selective pressure of predation. Noting that in bacteria and viruses mutation rate has been shown to increase when populations are exposed to intense selective pressure, we first investigated whether a similar increase in the mutation rate might occur in Drosophila, a sexually reproducing eukaryotic organism. Populations of Drosophila melanogaster were exposed to predation by the cellar spider Pholcus pahlangioides for 8 years, then samples from the populations were exposed to X-ray irradiation. The X-ray induced sex-linked recessive lethal mutation rate and reproductive sensitivity to mutation of these populations were then compared to those of flies from a control population. The results of these experiments indicate that there is no difference in induced mutation rate between flies under predation and stock flies, confirming theoretical models of mutation rate evolution in sexual populations. Next, we examined progeny production of flies under predation, a characteristic that in other organisms has been found to change in response to selective pressure. This experiment revealed that flies from one of the experimental populations produce on average 13 more progeny per pair than control flies, demonstrating that flies with increased reproductive potential may be selected for in the face of predation. We also examined the longevity of flies under predation to determine if the increased fecundity of one of these populations corresponded to a decrease in longevity, a possible evolutionary tradeoff. No sign of such a tradeoff was found; in fact, we observed a non-significant trend for flies under selective pressure to have a slightly longer lifespan than stock flies. This observation confirms previous studies that suggest that in the absence of selection, laboratory populations may deteriorate and become less representative of wild populations. 316 POSTERS: Evolution and Quantitative Genetics

671B Recurrent recruitment of retrogenes involved in nuclear transport. Mansi Motiwale, Charles Tracy, Xavier Río, Esther Betrán. Biology, University of Texas at Arlington, Arlington, TX. Drosophila nuclear transport factor-2 (Dntf-2) and Ran have given rise to retrogenes three times independently in different Drosophila lineages. Dntf-2 gave rise to a retrogene in the lineage leading to D.melanogaster, D.simulans, D.sechellia and D.mauritiana. Two additional independent retrogenes originated in the D.ananassae lineage and D.grimshawi lineage. Ran has also given rise to retrogenes independently in the melanogaster subgroup, the D.ananassae lineage and the D. grimshawi lineage. Interestingly, all these retroposition events involve retroposition from X chromosome to autosomes. It is known that retrogenes involving retroposition from X chromosome to autosome often develop male germline specific expression pattern. In D.melanogaster both the retrogenes have developed a testis biased expression pattern. Previously, it has been established that the proteins encoded by Dntf-2 and Ran interact with each other during transport of proteins to the nucleus. This fact and the fact that both are X linked genes suggest that there may be high selective pressure on the autosomal copies to develop male germline specific expression, where retrogene encoded proteins may interact with each other during spermatogenesis. We are currently studying the expression pattern of retrogenes in all the lineages, their sequence evolution and the role of co-evolution in the recurrent recruitment of retrogenes from Dntf-2r and Ran.

672C Selective Constraints Suggest Most of Drosophila Genome Comprised of Non-coding RNAs and cis-Regulatory Elements. Daniel Pollard1, Daniel Halligan2, Casey Bergman3, Peter Keightley2, Michael Eisen1. 1) Dept Genome Sci, LBNL, Berkeley, CA; 2) Inst of Evol Bio, Univ of Edinburgh, UK; 3) Life Sci, Univ of Manchester, UK. Selective constraint, the fraction of mutations eliminated due to selection, is ubiquitiously distributed throughout the Drosophila melanogaster genome, with the majority of constrained sequences falling outside of protein coding genes. Cis-regulatory sequences involved in the control of gene expression as well as non-coding RNAs involved in a broad array of cellular processes have been proposed to explain the high level of non-coding constraint, yet the patterns of selection acting on these sequences and their overall abundances are poorly understood. We have measured selective constraints in experimentally identified transcription factor binding sites, cis-regulatory modules and several classes of non-coding RNAs in D. melanogaster, by comparison with D. simulans and D. yakuba. We find that 6.4 out of 10 mutations in cis-regulatory modules, 7.7 out of 10 mutations in transcription factor binding sites and 8.6 out of 10 mutations in non-coding RNAs are lost due to selection, compared to 5.5 out of 10 for all non-coding DNA and 8.6 out of 10 for non-degenerate coding sites. Constraint in cis-regulatory sequences extends into 5' and 3' flanking sequences, falling to non-coding averages at a mean distance of 35 base pairs, while constraint is very low in sequences flanking non-coding RNAs. Constraints vary significantly across loci and chromosomes, with lower constraints on the X chromosome relative to the autosomes. Finally, we estimate that the percentage of the non-coding genome that would fall within each class of sequence, were it to be the only contributor to non-coding constraint, is 86% for cis-regulatory modules, 71% for binding sites and 64% for non-coding RNAs. These results suggest that constraints on functional non-coding elements are similar to those on protein coding genes and that it is reasonable to propose that most non-coding regions of the genome consist of cis-regulatory modules and non-coding RNAs.

673A Evolution of genic content in the innate immune system of Drosophila. Timothy Sackton1,2, Andrew Clark2. 1) Field of Ecology & Evolutionary Biol, Cornell Univ, Ithaca, NY; 2) Dept of Molecular Biology and Genetics, Cornell Univ, Ithaca, NY. The signal transduction components of insect innate immune pathways are deeply conserved across insect orders, and many immune signaling proteins have recognizable homologs in mammals. However, the recognition and effector components of innate immune pathways are much more diverse across species, because of both gene turnover within conserved families and the acquisition of novel gene families. In Drosophila, computational comparison of the 12 sequenced genomes revealed rapid turnover of gene content in several antimicrobial peptide families and recognition components, as well as the presence of antimicrobial peptide families and other effectors that appear to be completely novel with a subset of lineages (with no recognizable homologs outside the melanogaster group). This computational analysis has two important limitations: it cannot identify components of the immune system that are present in non-melanogaster species but absent from D. melanogaster, nor can it verify that gene expression patterns are consistent with a role in immunity for paralogs of known immune components. These limitations can be overcome by sequencing cDNA from naïve and infected flies to identify transcripts upregulated upon infection. Here, we present the results of such a sequencing screen, comparing infected and uninfected cDNA pools in D. virilis and D. mojavensis by short-read sequencing to identify novel components of the innate immune pathway. We also present data from species-specific microarrays on the transcriptional dynamics of genes regulated by infection in non-melanogaster species, and relate divergence in transcriptional profiles to divergence in genome sequence. POSTERS: Evolution and Quantitative Genetics 317

674B Genomic Rearrangements Inferred from Gene Order Data Collected from Ten Species of Drosophila. Stephen Schaeffer1, Arjun Bhutkar2,3, Mu Xu1, Susan Russo2,4, Temple Smith3, William Gelbart2,4. 1) Dept Biol, Pennsylvania State Univ, University Park, PA; 2) Dept Mol and Cell Biol, Harvard Univ, Cambridge, MA; 3) BioMolecular Engineering Research Center, Boston Univ, Boston, MA; 4) FlyBase, The Biological Laboratories, Harvard Univ, Cambridge, MA. Rates of chromosomal rearrangement can be inferred from gene order data using adjacency information at the boundaries of conserved linkage groups. A conserved linkage group is a set of genes on a chromosome that are in the same order among two or more species. The boundaries of conserved linkage groups provide information about the set of breakpoints that were used in ancestral inversion events. We used gene order data from ten Drosophila species with complete genome sequence to infer the rates of chromosomal inversion using the adjacency information in a linkage chain analysis. A linkage chain with n breakpoints infers n- 1 inversion events. The inversion rate is lower in the Drosophila subgenus compared to the Sophophoran subgenus. Rearrangement breakpoints show a high rate of re-usage, where the breakpoint is defined as the interval between two different conserved linkage groups. A model of random breakage is inconsistent with the average rate of breakpoint re-usage that is observed, however, a model of hot-spots and cold spots for chromosomal breakage fits the observed data best. We consider gene expression as a possible explanation for why some blocks of genes lack a rearrangement breakpoint in the history of these Drosophila species. Some chromosomal arms show elevated inversion rates in some species that is correlated to the observed gene arrangement polymorphism levels.

675C Contrasting features of sex and autosome chromosomal evolution in malaria mosquitoes. Igor Sharakhov1, Ai .Xia1, Maria Sharakhova1,2, Zhijian Tu2, Yogesh Shouche3. 1) Department of Entomology, Virginia Tech, Blacksburg, VA, USA; 2) Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA; 3) National Centre for Cell Science, Ganeshkhind, Pune 411 007, India. Chromosomal rearrangements are often associated with differential adaptations of malaria mosquitoes to various environments. Polymorphic inversions tend to cluster in on 2R arm suggesting existence of hot spots for generating and maintaining rearrangements. Localization of hot and cold spots for rearrangements could be useful for identification of genes involved in ecological adaptations and biologically important gene clusters. The local adaptation model predicts parallelism between the extent of chromosomal polymorphism and evolutionary rates of inversion fixation. Physical maps are useful tools for studying the genome evolution and for relating sequence information to the chromatin structure. We develop a physical genome map of Anopheles stephensi by mapping 206 A. stephensi, A. gambiae, and A. funestus cDNA and BAC clones to 239 chromosome sites. We compared physical maps of A. stephensi, A. funestus, and A. gambiae which belong to subgenus Cellia. The gene order comparison at the 1.6 Mb resolution has been performed using the Multiple Genome Rearrangements (MRG) and Sorting Permutation by Reversals and block-INterchanGes (SPRING) programs. We have found that inversions fixation rates vary significantly among the chromosomal arms. The small and large blocks of the conserved gene order have been identified among A. stephensi, A. funestus, and A. gambiae. The smallest conserved blocks are found on 2R and X chromosome. The largest conserved blocks (up to 6.2 Mb long) have been found on the chromosomal arms 3R and 3L of A. gambiae. Interestingly, these genomic regions are free from polymorphic inversions in the three species. In contrast, X chromosome has the highest rates of inversion fixation but does not have any polymorphic inversions in A. stephensi, A. funestus, or A. gambiae. Thus, we found parallelism between the extent of chromosomal polymorphism and evolutionary rates of inversion fixation for the autosomes but not the sex chromosome.

676A Fine-scale recombination rate estimation in D. melanogaster. Nadia D. Singh, Charles F. Aquadro, Andrew G. Clark. Molecular Biology and Genetics, Cornell University, Itahca, NY. Accurate assessment of local, fine-scale recombination rate variation is important for understanding the recombination process and for determining the impact of natural selection on linked sites. Classical half-tetrad analysis is not easily applied to any arbitrary chromosomal location, and has been used to produce fine-scale maps of only a few genomic regions. Statistical estimation of local recombination intensity (cM/Mbp) has employed the local slope of the relation between the physical and genetics maps, but the resolution of these methods is limited by the genetic map, and the statistical methodology leaves considerable uncertainty about the scale at which rate variation occurs. Moreover, these approaches differ markedly in their predictions with respect to recombination intensity in several regions of the genome. Further, tests of highly localized recombination hotspots in Drosophila have not been adequately done. The question of fine-scale variation in recombination rate has been addressed both directly and indirectly in humans; these studies are suggestive of extraordinary variation in local recombination rates. While there is some evidence in D. pseudoobscura suggesting several-fold variation in the rate of crossovers at a local scale, little is known about the fine-scale structure of recombination rate variation in D. melanogaster. Here we present the first attempt to quantify heterogeneity in recombination rate at a local scale in D. melanogaster. We generated a strain carrying both white and echinus mutations; these X-linked genes are separated by approximately 1.2 Mb and 4 cM. Using a two-step crossing scheme, we were able to screen hundreds of thousands of flies to identify thousands of males containing a single recombination event in this region. By genotyping SNP markers across this region, we will obtain a detailed picture of the recombinational landscape in this region. These data will help inform appropriate scales for local regression in traditional recombination rate estimators. 318 POSTERS: Evolution and Quantitative Genetics

677B Study of testes-specific proteasome retrogenes. Mehran Sorourian, Esther Betrán. Biology, University of Texas at Arlington, Arlington, TX. Proteasome is protein degrading machinery mediated by ubiquitin pathway. This large, multisubunit complex is made of 19S regulatory cap and 20S particle that plays major role in hydrolyzing peptide bonds. In Drosophila melanogaster, out of the 32 subunits comprising the proteasome, 10 have duplicates all of which show male specific expression. Having duplicates that express only during late spermatogenesis, suggests that after meiosis there is need for a testes specific proteasome which plays major role in sperm individualization and compaction. In the lab we are studying three genes that have retroposed isoforms. Pros28.1, pros29, and pros 35 are three parental genes that gave rise to pros28.1A, prosα3T, and prosα6T correspondingly. In addition to pros28.1A, pros28.1 has another duplicate pros28.1B which shows testes specific expression as well. We are investigating the evolution of these retrogenes as well as studying how they acquired testis-specific regulatory regions.

678C Demasculinization of X chromosomes in the Drosophila genus. David Sturgill1, Yu Zhang1, Michael Parisi2, Brian Oliver1. 1) Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD; 2) Department of Biology, University of Pennsylvania, Philadelphia, PA. X chromosomes evolve differently from autosomes. Since females have twice as many X chromosomes as males, genes that benefit one sex are likely to experience different selection pressures when X linked than when autosome-linked. For example, since polymorphisms in hemizygous males are immediately available for selection, one would expect genes favoring males to accumulate on the X. Contrary to this expectation, we found that the X is a disfavored location for genes favoring males in D. melanogaster. We examined whether this pattern is simply a property of the D. melanogaster species, by extending these observations to six additional Drosophila species. In direct global profiling experiments using species-specific microarrays, we found a nearly identical paucity of genes with male-biased expression on D. melanogaster, D. simulans, D. yakuba, D. ananassae, D. virilis and D. mojavensis X chromosomes. We also observed the same under-representation on the neo-X of D. pseudoobscura, a chromosome arm which is autosomal in the other species. These results show that the under-representation of genes with male biased expression on the X is a generic property of X chromosomes throughout the Drosophila lineage, rather than just Muller A in D. melanogaster. We show that X chromosome genes with male-biased expression are under-represented in somatic cells and in mitotic male germ cells. These data are incompatible with simple X-chromosome inactivation models. Using expression profiling and comparative sequence analysis, we show that selective gene extinction on the X chromosome, creation of new genes on autosomes and changed genomic location of existing genes contribute to the unusual X chromosome gene content.

679A Chromosome movement of retrogenes and X inactivation during Drosophila spermatogenesis. Maria D Vibranovski1, Hedibert F Lopes2, Timothy L Karr3, Manyuan Long1. 1) Department of Ecology and Evolution, The University of Chicago, Chicago, IL; 2) Graduated School of Business, The University of Chicago, Chicago, IL; 3) Department of Biology and Biochemistry, University of Bath, Bath, UK. One important observation regarding the evolution of sex chromosomes in Drosophila is the excessive movement of retrogenes out of the X chromosome to the autosomes. Interestingly, those retrogenes are expressed in testis. An explanation for such movement is the possible X chromosome inactivation during spermatogenesis. Although there is some evidence suggesting the occurrence of meiotic X inactivation, this phenomenon has not been conclusively investigated in Drosophila through a global expression analysis of X- and autosome-linked genes during spermatogenesis. Moreover, even if the X becomes inactive, retrogene movement may not necessarily be driven by the decrease of expression levels during meiosis. Here we investigate meiotic X-inactivation in D. melanogaster by monitoring X- and autosome-linked gene expression during developmental phases of spermatogenesis: spermatogonias, the mitotic cells; spermatocytes, the cells in meiotic division; and spermatids, the post-meiotic phase. Multiple hypothesis testing and inference were conducted by Bayesian hierarchical modeling (BHM) in order to classify gene products as over, under or equally expressed. In complete agreement with the X inactivation hypothesis, we observed depletion and enrichment of X-linked genes over and under expressed during meiosis, respectively. In order to investigate if X-inactivation is a selective force driving the excessive retrogene movement out of the X chromosome, we compared spermatogenesis gene expression of 90 pairs of retrogenes and their parental genes. Retrogenes moving out of the X-chromosome have higher posterior probabilities of being underexpressed in meiosis than genes moving out of the autosomes. This is the first evidence of meiotic X chromosome inactivation as a driving force of retrogene movement out of the X chromosome. POSTERS: Evolution and Quantitative Genetics 319

680B Constraint and turnover in sex-biased gene expression in the genus Drosophila. Yu Zhang1, David Sturgill1, Michael Parisi1,3, Sudhir Kumar2, Brian Oliver1. 1) LCDB/NIDDK/NIH, Bethesda, MD; 2) Center for Evolutionary Functional Genomics, Biodesign Institute, Arizona State University, Tempe AZ; 3) Department of Biology, University of Pennsylvania, PA. Both genome content and deployment contribute to phenotypic differences between species. Sex is the most important difference between individuals in a species and has long been posited to be rapidly evolving. Indeed, in the Drosophila genus, traits such as sperm length, genitalia, and gonad size are the most obvious differences between species. Comparative analysis of sex-biased expression should deepen our understanding of the relationship between genome content and deployment during evolution. Using existing and newly assembled genomes, we designed species-specific microarrays to examine sex-biased expression of orthologues and species-restricted genes in D. melanogaster, D. simulans, D. yakuba, D. ananassae, D. pseudoobscura, D. virilis and D. mojavensis. We show that averaged sex-biased expression changes accumulate monotonically over time within the genus. However, different genes contribute to expression variance within species groups compared to between groups. We observed greater turnover of species-restricted genes with male-biased expression, indicating that gene formation and extinction may play a significant part in species differences. Genes with male-biased expression also show the greatest expression and DNA sequence divergence. This higher divergence and turnover of genes with male-biased expression may be due to high transcription rates in the male germline, greater functional pleiotropy of genes expressed in females, and/or sexual competition. Comparative expression studies facilitated by genome sequencing projects will provide abundant data for testing old ideas and will provide new insights on the contributions of change, chance, and selection in evolution and population biology.

681C Bacteria associated with natural populations of the cactophilic species Drosophila aldrichi and D. arizonae. Vanessa Corby- Harris, Jorj Wagner, Therese A. Markow. Dept. of Ecology and Evolution, University of Arizona, Tucson, AZ. Cactophilic Drosophila are a preeminent ecological genetic model system for studying the biology of speciation and adaptation. Little is known, however, about the bacterial communities that associate with these insect hosts in nature. In order to characterize these bacterial communities, we analyzed 16S rDNA gene sequences recovered from two species of cactophilic Drosophila endemic to the southwest, D. aldrichi and D. arizonae. Both male and females of both species were collected from rotting fruits of Opuntia sp. host plants in Tucson, Arizona, and were each subjected to a series of treatments. These treatments facilitated the analysis of the bacterial communities associated with the insect’s exterior or interior, in addition to those bacteria occurring in the insect’s gastrointestinal tract. Our analysis adds to the growing body of literature characterizing Drosophila-bacteria interactions and offers insight into the unique ecology of cactophilic Drosophila.

682A Post-mating gene expression in tissues of the lower female reproductive tract in Drosophila pseudoobscura. Dean A Croshaw, Dalziel Dominguez, Carlos A Machado. University of Arizona, Tucson, AZ. Post-mating molecular interactions between male ejaculate and female reproductive tissues are likely important in determining fitness of both sexes in promiscuous species. The importance of these interactions is suggested by the fact that female genotype clearly affects paternity outcomes, both within and between species. Also, because of the presumed ubiquity of sexual conflict, genes involved in these types of interactions may experience sexually antagonistic selection, which could itself result in reproductive isolation among closely related lineages. Although there has been much work characterizing male ejaculate components and their effects on females, we know very little about female-expressed genes that are involved in mediating post-mating sexual interactions. We collected microarray data to identify candidate genes whose expression in female reproductive tissues may change in response to mating in Drosophila pseudoobscura. We used lower female reproductive tracts of virgin females and mated females that were dissected at several different time points after mating. When compared to sexually mature adult females, thousands of genes were differentially expressed at some time point post-mating. Gene ontologies involving immunity and defense were significantly overrepresented in the set of genes that changed in expression. Most differences occurred at 6 hours following mating and there was little overlap with the smaller set of genes whose expression changed at 0 or 2 hours. Our data provide a starting point for further study of post-mating molecular interactions between the sexes in a new model system. 320 POSTERS: Evolution and Quantitative Genetics

683B Identification and expression analysis of putative mRNA-like non-coding RNAs (mlncRNAs) in Drosophila pseudoobscura . Zifeng JIANG, Carlos Machado. Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ. It is becoming increasingly clear that non-coding RNAs (ncRNAs), i.e. RNAs with little protein coding potential, have a widespread distribution in both prokaryotes and eukaryotes, and fulfill vital functions in several cellular processes. Although we do not know the precise function of the vast majority of identified ncRNAs, this group of RNAs may be quite important in developmental processes and are thus promising molecules that may help explaining the complexity of diverse living organisms. ncRNAs which are similar to messenger RNAs (i.e. they have introns and are polyadenylated) but have limited or no protein-coding ability are termed mRNA-like non-coding RNAs (mlncRNAs). The functions of almost all mlncRNAs are currently unknown, and computational methods to identify them are still in their infancy. Using expression data from a cDNA-based microarray we have identified 38 novel mlncRNA candidates in Drosophila pseudoobscura. Homologues of all those mlncRNA sequences are present in D. persimilis, the closest relative of D. pseudoobscura, but could not be found in D. melanogaster or any other sequenced Drosophila species. All those mlncRNAs are expressed on the cDNA array. This suggests that these mlncRNAs possibly possess important functions.

684C Evolutionary relationships, timescales, and selective pressures in the 12 fruit fly genomes. Sudhir Kumar1,2, Sonja Prohaska1,3, Alan Filipski1,2. 1) Center for Evolutionary Functional Genomics, Biodesign Institute, Arizona State Univ, Tempe, AZ; 2) School of Life Sciences, Arizona State Univ, Tempe, AZ; 3) Department of Biomedical Informatics, Arizona State Univ, Tempe, AZ. The sequencing of the genomes of twelve species in the genus Drosophila provides a unique opportunity to establish a robust chronology and the ordering of the speciation events that led to the origin of Drosophila melanogaster. We have analyzed over 9500 orthologous protein coding gene alignments for inferring the evolutionary relationships among these species using genomic mutational distances (GMD) as well as exclusively nonsynonymous substitutions. In GMD we convert the observed difference between genomes to a mutational distance by considering the effects of codon usage bias within genomes, inequality of base composition bias among species, and multiple substitutions under a sophisticated model of nucleotide substitution. The topology inferred using GMD was identical to that obtained using a average genomic distance at second codon positions as well as the extended majority rule consensus of gene trees based on pairwise distances derived from fourfold-degenerate and second codon positions. Our analysis of the landmark speciation events leading to the evolution of D. melanogaster confirm our previous estimates based on only a fraction of protein-coding genes. This includes an estimate of the most recent common ancestry of D. melanogaster with D. simulans+D. sechellia (~5 million years ago), which is two times older than estimates arrived at by other groups. We attribute this discrepancy to the dramatic difference in the codon usage bias between D. melanogaster and the Hawaiian species. A comparison of estimates of per site mutation input and the number of nonsynonymous substitutions reveals that the natural selection has eliminated ~90% of all mutations. As expected, the gene-specific mutational distances show much better than correlation with divergence times between species as compared to the nonsynonymous distances.

685A Population size is not a major determinant of rates of protein adaptation in Drosophila. Doris Bachtrog. Asst Prof, Div Biological Sci, Univ California, San Diego, La Jolla, CA. Adaptive protein evolution is common in several Drosophila species investigated. Some studies point to very weak selection operating on amino-acid mutations, with average selection intensities on the order of Nes ~ 5 in D. melanogaster and D. simulans. Species with lower effective population sizes should undergo less adaptation since they generate fewer mutations and selection is ineffective on a greater proportion of beneficial mutations. I analyze patterns of polymorphism and divergence at 92 X-linked loci in D. miranda, a species with a roughly 5-fold smaller effective population size than D. melanogaster. Surprisingly, I find a similar fraction of amino-acid mutations being driven to fixation by positive selection in D. miranda and D. melanogaster. Genes with higher rates of amino-acid evolution show lower levels of neutral diversity, a pattern predicted by recurrent adaptive protein evolution. I fit a hitchhiking model to patterns of polymorphism in D. miranda and D. melanogaster and estimate an order of magnitude higher selection coefficients for beneficial mutations in D. miranda. This argues against effective population size being a major determinant in rates of protein adaptation. Instead, the distribution of fitness effects for beneficial mutations might differ vastly between different species or populations. Alternative explanation such as biases in estimating the fraction of beneficial mutations or slightly deleterious mutation models are also discussed. POSTERS: Evolution and Quantitative Genetics 321

686B Molecular evolution and population genetics of two Drosophila mettleri cytochrome P450 genes involved in host plant adaptation. Jeremy M Bono, Luciano M Matzkin, Therese A Markow. Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ. Understanding the genetic basis of adaptation is one of the primary goals of evolutionary biology. The evolution of xenobiotic resistance in insects has proven to be an especially useful arena for investigating the genetic basis of adaptation, and resistant phenotypes are known to result from both coding and regulatory changes. In this study, we examine the evolutionary history and population genetics of two Drosophila mettleri cytochrome P450 genes that are putatively involved in the detoxification of alkaloids present in two cactus hosts: saguaro (Carnegiea gigantea) and senita (Lophocereus schottii). Previous studies demonstrated that CYP28A1 was highly upregulated following exposure to rotting senita tissue, while CYP4D10 was highly upregulated after exposure to rotting saguaro tissue. Here, we show that amino acid sites in CYP28A1 also show a signature of positive selection specifically in the D. mettleri lineage, thus suggesting that both coding and regulatory changes have been important in shaping this adaptation. In contrast, we did not find evidence for positive selection on sites in CYP4D10, though this certainly does not preclude its involvement in host plant use. A surprising result that emerged from our population genetic analyses was a strong non-random association among haplotypes at three different loci in the Organ Pipe National Monument population, which indicates significant population subdivision within a single location.

687C Natural selection shapes genome wide patterns of copy number polymorphism in D. melanogaster. Margarida Cardoso- Moreira1,2,3, J.J. Emerson1, Justin O. Borevitz1, Manyuan Long1. 1) Dept Ecology & Evolution, Univ Chicago, Chicago, IL; 2) Graduate Program in Areas of Basic and Applied Biology, Universidade do Porto, Portugal; 3) Faculdade de Ciências, Universidade do Porto, Portugal. Copy-number mutations are an important source of genetic change. Yet, the role natural selection plays in governing the locations and early evolution of these mutations remains largely unexplored. Here we employ high-density full-genome tiling arrays to create a fine-scale genomic map of copy number polymorphisms (CNPs) in Drosophila melanogaster. We show that CNPs are ubiquitous in the fruitfly, inferring a total of 2,751 independent events, 57% of which overlap genes. The location and frequencies of CNPs are strongly shaped by purifying selection, particularly for deletions. Moreover, population analyses suggest that duplications overlapping coding regions or affecting the X-chromosome are subject to stronger purifying selection than other duplications. We also identify some classes of genes that might be targets of positive selection, which include toxin-response genes. Finally, we investigate the non-random genomic distribution of CNPs and the role transposable elements and other structural variants play in their formation.

688A Reduced nucleotide diversity on neo-Y chromosome of Drosophila albomicans. Hsiao-Han Chang1, Chau-Ti Ting2, Hwei-yu Chang3. 1) Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan 300, ROC; 2) Department of Life Science, Institute of Ecology and Evolutionary Biology, & Institute of Zoology, National Taiwan University, Taipei, Taiwan 106, ROC; 3) Department of Entomology, National Taiwan University, Taipei, Taiwan 106, ROC. Recombination is one of the major forces shaping genome evolution. Empirical study in Drosophila melanogaster has shown a positive correlation between recombination rate and nucleotide diversity. These results suggest that reduced nucleotide diversity across loci in low-recombining region could be due to both hitchhiking and background selection. On the other hand, it is inferred in recent study that selection efficacy could be maintained with minimal recombination rate. Therefore, it is ideal to identify non- recombining chromosomal regions, such as Y chromosome, to study the inference of recombination on nucleotide diversity. However, most of the Y chromosome genes lack homologous X-linked counterparts as reference. To compare genetic diversity of loci with or without recombination, we chose genes on a pair of neo-sex chromosomes derived from fusion events between sex chromosomes and autosomes less than half million years ago. Our data shows that polymorphism on neo-Y chromosome is much lower than that on neo-X chromosome after the correction for different effective population sizes. This result supports the hypothesis of reduced polymorphism on a non-recombining chromosome. In other words, positive and/or negative selection play a role on shaping polymorphism and the influence is more severe on a non-recombining chromosome as theory predicted. 322 POSTERS: Evolution and Quantitative Genetics

689B Evolution of a Female Reproductive Protease Gene Family in Cactophilic Drosophila. Erin Kelleher, Therese Markow. Dept EEB, Univ Arizona, Tucson, AZ. Protein components of the Drosophila male ejaculate are critical modulators of reproductive success, several of which are known to evolve rapidly. Recent evidence of adaptive evolution in female reproductive tract proteins suggests this pattern may reflect intersexual coevolution at the molecular level. Physiological evidence of ejaculate-female coevolution, paired with a promiscuous mating system, make the repleta species group and exciting model system in which to empirically test this hypothesis. We have identified five lineage-specific gene families of female reproductive tract proteases in the repleta species group. We present data on the evolutionary history and patterns of polymorphism for one of these gene families. The proteins show a history of ongoing gene duplication and adaptive evolution, and further exhibit dynamic patterns of pseudogenation, copy number variation, gene conversion, and selection within geographically isolated populations of D. mojavensis. This intriguing pattern of evolution has never before been demonstrated in a reproductive protein, and suggests involvement in a coevolutionary arms race.

690C Factors impacting Y chromosome evolution in Drosophila melanogaster. Amanda Larracuente, Andrew Clark. Dept Molecular Biol & Genetics, Cornell University, Ithaca, NY. Y chromosomes are subject to unique evolutionary pressures due to their lack of recombination, lower effective population size and male-male transmission. Most notably, the efficacy of natural selection is reduced on the Y due to interference between linked selected loci causing a retardation in the spread of adaptive mutations and an acceleration in the fixation of deleterious mutations, contributing to the heterochromatinization and degeneration of this chromosome. Thus, models of interference predict that the Y chromosome will have reduced levels of adaptation compared to autosomal or X-linked loci. Several competing evolutionary forces are responsible for shaping Y chromosome evolution. Several models of Y chromosome degeneration exist including those of positive selection, background selection, and Muller’s ratchet, all of which also predict a reduction in the polymorphism on the Y. We surveyed polymorphism at four Y-linked loci (kl-2, kl-5, kl-3 and ORY) in D. melanogaster in individuals from inbred lines originating from six populations from around the world. The site frequency spectra reveal a severe reduction in variation on the Y chromosome and are consistent with weak purifying selection. Neither the site frequency spectrum nor the patterns of LD provide compelling examples of recent positive selection, but past bouts of positive selection may nevertheless be a potent force shaping patterns of variation on this heterochromatic chromosome.

691A Population genetics in the Drosophila simulans sibling, D. sechellia. New insights into its evolutionary history based on selected and neutral polymorphisms. Delphine Legrand, Da Lage Jean-Luc, Lachaise Daniel, Cariou Marie-Louise. Laboratoire Evolution Génomes et Spéciation, CNRS, Gif-sur-Yvette, France. D. sechellia, a close relative of D. simulans, is endemic to the Seychelles archipelago. The species, among the youngest in the melanogaster subgroup, is estimated to have originated some 500,000 years ago. Although it has attracted considerable attention, little is known about its natural populations and demographic history. The general pattern of genetic variation in D. sechellia (10 populations) based on polymorphism data on 10 nuclear genes and microsatellites (17 loci) is documented. Compared to other Drosophila species, D. sechellia has much less nucleotide sequence variation and an extremely low effective population size, 15- 100 fold lower than that of D. simulans, that makes D. sechellia the least genetically diverse Drosophila species. There is no major population subdivision in D. sechellia despite its fragmented geographic distribution on different islands. Based on neutral genes, demographic scenarios in relation with the Holocene marine transgression that ended 10,000 years ago and ecological history of D. sechellia have been tested considering different ancestral population size. Polymorphism mostly consisted of shared polymorphisms among populations, and contrasting selective patterns were evidenced for a few genes. We discuss these findings in relation to adaptive traits for their implication in a particular life-history strategy of D. sechellia. POSTERS: Evolution and Quantitative Genetics 323

692B Molecular variation in the Lim3 locus controlling neuron development is associated with Drosophila melanogaster lifespan. Olga Y. Rybina, Elena Pasyukova. Institute of Molecular Genetics of RAS, Moscow, Russian Federation. Lim3 is an essential gene coding for RNA polymerase II transcription factor and playing a key role in neuron specification during Drosophila development. Lim3 was previously nominated as a candidate gene affecting Drosophila lifespan (Roshina, Pasyukova, 2007). Fifty substitution D. melanogaster lines containing natural second chromosomes in homozygous genetic background and significantly differing in lifespan (De Luca et al., 2003) and one wild type D. simulans reference line were used to analyze sequence variation in a 2 kb DNA fragment including 5’ surrounding (presumably regulatory) region and 5’ untranslated region, first exon and first intron of the major Lim3 transcript. Transcription start was determined using 5’RACE. 41 polymorphic sites were revealed, including 38 SNPs and 3 indels. Non-uniform distribution of SNPs was observed among different functional regions: the remote 5’ region and intron are the most variable (π = 0.01359, θ = 0.01191; π = 0.00901, θ = 0.00835), the 5’ region adjacent to the gene is considerably less variable (π = 0.00369, θ = 0.00209), only one SNP was found in 330 bp 5’ untranslated region and no SNPs were found in 109 bp exon. After Bonferroni correction for multiple tests, one SNP was significantly associated with variation in lifespan (P=0.0009). This A-T substitution is 512 bp apart from the structural gene, and results in five days difference in life span. Five more SNPs demonstrated significant associations with life span, which did not survive Bonferroni correction. However, three of these SNPs (two are situated in 5’ regulatory region and one in 5’ untranslated region) form a haplotype significantly associated with lifespan (P=0.0009). Given location of significant SNPs, it was reasonable to evaluate expression level of Lim3 in lines with different haplotypes. Preliminary results of RT-PCR show that at least one haplotype is characterized by lower expression of Lim3. Results of formal neutrality tests are discussed in connection with data on association between molecular variation and variation in life span and Lim3 expression.

693C Detecting selective sweeps using Hidden Markov Modells on sequence data. Christian Schloetterer1, Simon Boitard2, Andreas Futschik2. 1) Inst Tierzucht, VMU Wien, Wien, Austria; 2) Dept. of Statistics, University of Vienna. Detecting and localizing selective sweeps based on SNP data has received considerable attention recently. We introduce the use of Hidden Markov Models (HMMs) for the detection of selective sweeps in DNA sequences. Like previously suggested methods, our HMMs also use the site frequency spectrum to identify selection. In contrast to these earlier approaches, our HMMs do not treat linked sites as independent, but model the correlation of linked sites. The power of HMM is similar to competing methods, but the selected site is mapped with a higher precision. Expanding one of the introduced HMMs, we are able to distinguish between selection and demography with a high accuracy.

694A Understanding the effects of cis-polymorphisms on gene expression variation. Aaron Tarone1, Matthew Hahn2, Anne Genissel3, Joseph Dunham1, Sergey Nuzhdin1. 1) Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA; 2) Department of Biology and School of Informatics,Indiana University,Bloomington, IN; 3) Section of Evolution and Ecology, University of California at Davis, Davis, CA. In the last decade, the publication of multiple genome sequences has led to the ability to analyze whole genome expression patterns and to compare such patterns within and between species. With the development of these tools, it has become clear that a large portion of natural variation within and between species is due to differences in the regulation of genes. Understanding the sources of gene expression variation are not as simple as the evaluation of gene sequence, as cis elements, trans elements, and their interactions can all influence the expression of a gene. The relative contributions of these sources of variation to expression polymorphism are important for understanding medically, agriculturally, and evolutionarily important gene expression traits. The experiments presented here have been designed to understand cis-effects on the expression levels of 11 genes (abd-A, Ance, Ddc, en, ftz, gl, ninaE, salm, so, w, and z), which have been identified in previous experiments as loci that differ in their embryonic expression levels among nearly-isogenic lines (NILs) from the same population in Winters, CA. To determine the effects of cis- variation on the expression levels of these genes, 200 NILs from previous gene expression research will be resequenced at these loci. Large regions of genomic sequence (~10 Kb) spanning these loci will be amplified with long PCR and hybridized to resequencing arrays specific to these regions. A comparison of these sequences will enable the evaluation of linkage disequilibrium, patterns of evolution, cis-regulatory SNPs that influence gene expression at these loci (and several nested loci), and the identification of trends in cis-effects on expression. 324 POSTERS: Evolution and Quantitative Genetics

695B Genomic analysis of local adaptation in Drosophila melanogaster. Thomas Turner. Center for Population Biol, Univ California, Davis, Davis, CA. Drosophila melanogaster has rapidly diversified to exploit many natural and man-made resources. This fly feeds on microorganisms growing on a wide range of fruits, wine, and beer, and each of these food sources presents unique challenges to the fly’s metabolism. As a consequence, Drosophila populations are locally adapted to a diversity of environments, providing an opportunity to study how environmental changes create selection pressure for genetic changes, and how populations are able to change appropriately. I am currently investigating genomic and phenotypic differentiation between flies collected in wine cellars in Napa Valley, CA and those collected in citrus orchards in Winters, CA. I will present the results of a genomic analysis of differentiation obtained by hybridizing DNA from these locally adapted populations to Affymetrix tiling arrays.

696C Developmental and ecological determinants of life-history in Drosophila melanogaster. Alan Bergland, Marc Tatar. Dept Ecology & Evolution, Brown Univ, Providence, RI. In Drosophila melanogaster, the quality of nutrition during larval growth affects adult fecundity. We investigated the relationship between larval nutrition and adult fecundity in a set of 12 recombinant inbred lines derived from a wild population in Winters, CA. We reared flies under low yeast (0.2% yeast by volume, YBV) and high yeast (0.6% YBV) treatments and measured fecundity of individual flies for the first two and a half weeks of adult life. The genetic correlation between total fecundity and either ovariole number or thorax length was low (0.05 and 0.13, respectively) in the low yeast treatment and high (0.56 and 0.55) in the high yeast treatment. There did not appear to be any tradeoff between early and late fecundity with respect to the larval environment. These results suggest that larval nutrition affects adult fecundity through modifications of ovary and body size as well as other aspects of adult morphology and behavior. We tested this hypothesis by examining other histological aspects of the ovary and by measuring adult feeding rate in flies reared in low and high yeast environments. Further, we corroborate that the distributions of ovary and body size we obtained in our treatments reflect natural environments by rearing a subset of these lines on apples allowed to rot for various amounts of time. Results are discussed in the context of ecologically relevant aspects of development.

697A Evolution and development of natural variation in abdominal pigmentation. Ryan D Bickel1,2, Sergey Nuzhdin1, Artyom Kopp2. 1) Molecular and Computational Biology, University of Southern California, Los Angeles, CA; 2) Section of Evolution and Ecology, University of California-Davis, Davis, CA. Our goal is to identify genetic variation in the bric-a-brac (bab) region responsible for phenotypic variation in abdominal pigmentation. Previous studies have shown that within a natural population of Drosophila melanogaster, the bab locus is responsible for approximately 60% of the variation in abdominal pigmentation. We sequenced the 148 kb bab locus from 96 wild-caught inbred lines of Drosophila melanogaster. This region contains two candidate genes, bab1 and bab2, both of which are necessary for producing the abdominal pigmentation pattern. We are taking a three pronged approach to analyzing the region. First, we used population genetic methods to understand the evolutionary forces affecting the bab region. We have identified ~7000 polymorphisms at the bab locus. Using sliding window analysis of Tajima’s D, we have determined that a region near the 3’ end of bab2 is not evolving neutrally. This region also exhibits significant differentiation between two of our population samples that have different phenotypic distributions. Second, we used association mapping to identify regions that are responsible for variation in pigmentation. Using hierarchical modeling of linkage disequilibrium, we have identified ~40 polymorphisms associated with pigmentation variation. Third, we quantified transcription levels in the bab genes and other downstream pigmentation genes. Using quantitative PCR and allele specific pyrosequencing, we have measured the transcript levels of bab1 and bab2, as well as the downstream pigmentation genes, yellow, ddc, ebony, and tan. These data are combined to model how natural variation, transcription, and the genetic network interact to produce the pigmentation pattern. Thus, we can understand how natural variation at the bab locus affects the transcription of the bab genes, how this variation propagates through a genetic network to downstream genes, and ultimately how this results in phenotypic differences. POSTERS: Evolution and Quantitative Genetics 325

698B Identification of quantitative trait loci function through analysis of multiple cuticular hydrocarbons differing between Drosophila simulans and D. sechellia. Jennifer M. Gleason1, R. Andrew James2, Claude Wicker-Thomas3, Michael G. Ritchie2. 1) Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045; 2) School of Biology, University of St. Andrews, St. Andrews, Fife KY16 9TH, Scotland; 3) Laboratoire des Mecanismes de Communication, NAMC UMR-CNRS, Université Paris-XI Sud, 91405 Orsay, France. Identifying genes contributing to adaptive divergence and reproductive isolation between species can be informative about the process of speciation. Sexual isolation is a cause of reproductive isolation but few behavioral genes are known to influence speciation. Pheromones, such as Drosophila cuticular hydrocarbons (CHCs), can have potentially large effects on sexual isolation. Monomorphic species ( e.g. Drosophila simulans) have high levels of monoenes, especially 7-tricosene (7-T). Sexually dimorphic species (e.g. D. sechellia ) have females with dienes, primarily 7,11-heptacosadiene (7,11-HD). Males of monomorphic species do not court females of dimorphic species; thus the 7-T vs. 7,11-HD difference between females plays a role in sexual isolation. In a previous study we used a quantitative trait locus (QTL) approach to examine differences in the amount of 7-T and 7,11-HD in female D. simulans and D. sechellia (Gleason et. al. 2005). To refine the mapping, we added marker loci in candidate genes. In addition, to understand the nature of the QTL for 7-T and 7,11- HD, all CHCs differing between these species were included to examine the relative production of monoenes vs. dienes as well as differences in carbon chain length. Analysis of additional CHCs provides insights not only into the location of significant QTL for the differences between these species, but also the function of the genes underlying the QTL. In particular, a desaturase and an elongase, the former on 3L and the latter on 3R are implicated in sexual isolation. Gleason, J. M. et. al., 2005. Genetics 171: 1789-1798.

699C Functional Regulatory Divergence of the Innate Immune System in Interspecific Drosophila Hybrids. Erin Hill, Andrew Clark. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY. In order to investigate divergence of immune regulation among Drosophila species, we have engaged in a detailed study of innate immune function in F1 hybrids of D. melanogaster and D. simulans. If pathways have diverged such that incompatibilities have arisen between interacting components of the immune network, we expect the hybrids to display dysregulation of immune genes. We have quantified gene induction in hybrid and parental flies in response to bacterial infection. These results show that while the hybrid flies do not suffer widespread immune breakdown, they show significantly different regulation of many immune genes relative to the parents. We examine this divergence in terms of additivity and expression differences among genes, observing distinct patterns of dysregulation among various functional groups within the pathways of the innate immune system. The functional groups most sensitive to misexpression in the hybrids are the downstream components of the network, indicative of some propagation of dysregulation throughout the immune pathways. Interestingly, this dysregulation does not appear to associate with phenotypic differences in bacterial load post-infection in hybrids, possibly highlighting some robustness of function of innate immune response to perturbations like hybridization. To further inspect the sensitivity of the system to divergent genetic interactions, we examine immune responses of hybrids bearing null mutant alleles for one copy of certain immune pathway components to determine the effects of interactions between the remaining allele and diverged interacting proteins on immune function. With these, we aim to pinpoint not only divergent regulation, but also divergent protein interactions throughout the immune response in these Drosophila species.

700A Relating among species divergence to mutation and standing variation. David Houle, Kim van der Linde. Biological Science, Florida State University, Tallahassee, FL. Despite the knowledge that the process of mutation potentially causes standing variation within populations, and that the divergence among species is generated from that standing variation, it is still rare to estimate and compare variation at these three levels. We used an automated system for measuring wing morphology to characterize genetic variation due to mutation, standing variation and among species divergence within the family Drosophilidae. There is surprising similarity between the variance-covariance matrices at the three levels of variation. Differences among species are primarily along vectors with large amounts of genetic and mutational variation. This suggests either that mutation controls the pattern of among species difference in wing morphology, or that the pattern of mutational effects has been shaped by selection to be similar to among-species variation. 326 POSTERS: Evolution and Quantitative Genetics

701B The influence of diet on genetically based variation in the cost of reproduction. Mary F. Kaminski, Jeff Leips. Dept Biological Sciences, UMBC, Baltimore, MD. Trade-offs involving reproduction are among the most important in life history evolution due to their direct effects on fitness. While reproduction has been shown to produce trade-offs among traits such as longevity and immune response, the nature of these trade- offs may depend on the quantity and quality of available resources. As variation in resource quality and availability is common in natural populations, the plastic response of energy allocation to different traits in response to such variation is likely to have important effects on fitness. In this study we characterized the genetically based variation in the cost of reproduction to immune response and life span and assessed the plasticity of these costs under ad lib and restricted yeast diets. We used chromosome III substitution lines derived from the natural population in Raleigh, NC, crossing females from each of six CIII lines to males of an unrelated CIII line. We measured age-specific immune response and life span of virgin and mated flies under two food conditions: regular cornmeal based fly food and the same food with a 60% reduction in yeast. We will combine these data with previously obtained data on age-specific fecundity to begin to explore the genetic basis of variation in the cost of reproduction in immune response and life span, and the plastic response of these traits to dietary restriction.

702C Structural equation modeling of pigmentation in Drosophila. Brooke A. LaFlamme1, Jason G. Mezey2, ACERT National ESR Center. 1) Molecular Biology and Genetics, Cornell University, Ithaca, NY; 2) Biostatistics and Computational Biology, Cornell University, Ithaca, NY. The use of structural equation models (SEM) can be a powerful tool in describing the relative contributions of the components of a genetic regulatory network to an observable, quantitative phenotype. Structural equation modeling is preferable to univariate methods such as ANOVA for this purpose because it allows one to assess more complex interactions between dependent and independent variables, including those that cannot be measured directly. Here we present a comparison between simple SEM and ANOVA models using preliminary data on Drosophila pigmentation pathway components. Gene expression data, in the form of quantitative real-time PCR measurements, is shown for three species: Drosophila melanogaster, D. yakuba, and D. santomea. Drosophila santomea is unique among the melanogaster group species in having virtually no pigmentation in the males and very reduced pigmentation in the females. This species also lacks the characteristic sexually dimorphic pigmentation pattern seen in the other eight melanogaster group species, though it is very closely related to D. yakuba with which it coexists in part of its habitat. Total RNA for gene expression analysis of seven genes affecting the production of melanin was obtained from the abdominal cuticles of late-stage pupae, which have yet to develop pigmentation. In addition to gene expression data, measurements of total melanin in the adult abdominal cuticles for D. yakuba and D. santomeaa are presented. These measurements are based on electron spin resonance (ESR) spectra obtained from the cuticle samples. Amplitude of the characteristic melanin signal is used as a proxy for total melanin content. Various models, from very simple to more complex, are explored using SEM and the results are compared to those obtained by univariate linear models. We provide evidence that structural equation models are better equipped to describe the relationships between components of the pigmentation pathway and their effects on total melanin content.

703A Generation of quantitative variation in wing vein position by the Hedgehog and Decapentaplegic signaling pathways. James Lorigan, Fangfei Ye, Jason Mezey. Dept Biological Statistics, Cornell Univ, Ithaca, NY. The pattern of venation in the wings of Drosophila is a complex trait, which displays quantitative variation in the relative position of veins both among and within species. Many loci as well as environmental variation play a role in generating the vein position variation observed in natural populations. The Hedgehog and Decapentaplegic signalling pathways have been suggested to play an important role in generating quantitative variation in the distance between wing veins. Genotypic variation in, and differential expression of, genes in these pathways is therefore of interest in explaining the phenotypic variation found in wing vein position. We have identified polymorphisms from a natural population in both coding and regulatory regions of pathway members, and used qPCR to quantify Hedgehog and Decapentaplegic pathway gene expression in wing imaginal discs of third-instar larvae, and have quantified vein position along with cell size and number traits for wings of adult flies. Using an association testing approach, we have detected both cis- and trans- effects on gene expression during development and associated effects on adult wing phenotypes. Identifying polymorphisms and expression differences which appear to have major effects on vein position provides insight into the developmental mechanisms by which signaling pathways generate differences in adult phenotypes. These results will be used in ongoing refinement of models which can explain how development gives rise to quantitative variation. POSTERS: Evolution and Quantitative Genetics 327

704B Quantitative Trait Loci Affecting the Intraspecific Difference in Male Abdominal Pigmentation in Drosophila malerkotliana. Chen-Siang Ng1,2, Andrew Hamilton1, Amanda Frank1, Artyom Kopp1,2. 1) Section of Evolution & Ecology, University of California, Davis, CA 95616; 2) Center for Population Biology, University of California, Davis, CA 95616. Pigmentation is an important fitness trait as it plays a critical role in mimicry, sexual selection, species recognition, and thermoregulation in many groups of animals. Pigmentation also evolves rapidly among closely related Drosophila species, presenting a good opportunity to dissect the genetic changes underlying morphological differentiation at the molecular level. We investigated the genetic basis of intraspecific and interspecific differences in sexually dimorphic abdominal pigmentation in the D. bipectinata species complex. In this study, we mapped the QTLs responsible for color pattern differences between two subspecies of D. malerkotliana to the Muller element E, which is homologous to chromosomal arm 3R of D. melanogaster. We ruled out candidate genes known to be involved in pigmentation differences in other Drosophila species, as well as in pigment patterning and synthesis in general. We conclude that intraspecific pigmentation differences in D. malerkotliana are controlled by unknown genes. This study provides a framework for identifying these new genes, which are clearly essential for the development of pigmentation, by combining population-genetic, developmental, and genomic approaches. Characterization of the molecular changes underlying variation in pigmentation and sexually dimorphic traits in general will help link molecular genetics to ecology and improve our understanding of how selection shapes the evolution of developmental pathways within and between species.

705C Age-Specific Variation in Immune Response and its Correlations with Lifespan, Reproduction, and Lipid Storage. Adrienne Starks, Jeff Leips. Dept Biological Science, UMBC, Baltimore, MD. Senescence, the age related decline in physiological performance, is reflected in a number of traits including reproduction, susceptibility to disease, and mobility. When senescence is observed in an organism, it is unclear if there is a general decline in function across all traits or if certain traits decline at varied rates. Most studies that have attempted to address this phenomenon have used inbred lines or mutational studies to identify genes associated with aging, and these have been successful in identifying genes that influence senescence in traits. However, it is not known if genetic variation at these loci influence the variation observed in natural populations as the genes identified are typically essential to the survival or fitness of the organism. This study investigates the genetic effects of age on a host of quantitative traits including: immune response, fecundity (reproduction), triglyceride level (energy storage), and lifespan in Drosophila melanogaster. This approach to study traits collectively will also identify interactions between competing traits due to energy allocation. Allocating energy to one trait comes at the expense of other traits producing trade-offs, benefits and costs associated with energy distribution. Understanding the effect of age on allocation patterns among traits and the genetic basis of natural variation in age-specific allocation will greatly contribute to our understanding of the aging process. For each trait (except lifespan) I measured at an early and a late age to identify age related changes for each trait. Analysis of this data provides: (1) the relative amount of age-specific genetic variation for each trait and the degree of genetic correlation for each trait at each age, (2) genetically based age related differences within each trait, and (3) age-specific genetic correlations for each trait.

706A Investigating the interactions between the hybrid incompatibility protein LHR and heterochromatic proteins HP1 and HP6. Nicholas Brideau, Daniel Barbash. Dept Molecular Biol & Genetics, Cornell Univ, Ithaca, NY. Crossing D. melanogaster females to D. simulans males produces inviable F1 hybrid males and sterile, semi-viable F1 hybrid females. The gene Lethal hybrid rescue (Lhr) contributes to hybrid male lethality, because a loss of function mutation in D. simulans Lhr rescues the inviability. D. melanogaster LHR has been previously shown to interact with Heterochromatin Protein 1 (HP1) in a yeast two-hybrid assay. Consistent with this result, we have found that a MYC::LHR fusion co-immunoprecipitates with HP1 from Drosophila Kc cells, and that the chromo-shadow domain of HP1 mediates the interaction with LHR. We have also determined that a YFP::LHR fusion protein colocalizes with HP1 at heterochromatic regions of polytene chromosomes, supporting the interaction data. Furthermore, in addition to LHR from D. melanogaster, LHR from D. simulans, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, and D. virilis also interact with D. melanogaster HP1 in a yeast two-hybrid assay. These results show that the function of HP1 binding remains conserved despite the high sequence divergence of Drosophila LHR orthologs. We also tested whether the rapidly evolving protein Heterochromatin Protein 6 (HP6, also called Umbrea) interacts with LHR in manner similar to the well-conserved HP1. HP6 is a small chromo-shadow domain protein that was previously reported to interact with LHR. We have discovered that the seven Lhr orthologs mentioned above also interact with both D. melanogaster and D. simulans HP6 in a yeast two-hybrid assay. The conservation of the interaction between LHR and HP6 and the high sequence divergence of both proteins suggest the possibility co-evolution. To identify the functional domains in LHR we have undertaken domain-mapping analysis. The regions within LHR responsible for interacting with both HP1 and HP6 are currently under investigation using deletions and site- directed mutagenesis. 328 POSTERS: Evolution and Quantitative Genetics

707B Functional Studies of the Hybrid Male Sterility Gene, Odysseus (OdsH), in Drosophila melanogaster. Ya-Jen Cheng1, Niapm H. Patel2, Chau-Ti Ting1,3. 1) Inst. of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC; 2) Dept. of Molecular and Cell Biology, and Dept. of Integrative Biology, and HHMI, UC Berkeley, CA, USA; 3) Dept. of Life Science, Inst. of Ecology and Evolutionary Biology, and Inst. of Zoology, National Taiwan University, Taipei, Taiwan, ROC. Molecular characterization of genes contributing to hybrid incompatibility between species is one of the major tasks in evolutionary biology. In the past decades, several genes responsible for hybrid incompatibility between closely related Drosophila species have been characterized at the molecular level. Among them, Odysseus (OdsH) was characterized as one of the hybrid male sterility genes between D. mauritiana and D. simulans. Previous studies showed that the testes-specific transcript OdsH is a paralogous copy of unc-4, a neuronal transcription factor. However, the molecular function of OdsH in spermatogenesis is still unknown. Here, we took a candidate gene approach to identify genes downstream of OdsH by genetic and transcriptional analyses. Our data showed that OdsH is expressed in the early stage of spermatogenesis. The process of spermatogenesis is altered in both knockout and hybrid backgrounds. These results not only reveal molecular function of OdsH in spermatogenesis but also suggest interacting loci as predicted by Dobzhansky-Muller model.

708C Symbiotic Bacteria Affect Mating Choice in Drosophila melanogaster. Alex Kulikov1, Alexander Markov2, Oleg Lazebny1, Irina Goryacheva3, Maxim Antipin3. 1) Dept Genetics, Koltsov Institute of Developmental Biology, RAS, Moscow, Russian Federation; 2) Institute of Paleontology, RAS Moscow, Russian Federation; 3) Vavilov Institute of General Genetics, RAS, Moscow, Russian Federation. The aim of the study was to investigate dependence of mating preferences on Wolbachia infection in two genotypically different lines of Drosophila melanogaster by means of competition tests of different designs. Males of line “Red eyes”, genetically closer to the wild type and carrying less deleterious mutations were more successful than males of line “White eyes”. Wolbachia infection increased the mating ability of “White eyes” males but did not affect or slightly decreased mating ability of “Red eyes” males. Line “White eyes” exhibited positive assortative mating with regard to the infection status; in addition, infected “White eyes” females preferred similar (“friend”) genotypes. Unlikely, negative assortative mating for infection was observed in line “Red eyes”, and uninfected “Red eyes” females also preferred opposite (“foe”) genotypes. These results support the recently formulated hypothesis that mating choice might involve testing the partner for “friend” or “foe”, based on chemoreception with possible participation of immune system components. According to this hypothesis, mating partners with an optimal level of genetic or biochemical similarity have the highest degree of attractiveness. Under stress, the optimum may shift toward “friend” mating partners avoiding “washing out” of advantageous modifications in critical conditions, and in beneficial environments, toward “foe” ones reducing inbreeding of the offspring. In the present study, the internal stress factors were (1) Wolbachia infection and (2) belonging to line “White eyes”. Consequently, the most stressed flies (infected “White eyes”) showed high preference of “friend” partners, while the least stressed ones (uninfected “Red eyes”), preference of “foe” partners. Supported by Russian Foundation for Basic Research grant 06-04- 49702.

709A Two divergent island populations of the Drosophila simulans sibling, D. mauritiana. The splitting of a species into two? Delphine Legrand, Chenel Thomas, Lachaise Daniel, Cariou Marie-Louise. Laboratoire Evolution Génomes et Spéciation, CNRS, Gif-sur-Yvette, France. Evolutionary biologists have often suggested that ecology plays an important role in the origin of new species. A wide general acception is that adaptation to different ecological settings drives evolutionary divergence. Examples of ecological speciation are now becoming more abundant. In Drosophila most of the speciation studies have focused on reproductive isolation, hybrid sterility and inviability especially between closely related species focusing on mechanisms of postzygotic isolation and leading to the discovery of several genes responsible for hybrid incompatibilities, the so-called « speciation genes ». In contrast, the ecological basis of speciation in natural populations of Drosophila has been much less developped than in other taxa. D. mauritianais endemic to Mauritius and has diverged from its siblings of the simulans complex, D. simulansand D. sechellia, less than 0.5 My ago. A recent field study revealed two distinct populations of D. mauritiana showing contrasted ecological features (specialist versus generalist). This ecological divergence is associated with genetic differentiation at both the mitochondrial (mitochondrial types) and nuclear level. A population genetic analysis (4 nuclear genes) evidenced contrasted demographic parameters for the two populations and significant divergence, also confirmed by a microsatellite analysis (17 loci). Furthermore, experimental crosses between these two populations revealed asymetry in the reproductive success which does not seem totally linked to infection by Wolbachia, the cytoplasmic endosymbiont bacteria known to partially infect the species. All together, the results strongly suggest speciation in progress within D. mauritiana, this unsusual situation is discussed. POSTERS: Evolution and Quantitative Genetics 329

710B Genome-wide expression studies of phenotypic divergence and hybrid dysfunction in species of the Drosophila pseudoobscura group. Carlos Machado, Zifeng Jiang. Dept Ecology/Evolutionary Biol, Univ Arizona, Tucson, AZ. Changes in the timing and the level of gene expression have been long suggested to be fundamental for generating evolutionary change and to play a major role in the adaptation process. Furthermore, changes of gene expression patterns due to gene incompatibilities in the genome of hybrid individuals from interspecific crosses have been suggested to play a major role in the generation of reproductive isolation. Here we describe results of genome-wide expression studies of phenotypic divergence and hybrid dysfunction in Drosophila pseudoobscura and its close relatives D. persimilis and D. p. bogotana. Using an oligo array that includes probes for 18,000 predicted genes we have identified loci showing significant differences in their expression levels in adults of the three species, and a different set of loci showing aberrant expression patterns in adult hybrids.

711C High-resolution Genome-wide Dissection of the Two Rules of Speciation in Drosophila. John P. Masly1, Daven C. Presgraves2. 1) Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA; 2) Department of Biology, University of Rochester, Rochester, NY. Postzygotic reproductive isolation is characterized by two striking empirical patterns. The first is Haldane’s rule— the preferential inviability or sterility of species hybrids of the heterogametic (XY) sex. The second is the so-called large X effect— substitution of one species’ X chromosome for another has a disproportionately large effect on hybrid fitness compared to similar substitution of an autosome. Although the existence of Haldane’s rule has been well-established and is thought to result from the general recessivity of hybrid incompatibilities (dominance theory) and the more rapid accumulation of incompatibilities that cause hybrid male sterility (faster-male theory), the causes—and even the existence—of the large X effect remain controversial. We dissect the genetic causes of these two rules of speciation using a genome-wide introgression analysis of Drosophila mauritiana chromosome segments in an otherwise D. sechellia genetic background. We find that recessive hybrid incompatibilities outnumber dominant ones and that hybrid male-steriles outnumber all other types of postzygotic hybrid incompatibility, consistent with the dominance and faster-male theories of Haldane’s rule, respectively. Importantly, we also find that, although X-linked and autosomal introgressions are of similar size, most X-linked introgressions cause hybrid male sterility whereas few autosomal introgressions do. Our results thus confirm the large X effect and identify its proximate cause: incompatibilities causing hybrid male sterility have a higher density on the X chromosome than on the autosomes.

712A Thermotolerance and temperature preference are important factors in habitat isolation between Drosophila yakuba and D. santomea. Daniel Matute, Jerry A Coyne. Ecology and Evolution, University of Chicago, Chicago, IL. D. yakuba is widespread throughout sub-Saharan Africa. The species occupies open habitats, but is not found in rainforests. In contrast, D. santomea is endemic to São Tomé, a volcanic island off the coast of Gabon that also harbors D. yakuba. On the slopes of the highest volcano, Pico de São Tomé (el. 2024 m), D. yakuba lives at elevations below 1450 m and D. santomea at elevations above 1150 m. Between these elevations the species ranges overlap and one finds a low frequency of hybrids. Although both species coexist, reproductive isolation is enforced by habitat isolation: the species live at different altitudes. An aberrant hybrid zone has been found at the top of the volcano, outside the range of both parental species. To determine whether the species’ distributions reflect genetic preferences and/or tolerances of the species to environmental factors, we measured several fitness components at different stages in the life of the two species. We found adaptive difference in two categories: habitat preference and thermal tolerance. First, different genotypes prefer different temperatures: D. santomea prefers cooler environments than D. yakuba. Such preference might be important for the choosing micro geographic habitats and in the pattern of distribution of the two species in the volcano. Further studies show that the two species also show differences in their temperature tolerance. At high temperatures, the survival rate of larvae in D. santomea is virtually zero, whereas high temperature does not reduce the viability of D. yakuba. To roughly characterize the genetics of these differences, we made interspecific crosses and measured the survival of hybrid larvae at the same temperatures. The hybrids carrying the X chromosome and the cytoplasm of D. santomea have lower survival rate than the reciprocal cross at all temperatures. The only exception was at low temperatures, where these individuals have a better survival rate than the reciprocal cross and the pure species. These data provide an ecological context for studies of thermal stress these two sibling species. 330 POSTERS: Evolution and Quantitative Genetics

713B Intrinsic reproductive isolation barriers between Drosophila yakuba and D. santomea. Daniel R Matute, Jerry A Coyne. Ecology and Evolution, University of Chicago, Chicago, IL. Intrinsic reproductive isolation barriers between Drosophila yakuba and D. santomea. Reproductive isolating barriers are traits that prevent gene flow between groups of individuals, comprising key components of in speciation. Most of the research on reproductive isolation has focused on factors that affect pre-mating isolation. Nevertheless, postmating (pre- and postzygotic) barriers have also been shown to be important in maintaining sympatric species as distinct genetic entities. This study identifies several intrinsic isolating barriers that may reduce gene flow between Drosophila yakuba and D. santomea in the wild. These two sister species have overlapping ranges on the island of São Tomé off the coast of West Africa. Previous studies have shown that these two species are strongly sexually isolated. However, the level of sexual isolation observed in the laboratory is too weak to explain the very low frequency of hybrids (and dearth of introgressed genes) found in nature. We found several forms of postmating, prezygotic isolation: First, fewer eggs are produced when females mate with conspecific compared to heterospecific males. Second, the longevity of females is reduced when D. santomea females mate with heterospecific compared to conspecific males. This reduction in longevity is not symmetrical, as the longevity of D. yakuba females does not change when mated with D. santomea males. Third, sperm in females inseminated with interspecific males is exhausted faster than in conspecific crosses. Finally, developmental time from egg to first-instar larva is delayed in F1s hybrid individuals compared to pure species embryos. These forms of isolation may contribute to the integrity of these two species where they coexist in the wild.

714C Genomic differentiation between ecologically divergent forms of Drosophila melanogaster that coexist in Brazzaville, Congo. Carolyn McBride1, Tom Turner1, Pierre Capy3, Sergey Nuzhdin2. 1) Center for Population Biology, Univ California, Davis, Davis, CA; 2) Molecular and Computational Biology, Univ Southern California, Los Angeles, CA; 3) Laboratory of Evolution, Genomes, and Speciation, Centre Nacional de la Recherche Scientifique, Gif-sur-Yvette, France. Derived cosmopolitan populations of Drosophila melanogaster appear to have come into secondary contact with ancestral African populations in the city of Brazzaville, Congo. The cosmopolitan descendants have high tolerances for ethanol and subsist on beer- residues in breweries, while the native African flies have low tolerances and exploit fruits and vegetables in nearby markets and surrounding farms. The cosmopolitan descendants also differ from the native African flies in their cuticular hydrocarbon profiles and mating preferences. By hybridizing genomic DNA to whole-genome tiling arrays, we have scanned the genomes of these two incipient species for regions that remain differentiated (have not been homogenized by gene flow) and are thus likely to harbor the loci responsible for the persistent ecological and sexual differences. We describe the number, size, and gene content of such regions and ask whether they correspond to segregating chromosomal inversions.

715A Role of polymorphism in putative pheromone binding receptor Gr68b in mate recognition of D. virilis species group. Nikolai Mugue1, Varvara Vedenina2. 1) Inst Developmental Biology, Moscow, Russian Federation; 2) Institute of Information Transmission Problems, Moscow, Russian Federation. Gustatory receptor Gr68a was shown to play an important role in the first step of D. melanogaster courtship and believed to be a pheromone-binding receptor playing a crucial role in conspecific female pheromone recognition (Bray and Amrein, 2003). Previously we have shown that in all twelve species of D. virilis group a Gr68b gene (alternative splice form of Gr68a and homolog of Gr32a in D. melanogaster) have remarkable sequence variation in the first and second extracellular domains. Here we report a large -scale survey of sequence polymorphism of Gr68b gene in a number of geographically distinct populations in several wide distributed species of D. virilis group. A very little variation has been found in extracellular domains of Gr68b gene among populations of each species studied, indicating that this gene is under stabilizing selection. To assess the role of Gr68b receptor in courtship behavior, we performed QTL analysis of mate recognition during the touching stage of courtship. When male approach heterospecific female, in most cases courtship terminates after initial “touching” female with male’s forelegs and do not proceed to more advance stages such as singing, licking, circling and copulation. Hybrids of D. americana and D. littoralis have been produced and courtship behavior and mate preference of F2 hybrid males was assessed with females of both parental species. Preferences to either one parental species were scored and compared with Gr68b allele composition (americana or littoralis homozygotes and heterozygotes) and with microsatellite loci markers for all chromosomes. POSTERS: Evolution and Quantitative Genetics 331

716B Role of Gr32a sequence variation in courtship and mate recognition in D. virilis species group. Nikolai Mugue1, Varvara Vedenina2. 1) Inst Developmental Biology, Moscow, Russian Federation; 2) Institute of Information Transmission Problems, Moscow, Russian Federation. Gustatory receptor Gr68a has been shown to be male-specific and expressed in a few sensitive bristles of forelegs of D. melanogaster male. Expression of this receptor is thought to be important in first steps of mating -touching and assessment of female by conspecific male. We have sequenced complete Gr68a gene and its alternative spliceform Gr32a (Gr68b) in all 12 species of D. virilis group - D. litoralis, D. ezoana, D. lacicola, D. borealis, D. montana, D. flavomontana, D. kanekoi, D. virilis, D. a. americana, D. a. texana, D. novomexicana, and D. lummei. Every species of the group possess a unique type of Gr32a receptor, with majority of amino acid variation concentrated in the first and second extra cellular regions, corresponding with ligand-binding position. Heterospecific mate choice experiments were conducted on D. virilis, D. lummei, D. americana, D. littoralis, D. montana, and D. ezoana in different combinations. Majority of heterospecific courtships terminated on the “touching” stage and courtship did not proceed either to “licking” or to “singing” stages. For example, 83 % of courtships between montana females and littoralis males and 70 % of courtships between littoralis females and ezoana males terminated after touching. For some species combinations, however, heterospecific courtships proceeded to more advance stages, e.g. singing and circling. The most unselective males were found to be the males of D. americana: in experiments with littoralis females, 80 % of courtships proceeded to singing and circling. The results of behavioral experiments are compared with degree of sequence variation in Gr32a in different species of D. virilis group.

717C Incomplete lineage sorting in the Drosophila simulans clade. Yu-Ping Poh1,2, Chau-Ti Ting3, Shun-Chern Tsaur2. 1) Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan; 2) Research Center for Biodiversity, Academia Sinica, Taipei, Taiwan; 3) Department of Life Science, Institute of Ecology and Evolutionary Biology, & Institute of Zoology, National Taiwan University, Taipei, Taiwan. Gene tree does not always reconcile with species tree, especially when a series of speciation events occurred in a narrow time frame. The discordance is the result of shared alleles inherited from the common ancestor, and is known as incomplete lineage sorting. According to the genic view of speciation, gene flow of alleles responsible for reproductive isolation between species would cease in the early stage of speciation and these genes could reveal the true phylogeny. However, identifying the genes diverged at early stage of speciation and examining the linkage range of the fixed adaptive alleles remain major issues. The phylogenic relationship among the Drosophila simulans clade provides an ideal model for studying incomplete lineage sorting. All three possible groupings have been reported based on different loci with some ambiguity. On the contrary, the genealogy of OdsH, a hybrid male sterility gene, reveals clear supporting of D. simulans-D. mauritiana grouping. By means of phylogenetic analysis among the three species of D. simulans clade, distinct levels of introgression are found across the whole third chromosome. The numbers of unambiguous and ambiguous sites are counted for each topology while considering polymorphism existing in D. simulans. The result shows the odds are on the grouping of D. simulans-D. mauritiana, which is congruent with the topology based on OdsH. This study provides a sophisticated survey of incomplete lineage sorting, and uncovers the historical imprints of genetic introgression during speciation.

718A Functional and cytological analysis of the Hybrid male rescue (Hmr) gene. Aruna Satish, Patrick M. Ferree, Daniel A. Barbash. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. Interspecific crosses between Drosophila melanogaster females and males of its sibling species, including D. simulans, produce F1 hybrid males that die as larvae. Mutations in the Hybrid male rescue (Hmr) gene of D. melanogaster suppress this hybrid male lethality. Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. Molecular analysis has revealed significant sequence divergence of Hmr among sibling species of the D. melanogaster subgroup that was driven by positive selection. These findings raise the questions: (1) what is the function of Hmr within the pure species, and (2) why is this gene rapidly evolving? A hypomorphic allele, Hmr1, partially suppresses hybrid male lethality but has no apparent phenotype within D. melanogaster. To further investigate the possible functions of Hmr in D. melanogaster, we are studying a deficiency allele, Df(1)Hmr- . Our genetic analysis reveals that Df(1)Hmr- reduces female viability and fertility. Df(1)Hmr- females lay significantly fewer eggs than Hmr+ females and the eggs that are laid show defects in eggshell structure. D. simulans and D. mauritiana Hmr+ transgenes transformed into D. melanogaster partially complement the deleterious effects of Df(1)Hmr-. This complementation suggests partial functional conservation of Hmr among sibling species. However, only Hmr+ from D. melanogaster and not from its sibling species causes hybrid lethality. This specificity of the D. melanogaster Hmr+ lethal effect in hybrids could be due to a) differences in mRNA expression, b) differences in protein localization, or c) amino-acid sequence differences which alter protein-protein or protein-DNA interactions in hybrids. To distinguish among these possibilities we have generated transgenic D. melanogaster flies carrying a FLAG epitope- tagged Hmr+ from D. melanogaster or from D. simulans. We will compare the mRNA levels and HMR protein localization patterns of these constructs in pure species and in hybrids. 332 POSTERS: Immune System and Cell Death

719B The Role of Caspases in Midgut Cell Death During Drosophila Metamorphosis. Donna Denton, Kathryn Mills, Sharad Kumar. Hanson Institute, IMVS, Adelaide, SA, Australia. During Drosophila metamorphosis obsolete larval tissues are removed by programmed cell death (PCD) and replaced by adult tissue. This process is regulated by pulses of the steroid hormone ecdysone. A pulse of ecdysone at the end of larval development triggers the destruction of the midgut (MG) and a subsequent ecdysone pulse about 12 hours after puparium formation triggers the destruction of the larval salivary glands (SG). Ecdysone binds its heterodimeric receptor EcR/Usp to regulate expression of components of the cell death machinery, including several caspases. Caspases are cysteine proteases that execute cell death by specifically cleaving a large number of proteins. There are seven caspases (Dronc, Dredd, Strica, Drice, Dcp-1, Decay, and Damm) in Drosophila. Of these Dronc, the main upstream cell death caspase, is essential for development. Dronc activity is regulated by binding to its adaptor Ark, subsequently activating downstream effector caspases such as Drice and Dcp-1. Our previous studies have shown that dronc expression is regulated by ecdysone in the MG and SG during metamorphosis, and overexpression of dronc is sufficient to mediate PCD. In dronc null mutants SGs persist indicating that Dronc is required for SG PCD. Interestingly, dronc is not required for PCD of the larval MG. Consistent with these observations ark mutants also show persistent SGs but normal PCD of larval MG. Using various caspase mutants we are now examining the mechanisms of Dronc-independent MG PCD. Our results so far reveal that dcp-1, drice and dronc triple mutants still undergo PCD in MG with similar levels of caspase activity compared to wild type. These data suggest that caspase activation and PCD in MG can occur by alternative pathways. We are currently exploring the potential roles of the remaining fly caspases in MG PCD.

720C Apoptosis independent role of DIAP1-Dronc in promoting cell proliferation. Celia Domingues, Hyung Don Ryoo. Dept Cell Biol, New York Univ, New York, NY. The coordination between cell death and proliferation needs tight regulation, in order to control the size of animal tissues. Across species, apoptosis is triggered by the initiator caspase and progresses through the activation of effector caspases. Drosophila IAP1 has a well stablished role as an anti-apoptotic molecule that directly inhibits both the initiator caspase Dronc and the effector caspases. Apart from their apoptotic roles, DIAP1 and Dronc show a strong mitogenic activity when the apoptotic outcome is blocked through a caspase inhibitor, p35, thus producing the so called “undead” cells. Activation of JNK signalling has been reported to be associated with the mitogenic role of DIAP1/Dronc, and both wg and dpp have been identified as two mitogens produced autonomously by the “undead” cell population. Although the requirement of Dronc and JNK kinase for the mitogenic activity of “undead” cells is well documented, no epistasis mechanism has been elucidated. We studied the relationship between Dronc and JNK in triggering the non-autonomous tissue growth, and found that abolishing Dronc blocks JNK activation in the undead cell population. Conversely, we found that ectopic activation of Dronc and Apaf1, both components of the apoptosome, results in JNK activation. Overall, this results confirm that Dronc lies upstream JNK in the pathway that leads to the production of the mitogens wg and dpp within the “undead” cells. In agreement with a requirement for JNK activity in this pathway, we show that IR transgene lines that abolish JNK and the upstream kinase hep, as well as the transcription factor c-fos, all considerably suppress the overgrown discs phenotype. However, two of the upstream kinases of JNK, dASK and dTAK, do not seem to play a role in JNK activation in our system, since impairing the function of either one of this two JNKKK does not have an effect in the overall size of discs. Taken together, these results show that Dronc and JNK are part of a linear pathway that culminates in non-autonomous tissue growth.

721A Imaging analysis of the spatiotemporal pattern of caspase activation during epithelial cell sheet replacement. Yuichiro Nakajima, Erina Kuranaga, Masayuki Miura. Department of Genetics, Grad. Sch. Pharm. Sci., University of Tokyo, Tokyo, Japan. Programmed cell death is one of the essential events for morphogenesis of multicellular organisms. Apoptosis serves many functions like sculpting structures and controlling cell numbers during animal development. Cell-death mechanisms executed by caspases are well conserved through evolution. Although cell-death signaling mechanisms have been extensively studied by using in vitro cultured cells, in vivo dynamics of cell death and its regulation during development are not well known. Cell-fate determination like cell death in cell community is considered as a result through cell-to-cell interactions. Therefore, combined approaches of in vivo live-imaging and genetics should be ideal to investigate the physiological function and regulation of cell death. We used a FRET- based indicator for caspase activation, named ‘SCAT3’ that can be applied for in vivo analysis of caspase-3-like activity. Epithelial morphogenesis accompanying cell proliferation and cell rearrangements is a general morphogenetic event. Replacement of larval tissues by imaginal ones is one of the fundamental events during metamorphosis in holometabolic insects. Abdominal epithelium is rearranged from larval epidermal cells (LECs) to abdominal histoblasts at pupal stage in Drosophila. We detected caspase activation in LECs, which are destined to be eliminated during epithelial rearrangement, and found the spatial and temporal patterns of caspase activation in each cell. We generated a transgenic fly expressing NLS-SCAT3, which was localized in the nucleus and quantified the order of cell death at abdominal segments during cell replacement. The pattern of cell death is abolished by caspase inhibition. Live imaging of caspase activation also suggests the interaction between proliferating histoblasts and dying LECs. We discuss the mechanism and the function of pattern of caspase activation that may be necessary for epithelial replacement. POSTERS: Immune System and Cell Death 333

722B JNK activity induces oncogenic transformation after stress events. Evgeny Shlevkov, Ginés Morata. Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, 28049 Madrid, Spain. In Drosophila, stresses such as x-irradiation or severe heat shock cause many epidermal cells to die by apoptosis. Recent studies have shown that compromising apoptosis with the caspase inhibitor P35 causes severe developmental aberrations. Caspase- inhibited cells that initiate apoptosis do not die. Instead, they persist in an “undead” state in which they ectopically express the signalling genes decapentaplegic (dpp) and wingless (wg) and induce abnormal growth and proliferation of surrounding tissue. In addition, they frequently show invasive behaviour and invade neighbour compartments. In this study we investigate the mechanisms responsible for these effects. We show that dpp and wg are both induced by the JNK pathway, which, in turn, is activated by dronc. We also demonstrate that the JNK activity is part of a positive feedback loop that amplifies and sustains the apoptotic response. In addition, we find that JNK signalling promotes the invasive behaviour of undead cells, as well as the cell shape changes associated with this process. Our results suggest that once initiated the apoptotic program, the JNK pathway contributes to the formation of a feedback loop, presumably to ensure the death of the cell. When the execution of apoptosis is prevented by inhibiting the effector caspases, the undead cells express other functions associated with JNK signalling such as dpp activity and migratory behaviour.

723C A dual mechanism to promote Pseudomonas aeruginosa infectivity: KerB restricts elicitors and enhances suppressors of innate immunity. Yiorgos Apidianakis1, Sagi Sapira1, Guillaume Charriere1, Jianxin He1, Ding Ding An1, Michael Mindrinos2, Regina Baldini1, Laurence Rahme1. 1) Massachusetts General Hospital, Boston, MA; 2) Stanford University, Palo Alto, CA. Pseudomonas aeruginosa is human opportunistic pathogen of high clinical relevance, exhibiting its virulence potential in widely diverse hosts by using a shared set of virulence factors. The success of P. aeruginosa to survive in nature and in humans is attributed to the highly redundant virulence repertoire. In this report we show that KerB, a putative SAM methyltransferase, conserved among phytopathogenic, entomopathogenic and human opportunistic pathogen species of genus Pseudomonas is attenuated in virulence in various hosts. The attenuation in virulence was exhibited in Arabidopsis thaliana, Dictyostelium castaneum, Drosophila melanogasterand Mus musculus. Using the Drosophila-Pseudomonas system of acute infection, we show that KerB mutant unlike wild type bacteria do not increase their titer up to 24 hours following infection. Our results show that during the early phase of infection the expression of a set of virulence factors, including genes of the type III secretion system (T3SS) is reduced. In addition, KerB mutant expresses higher levels of nitric oxide and pyochelin regulation genes. We provide evidence that KerB maintains low levels of secreted immunity elicitors and high levels of T3SS, causing a suppression of antimicrobial peptides during infection.

724A Characterization of Tep genes during the innate immune responseDrosophila. Richard Bou Aoun, Nicolas Matt, Jules Hoffmann, Dominique Ferrandon. UPR9022 IBMC, CNRS, Strasbourg, Alsace, France. An analysis of the genome of Drosophila melanogaster led to the identification of genes sharing similarities with the C3/C4 (complement factors in mammalian immunity), which were named Teps for ThiolEster containing Proteins. These proteins also share some similarities with the α2-macroglobulins family of protease inhibitors. The thiolester active site typical of the C3/C4 and the α2-macroglobulins is present in the Tep proteins. There are 6 Tep proteins in the genome of Drosophila (Tep1-6), including Tep5 which is a putative pseudogene, and Tep6 (also named mcr: macroglobulin complement-related) in which the thiolester site is mutated. Tep proteins have also been studied in the mosquito Anopheles gambiae. aTep1 interferes with the life cycle of Plasmodium bergeï by limiting the development of the parasite during its invasion of the intestine of Anopheles. It constitutes an essential factor in the anti parasitic response: it is able to bind to the surface of ookinetes and facilitates their elimination. The expression of the Tep1 gene is induced during infections with bacteria and parasites. First, we investigated the expression patterns of the Drosophila Tep genes by performing in situ hybridization on whole mount (larvae and adults) and on sections (adults). We show that Tep genes are transcribed in a broad range of tissues that may be involved in the defense of the flies against microorganisms. Second, we confirm, by Q-RT PCR, that Teps transcription increases in adults after a microbial challenge. These findings indicate that Teps may play a role in the defense against microbes. In Drosophila melanogaster, very little is known about the molecular function of this family of proteins, with the exception of the Tep-like protein, Mcr. Mcr has been shown to be involved in the phagocytosis of Candida albicans by Schneider cells. We are studying the molecular function of Tep proteins in Drosophila using genetic analysis. 334 POSTERS: Immune System and Cell Death

725B Novel virus-like particles from Leptopilina boulardi, a parasite of fruit flies Drosophila melanogaster. Felix Castellanos, Jorge Morales, Shubha Govind. Biology Department. The City College of The City University of New York, 138th Street and Convent Avenue, New York, N.Y. 10031. We are using the model organism D. melanogaster and its parasites to study innate immunity, host pathogen interactions including mechanisms underlying pathogen virulence. Females of the endoparasitoid wasps L. boulardi parasitize D. melanogaster larvae. In this highly specific host-parasitoid interaction, wasp females lay one or more eggs. In wild type fly strains, the parasite egg easily overcomes the host’s cellular and humoral immune responses, developing into an adult wasp. Recent microarray analysis of wasp- infected hosts (Schlenke et al., 2007) suggests that the major genes and signaling cascades controlling humoral and cellular immune pathways are upregulated after L. boulardi infection. Yet, these immune reactions appear to be thwarted as the parasite almost always succeeds within the host. In contrast, when the same L. boulardi strain infects D. yakuba hosts, a strong encapsulation reaction ensues, ensuring the success of the host. To understand these differences between parasitoid-host interaction, we are characterizing the virulence factors that may interact with the cellular and molecular aspects of immunity pathways. Previous studies in similar wasp species have suggested that such factors reside in the long gland-reservoir complex of the female wasp. Our scanning and transmission electron microscopy studies reveal the presence of membrane-bound, 200-500 nm, virus-like particles of differing morphologies: spherical particles with spikes, spherical particles without spikes, and filamentous particles. Some of the VLPs have internal organization with electron-dense and electron-light regions. We will present results that will address the relationship between these three classes of structures and their potential role in immune suppression.

726C “Too much of a good thing” - extracellular adenosine effects in flies. Tomas Dolezal, Monika Zuberova, Milena Novakova, Michaela Fenckova. Faculty of Sciences, University of South Bohemia, Ceske Budejovice, Czech Republic. Extracellular adenosine is an important regulator of various physiological processes, including neuromodulation, immunomodulation and reaction to hypoxia. Faulty regulation of its levels has often drastic pathological consequences. In recent years, we have connected forward and reverse genetics to develop a fly model for adenosine effects in organism. First, we have used homologous recombination to knock out five genes from adenosine deaminase family (called ADGFs). Mutant in the ADGF-A gene has dramatically increased level of extracellular adenosine during larval stages leading to death. Most recently, we have performed a genetic screen to identify suppressors of this phenotype and among others we have isolated Phosphorylase kinase γ (PhKγ) as a strong suppressor of the adgf-a mutant phenotype. This showed us the real reason for larval lethality in presence of high adenosine levels - a progressive loss of energy reserves (process known as wasting). We have previously shown that the main regulator of extracellular adenosine levels are larval hemocytes and that adenosine genetically interacts with the main immune regulator - the Toll signaling. Thus we speculate that adenosine is one of the main signal, released from hemocytes, to re-allocate energy for the immune system during systemic infections. In case of the lack of the adenosine deaminase activity (as in the adgf-a mutant), the extracellular levels are kept high leading to constitutive energy re-allocation resulting in loss of energy reserves and eventually to death. This could be similar to what is happening during chronic infection, which is sensed by immune system (e.g. hemocytes in flies), leading to high levels of adenosine for longer time and hence to wasting. This model might be extremely important for biomedical research when similar processes, i.e. systemic infections leading to wasting, are being described in humans but the underlying cause is not understood.

727A Identification of Rift Valley Fever Virus cellular determinants using high throughput screening. Claire Marie Filone1, Robert Doms1, Sara Cherry1,2. 1) Department of Microbiology, University of Pennsylvania, Philadelphia, PA; 2) Penn Genomics Institute, University of Pennsylvania, Philadelphia, PA. Rift Valley Fever Virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is an emerging arthropod-borne virus transmitted by mosquitoes to both humans and domestic animals. RVFV infection of the insect host is non-pathogenic, but causes severe disease in mammals. The host and viral determinants of this difference are unknown. Furthermore, relatively little is known about the host determinants necessary for RVFV replication. To characterize the cellular determinants of infection and gain a better understanding of the differences between the replication cycles in these two hosts, we have performed small molecule screens in cell lines derived from each host. We screened a library of 1,200 pharmacologically active and well-defined small molecules to identify inhibitors of RVFV. This approach allowed us to compare the requirements of infection between insect and mammalian cells. Using this strategy, we discovered three classes of inhibitors. First, we found a subset than inhibited either human or Drosophila cells, while a third class inhibited replication in both cell types. Within the group that was active in both human and insect cells were a panel of calcium signaling inhibitors, making it a conserved cellular pathway required for RVFV infection. We are currently characterizing the mechanism of this requirement. Another strategy we are using to further elucidate the role of calcium signaling and other cellular pathways for infection is a high-throughput genome-wide RNAi screen for factors that impact RVFV replication in Drosophila cells. Altogether, these studies will provide insight into RVFV-host interactions as well as identify new targets for antiviral therapeutics against RVFV and perhaps other viruses in the Bunyaviridae family. POSTERS: Immune System and Cell Death 335

728B Undertaker, a Drosophila Junctophilin links Draper and dCed-6 to calcium homeostasis during phagocytosis. Nathalie Franc, Leigh Cuttell, Claire Escaron, Mark Lavine, Emeline Van Goethen, Jean-Pierre Eid, Magali Quirin. MRC Cell Biol Unit, MRC LMCB UCL, London, United Kingdom. Programmed cell death is important for the development of multi-cellular organisms. Phagocytes, such as macrophages, clear cells that are dying by apoptosis. In C. elegans, two pathways including the C. elegans death genes ced-1, -6 and -7, and ced-2, -5, -10 and -12, have been characterized that participate in apoptotic cell clearance. While the Ced-2/5/10/12 pathway leads to the activation of the small GTPase Ced10, which controls actin cytoskeleton rearrangement during phagocytosis, the role of the Ced-1/ 6/7 pathway remains unclear. Combining Drosophila genetics and S2 cell-RNAi screens for genes required in apoptotic cell clearance, we identified undertaker (uta) whose mutation led to a phagocytosis defect. uta encodes a junctophilin-related protein. In excitable cells, mammalian junctophilins form junctional complexes that couple Ca2+ channels at the plasma membrane and that of the endoplasmic/sarcoplasma reticulum Ca2+ stores, known as Ryanodine receptors, thereby regulating Ca2+ homeostasis. We found that the Drosophila Ryanodine receptor, Rya-r44F and extracellular Ca2+ are required for efficient apoptotic cell clearance. We placed Uta and Rya-r44F in the Ced-1/6/7 pathway by demonstrating genetic interactions between uta, rya-r44F, dCed-6 and the ced-1 homologue draper. Our results implicate junctophilins in macrophage function, and link Draper and dCed-6 to Ca2+ homeostasis during phagocytosis. We will present our latest results in understanding the role of this pathway in phagocytosis.

729C The hidden enemy: Insect endosymbionts and the evasion of host immunity. Harriet Harris1,2, Stephinie Geis1, Lesley Brennan1,2. 1) Dept Biol, Concordia Univ Col, Edmonton, AB, Canada; 2) Dept Biological Sci, Univ of Alberta, Edmonton, AB, Canada. Insects have efficient innate immune systems including cellular and humoral reactions that allow them to avoid infection by bacteria, viruses and fungi, to heal wounds, and to encapsulate parasitoid eggs. However, there are well known examples of organisms living within insects that successfully evade the host’s defenses: the colonization of mosquitoes and sandflies by protozoan parasites and the existence of endosymbionts living within the cytoplasm of host cells are just two examples. The obligate maternally inherited endosymbiont Wolbachia pipientis appears to be refractory to the host immune system. Previous work by Bourtzis et al, (Insect Molecular Biology 9, 635-639, 2000) and others have shown that Wolbachia do not activate or impair the imd or Toll pathways in insect hosts. Our lab has been investigating the nature of host-endosymbiont cellular interactions using cultured mosquito cells from Aedes albopictus infected with Wolbachia as a model system. We have used a proteomics approach to investigate the expression of proteins induced by Wolbachia and have identified several host proteins that are upregulated as a consequence of infection. Our results suggest that insect cells do, in fact, recognize the presence of endosymbiontic bacteria and they do mount an immune response which changes cell behaviour and may represent a cost for the host cell.

730A 1,4 1,4 1 Suppression of innate immune responses by elevated CO2 levels. Tomasz Krupinski , Iiro T Helenius , Dennis Wang , Doug Turnbull2, Neal Silverman3, Eric Johnson2, Jacob I Sznajder1, Peter Sporn1, Greg Beitel1. 1) Northwestern U; 2) U of Oregon; 3) U Mass; 4) co-first authors.

We are interested in understanding the mechanisms by which elevated levels of CO2 (hypercapnia) adversely affect outcomes in human lung diseases such as chronic obstructive pulmonary disease (COPD), currently the fourth leading cause of death in the US. To this end, we are investigating the effects of hypercapnia on Drosophila in order to define mechanisms by which non-neuronal cells sense and respond to elevated CO2. We have found that hypercapnia suppresses expression of subsets of antimicrobial peptides (AMPs) in both unchallenged and bacteria-infected whole flies, and in S2 cells challenged with bacterial cell wall petidoglycan (PGN). This suppression of AMPs appears to be physiologically significant, as hypercapnia accelerates mortality in flies infected with S. aureus and E. faecalis. We have also found that expression and release of host-defense-related cytokines are suppressed by hypercapnia in human macrophages. These results suggest that the immunosuppressive effects of hypercapnia are conserved and could contribute to the prevalence and pathogenesis of infections in COPD. CO2 suppression appears to act downstream of, or in Κ Κ Α parallel to, NF B/Rel activation because CO2 does not affect endotoxin-induced degradation of mammalian I B or the peptidoglycan- induced cleavage of Drosophila Relish in cell culture. Further, we find that the effects of CO2 on the innate immune system are independent of pH, nitric oxide, hypoxia, neuronal CO2 sensing, and general stress responses such as heat shock pathways. We therefore propose that the CO2 suppresses innate immune responses at the transcriptional level via a novel, but conserved pathway, resulting in impaired host defense against infection in flies and humans. 336 POSTERS: Immune System and Cell Death

731B Characterization of CG1837 protein as a candidate ligand for Draper, a Drosophila phagocytosis receptor. Takayuki Kuraishi1, Takashi Ishimoto2, Naoko Yamamoto2, Koichi Ueda2, Hiroshi Nakayama3, Masamitsu Yamaguchi4, Takeshi Awasaki5, Yumi Hashimoto1, Takeshi Moki2, Akiko Shiratsuchi1, Yoshinobu Nakanishi1. 1) Grad. Sch. Med. Sci., Kanazawa Univ; 2) Grad. Sch. Nat. Sci. & Tech., Kanazawa Univ; 3) Biomol. Charact. Team, RIKEN; 4) Dept. Applied Biol., Kyoto Inst. Tech; 5) Dept. Neurobiol., Univ. Mass. Med. Sch. Apoptotic cells are selectively and rapidly eliminated by phagocytosis. Target selectivity in this reaction owes to specific interaction between phagocytosis markers of targets and their receptors of phagocytes. We previously showed that Draper, a Drosophila homologue of the C. elegans phagocytosis receptor CED-1, is required for phagocytosis of apoptotic cells and pruning of neuronal axons. To identify a ligand for Draper, we affinity purified proteins that bound to the extracellular portion of Draper. We here report the characterization of one such protein encoded by CG1837. We found that CG1837 protein changes its subcellular distribution upon induction of apoptosis, and that l(2)mbn, a hemocyte cell line, phagocytosed latex beads coated with CG1837 protein as well as S2 cells that overexpressed CG1837 protein at their surfaces more efficiently than control target particles or cells. These results suggest that CG1837 protein is a phagocytosis marker that is recognized by and activates Draper. We are currently analyzing null mutants of CG1837 for the level of apoptotic cell phagocytosis and axon pruning.

732C Cellular immune response and JAK/STAT signalling pathway. Rami Makki1, Virginie Daburon1, Delphine Pennetier1, Joanna Krzemien1, Marie Meister2, Alain Vincent1, Michèle Crozatier1. 1) Centre de Biologie du Développement, Toulouse, France; 2) Musée de Zoologie, Strasbourg, France. The innate immune response is common to all multicellular animals and provides an immediate protection against pathogens by using “blood cells” specialised in their destruction. Drosophila “blood cells”, the haemocytes, originate from a specialized larval haematopoietic organ, the lymph gland. Larval haematopoietic progenitors (prohaemocytes) give rise to three types of haemocytes: plasmatocytes, crystal cells and lamellocytes. Lamellocytes, which are devoted to encapsulation of large foreign bodies, only differentiate in response to specific immune threats, such as parasitisation by wasps. We showed that a small cluster of signalling cells in the lymph gland, termed the PSC for Posterior Signalling Centre, controls the balance between multipotent prohaemocytes and differentiated haemocytes. The key role of the PSC in controlling blood cells homeostasis is reminiscent of micro-environmental stem cells niches that provide support for haematopoiesis in vertebrates (Krzemien et al., 2007; Mandal et al., 2007). Activation of the JAK/STAT pathway is required to maintain a pool of non differentiated prohaemocytes in physiological conditions. After an immune challenge such as wasps parasitisation the JAK/STAT signalling pathway is turned off and massive differentiation of lamellocytes occurs. Implication of the JAK/STAT signalling pathway in the specification of lamellocytes will be discussed.

733A Identification of host factors involved in Poxvirus infection using genome-wide screening approaches in Drosophila. Theresa Moser, Sara Cherry. Department of Microbiology, University of Pennsylvania, Philadelphia, PA. Vaccinia virus is the best studied member of the family Poxviridae, which includes variola virus, the causative agent of smallpox. Poxviruses are large double-stranded DNA viruses that have a complex lifecycle which includes a number of temporally regulated steps and morphological intermediates. A large number of the over 200 genes present in the vaccinia genome encode proteins that are nonessential for growth in culture, and instead have immunomodulatory functions which inhibit the anti-viral response. Many of these genes represent important determinants of poxvirus virulence and tropism. While numerous studies have investigated the roles of poxvirus-encoded proteins, less is known about the cellular factors that impact infection and pathogenesis. Our goal is to take advantage of Drosophila genetics and RNAi technology to dissect the interplay between virus and host by identifying cellular factors that affect viral pathogenesis. We have found that vaccinia initiates infection in Drosophila cells, but fails to complete later stages of the life cycle. We have developed a high-throughput assay to screen a genome-wide collection of dsRNA against the Drosophila genome for factors that impact vaccinia infection. We will identify factors that when missing, alter the course of infection in several ways, including progression to late stages of infection, as well as an increase or decrease in early infection. Small scale RNAi and small molecule inhibitor studies have suggested the importance of signaling cascades in vaccinia infection, and these findings will be discussed. POSTERS: Immune System and Cell Death 337

734B Melanotic mass formation and lamellocyte differentiation in lam mutant larvae. Maja Pavlovic1,2, Per Kylsten1, Mitch Dushay1,3. 1) Life Sciences, Södertörns högskola, 141 89 Huddinge, Sweden; 2) Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden; 3) Comparative Physiology, EBC, Uppsala University, Norbyvägen 18A, 752 36 Uppsala, Sweden. Lamins are intermediate filament proteins that form the nuclear lamina and affect cell differentiation and function in metazoans. We have shown that while the Drosophila lamin genes lam and lamC are expressed in patterns similar to vertebrate lamins B and A/C respectively, they are not orthologous and evolved independently, and that lam mutant larvae and surviving adults show phenotypes similar to muscular dystrophy and premature aging. We now report that loss-of-function lam mutations as well as overexpression of lam cDNA in hemocytes cause the formation of melanotic masses in larvae. These result from an autoimmune-like attack of hemocytes on self tissue similar to the encapsulation of parasite eggs. Like in parasitized larvae, there is differentiation in lam larvae of lamellocytes, the hemocyte type associated with encapsulation.

735C High-Throughput Screening of Sindbis virus in Disparate Hosts to identify novel antiviral targets. Patrick P Rose1, Rich W Hardy2, Sara Cherry1. 1) Department of Microbiology, University of Pennsylvania, Philadelphia, PA. 19104; 2) Department of Biology, Indiana University, Bloomington, IN 47405. Deciphering host-pathogen interactions can lead to a better understanding of both essential cellular pathways as well as the mechanisms underlying disease. Viruses in particular have developed highly effective strategies to hijack cellular machinery and to subvert host immune responses making them interesting from both the perspective of disease, and the fact that they can be used as a tool to probe biological functions. Despite the focus on studying virus-host interactions, the identification of cellular factors involved in viral replication and pathogenesis has been difficult due to the lack of virus-host systems open to genetic screening. In light of this, we chose to use a novel cell-based, genome-wide high-throughput RNAi screen in Drosophila cells to identify host factors that regulate pathogenesis, including both those factors hijacked by the virus for replication and those systems used by the host to combat the viral invasion. As a model system, we chose to dissect the host factor requirements of Sindbis virus, a member of the Togaviridae family of arboviruses. This class of viruses is transmitted to vertebrates by insects such as mosquitoes. Therefore, we are using Drosophila as a model for the insect host of this medically important pathogen. To compliment the genome-wide RNAi screen, we are also studying this virus-host interaction in human cells and have performed small molecule-inhibitor screening in both insect and human cells. By comparing the host factor requirements between insect cells and mammalian cells we hope to gain a comprehensive understanding of the differences and similarities between the cellular pathways required in these disparate hosts. Our findings suggest that there are a number of conserved cellular pathways required in insects and mammalian cells for viral propagation that have been previously unknown.

736A A role for autophagy in innate antiviral immunity: Lessons learned from Drosophila. Spencer Shelly, Nina Lukinova, Allison Berman, Sara Cherry. Microbiology, University of Pennsylvania, Philadelphia, PA. Autophagy, historically known as a cellular homeostasis pathway, has recently been recognized as a pathway involved in innate immunity to intracellular bacteria. Here we show an essential role for autophagy in controlling viral infection in Drosophila cells. Depletion of autophagy gene products by RNAi in cells leads to increased viral replication demonstrating an intrinsic role in controlling viral replication in cells. We are continuing to dissect the role of autophagy in antiviral immunity to identify the mechanism by which this pathway controls infection. Drosophila provides a tractable genetic system to study viral infection due to the powerful genetic tools available including forward genetics in animals and functional genomics in cell culture. One strategy we are using to further dissect the role of autophagy in antiviral innate immunity involves a high-throughput genome-wide RNAi screen to identify additional factors that control this autophagic innate antiviral response. Moreover, we are characterizing this pathway in vivo, by challenging animals mutant for components of the autophagy pathway to determine whether this pathway also plays an antiviral role at the organismal level. 338 POSTERS: Immune System and Cell Death

737B E3 ligase and proteasome pathway in phagocytosis of apoptotic corpses by embryonic macrophages. Elizabeth Silva, Nathalie Franc. MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London, United Kingdom. Programmed cell death is essential for the proper development of all metazoans and integral to this process is the engulfment of the apoptotic corpses that results. Failure to do so can result in inflammation and release of toxins, and defective apoptotic cell engulfment has been implicated in the development or exacerbation of auto-immune disorders such as Lupus Erythematosus and Rheumatoid Arthritis. We recently described a requirement for Pallbearer, a novel F-box domain containing protein, in the engulfment of apoptotic cells in the embryo (Silva et al., Immunity 27(4): 585-596). Pallbearer functions with SkpA, Cullin1 and the E2 Ubiquitin ligase Effete to form a unique SCF complex that ubiquitylates and mediates the degradation of an as yet unidentified substrate via the 26S proteasome. In the absence of this substrate degradation, embryonic macrophages are impaired in engulfment and elimination of apoptotic corpses. Here we further describe the phenotypes associated with Pallbearer and our efforts to characterize its substrate with the aim of clarifying its role, and that of proteasomal degradation, in the engulfment process.

738C Characterization of Apoptosis Inducing Factor (AIF) in the Drosophila visual system. Zachary Lemmon, Joseph O’Tousa. Biological Sciences, University of Notre Dame, Notre Dame, IN. We are investigating the localization and apparent activity of Apoptosis Inducing Factor (AIF) in both wild type photoreceptors and photoreceptors undergoing age dependent retinal degeneration. AIF is a phylogenically ancient flavoprotein that localizes to the mitochondria and has a putative role in electron transfer. AIF is also involved in apoptotic processes. Upon cellular stress it is subjected to proteolysis and released from the mitochondria. The AIF carboxyl domain is relocated to the nucleus, where it promotes chromatin alteration and cell death. Work with mammalian cultured cells suggests the mitochondrial localization and function of AIF is located within the amino terminus of the protein, while the apoptotic activity of AIF is dependent on the carboxyl terminus of the protein. To study AIF function in adult Drosophila photoreceptors, three mRFP-tagged AIF transgenic strains (full length AIF, AIF amino terminal domain, and AIF carboxyl terminal domain) were generated. As predicted by the cultured cell studies, we show that the amino terminal domain specifies mitochondrial localization in Drosophila photoreceptors. Characterization of the full length and carboxyl terminal domain AIF constructs is currently underway. Analysis of these constructs in wild type, as well as in norpA and other retinal degeneration mutations, will be used to further define the roles of the AIF domains and assess the changes of AIF localization during the degeneration process. We also aim to study the effect of AIF loss of function in photoreceptor development and degeneration using a recessive lethal AIF transposon insertion allele. To bypass the lethality of the allele, we are attempting to create flies lacking AIF function in the adult retina via construction of genetic mosaics. Histological analysis of these AIF mosaics will be presented.

739A DNaseII acts cell autonomously during nurse cell death in oogenesis. Kim McCall, B. Paige Bass, Elizabeth Tanner, Daniel Mateos San Martín. Department of Biology, Boston University, Boston, MA. The process of DNA fragmentation during programmed cell death has not been well-characterized in Drosophila. In mammals, caspase activated DNase (CAD) cleaves DNA into nucleosomal fragments in dying cells, whereas DNaseII, a lysosomal nuclease, completes the DNA degradation but acts within engulfing cells. Here we examine the requirement for CAD and DNaseII during two forms of cell death that occur in the fly ovary, starvation-induced death of mid-stage egg chambers and developmental nurse cell death in late oogenesis. Mutants of Inhibitor of CAD (ICAD or Rep1) and DNaseII were both found to display defective cell death in mid-oogenesis. Dying mid-stage DNaseII mutant egg chambers retained diffuse nurse cell nuclear material, resulting in an opaque appearance to egg chambers. Dying ICAD mutant egg chambers also retained nurse cell nuclear material however, nuclear remnants remained condensed and centered within egg chambers. These findings suggest that each nuclease is required for a distinct process during starvation-induced cell death. In contrast to cell death in mid-oogenesis, only DNaseII was found to disrupt developmental nurse cell death, as previously reported by Mukae et al. (2002). We detected persisting nurse cell nuclei in approximately half of stage 14 DNaseII mutant egg chambers. To determine the tissue specificity of DNaseII, germline clones were generated. Surprisingly, we found that DNaseII was required cell autonomously in the nurse cells during both mid and late oogenesis, indicating that it acts within dying cells. Consistent with this, we detected vesicles and increased lysotracker labeling in dying egg chambers during both mid and late oogenesis, indicating that lysosomes and/or autophagy are likely to play a role in programmed cell death in the ovary. POSTERS: Immune System and Cell Death 339

740B klumpfuss regulates expression of genes involved in retinal apoptosis. Jamie Rusconi, Erica Hutchins, Malcolm Schongalla, Sunil Ganesh. Dept Biological Sciences, University at Albany, Albany, NY. Proper apoptotic regulation is necessary in all organisms to refine the morphology of developing limbs and tissues, balance cell proliferation rates, and eliminate unnecessary or abnormal cells or structures. In Drosophila, the retina is a useful model in which to study the mechanisms of apoptotic regulation, where misregulation is easily detected at the cellular level. We have previously shown that a transcription factor, klumpfuss (klu), is necessary and sufficient for apoptosis in the retina. Recent microarray data to identify klu-regulated molecules identified homer, which is upregulated in response to klu overexpression. We demonstrate that Homer is required for apoptosis; retina from homer loss-of-function pupae display a decrease in apoptosis, resulting in extra interommatidial cells. Additionally, we show that Homer is dynamically expressed in these cells, those that will “choose” life or death in the retina, during retinal development. Ultimately, we are seeking to identify precisely how Homer interacts with the apoptotic pathway. Previously, Homer has only been suggested to function in an anti-apoptotic capacity through Akt signaling in neuronal cells. Our data indicate a novel role for Homer in apoptosis, and suggest another possible signaling pathway where Homer interacts with klu to affect apoptosis.

741C Drosophila Cbl is essential for control of cell death and cell differentiation during eye development. Yuan Wang1,2, Christian Werz3, Dongbin Xu1,2, Zhihong Chen1,2, Ying Li1,2, Ernst Hafen3, Andreas Bergmann1,2. 1) The University of Texas M.D. Anderson Cancer Center, Department of Biochemistry & Molecualr Biology; 2) The Genes & Development Graduate Program; 3) ETH. Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, as important as the activation of these receptors is their appropriate spatial and temporal down-regulation for normal development and tissue homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of receptor tyrosine kinases (RTKs), most notably the Epidermal Growth Factor Receptor (EGFR) through ubiquitin- mediated endocytosis and lysosomal degradation. Here, we report several mutant phenotypes of Drosophila cbl (D-cbl) during eye development. D-cbl mutants display overgrowth phenotype with enlarged adult heads and eyes, inhibition of apoptosis, increased ommatidial spacing and differentiation defects of extra numbers of photoreceptor, cone and pigment cells. Using genetic interaction and molecular markers we show that most of these phenotypes are caused by increased activity of the Drosophila EGFR, while Sevenless, another RTK in Drosophila is not regulated by D-cbl. Our genetic data also indicate a critical role of ubiquitination for D- cbl function, consistent with biochemical models. Taken together, these data may provide a mechanistic model for the understanding of the oncogenic activity of mammalian cbl genes.

742A Characterization of Mutants from Cell Death Screens for the 3rd Chromosome in Drosophila. Dongbin Xu, Andreas Bergmann. Dept. of Biochemistry & Molecular Biology, Genes & Development Graduate Program. University of Texas, MD Anderson Cancer Center, Houston, TX. In order to isolate recessive mutations in some essential apoptosis regulatory genes we performed GMR-hid ey-FLP (GheF) screens for the 3rd chromosome in Drosophila. Besides 4 dronc mutants, 1 drICE mutant and 6 cbl mutants, we established 8 additional complementation groups of the mutants from the screens. All mutants suppress the GMR-hid induced eye ablation phenotype. Some of them also show outgrowth of cuticle tissue around adult eyes when ey-FLP is used to generate clones. We identified several mutations in salvador, warts and mats, the essential components of Hippo pathway, which inhibits proliferation and promotes apoptosis. A group of two strong suppressors maps to a region near the drICE gene. Currently we are testing if these two suppressors are drICE mutants. Mapping and characterization of other mutants are on going and the updated results will be presented. 340 POSTERS: Immune System and Cell Death

743B Evolution of the morgue cell death gene in the phylum Arthropoda. Ying Zhou2, John Nambu1,2. 1) Department of Biology, University of Massachusetts, Amherst, MA; 2) Program of Neuroscience and Behavior, University of Massachusetts, Amherst,MA. Morgue gene expression enhances cell death induced by the RHG genes. The Morgue protein has a unique architecture and contains a zinc finger motif, an F box, and an ubiquitin E2 conjugase domain with a Glycine in place of the active site Cysteine. No other protein contains this combination of domains. The presence of an F box and a conjugase domain implies that Morgue acts in protein ubiquitination and the ability of Morgue to associate with the DIAPI suggests that Morgue may regulate PCD via ubiquitination and turnover of cell death regulators. To gain insight into the evolution of this unique protein and its roles in ubiquitination and PCD, we have initiated studies to identify and characterize related sequences from other species. Using bioinformatics and molecular approaches, we have found that Morgue distribution appears to be restricted to Arthropods. Thus far we have identified Morgue homologues in all the Drosophila species examined, as well as several other representative insects including the honeybee (Apis mellifera), silkworm (Bombyx mori), and red flour beetle (Tribolium castaneum). In addition, we have identified Morgue homologs in two crustacean species, the American lobster (Homarus americanus) and the water flea (Daphnia pulex). Each of these Morgue homologues contains a related zinc finger, F box, and variant conjugase domain in a similar organization. Furthermore, the Glycine substitution in the conjugase domain is conserved, suggesting specific functions for this residue. In several species the three domains are encoded by separate exons. Significantly, we have also identified a putative Morgue homologue from the wandering spider (Cupiennius salei)that appears to consist only a conjugase domain with the conserved Glycine substitution. Spider Morgue does not appear to contain a zinc finger or an F box. We are currently working to confirm this finding and identify Morgue-related zinc finger and F box sequences in the spider. Together, our data suggests that the morgue gene arose via exon shuffling events during divergence of the phylum Athropoda.

744C ATP-SENSITIVE POTASSIUM CHANNELS MEDIATE SURVIVAL DURING VIRAL INFECTION IN DROSOPHILA. Ioannis Eleftherianos, Safia Deddouche, Jules Hoffmann, Jean-Luc Imler. Institut de Biologie Moleculaire et Cellulaire, Strasbourg, Alsace, France. Drosophila melanogaster has been established as an excellent genetic model for the study of the innate immune system because of its genetic malleability, its lack of a traditional mammalian adaptive immune system and the conservation of immune signaling pathways with those of higher organisms. To date, innate immune responses against bacteria and fungi have been well characterized in flies. These responses occur via the Toll and immune deficiency (Imd) signaling pathways. The study of the response of Drosophila to RNA virus infections already identified two types of immune responses, which are also evolutionary conserved. A first antiviral mechanism is degradation of viral RNA by RNA interference (RNAi). A second response to viral infection is in part dependent on the JAK-STAT pathway and involves the induction of a large number of genes, some of which may counter viral infection. Interestingly, our data indicate that the transcriptional response induced by Drosophila C virus (DCV) or Flock House virus (FHV) is associated with the pathogenesis of the infection, and contributes to lethality, in a manner reminiscent to septic shock in mammals. We have now identified a potassium channel, which plays a critical role in the heart of Drosophila during viral infection. The SUR2 homologue of Drosophila, dSUR, is mainly expressed in the heart, where it plays a protective role against hypoxic stress. Transgenic dSUR RNAi flies crossed with a heart-specific driver GMH5-Gal4 showed a highly susceptible phenotype when infected with FHV. Our findings open the way for future studies on the role of stress-activated K+ channels in viral infection. Interestingly, these channels also play important homeostatic role during infection in mice.

745A Examining the Cross-Talk between the Insulin Signaling and Immune System Pathways in Drosophila melanogaster. Ingrid Hansen1,2, Scott Pletcher1,2,3. 1) Huffington Ctr on Aging, Baylor Col Medicine, Houston, TX; 2) Interdepartmental Program in Cell and Molecular Biology, Baylor Col Medicine, Houston, TX; 3) Department of Molecular and Human Genetics, Baylor Col Medicine, Houston, TX. The transcription factor dFOXO is an important component in the insulin signaling pathway of the fruit fly Drosophila melanogaster. In the absence of insulin signaling dFOXO is active in the nucleus. When insulin signaling is present, dFOXO is rendered inactive by phosphorylation and translocation to the cytoplasm. Recent evidence has suggested cross-talk between the insulin signaling pathway and the innate immune system of Drosophila. To determine how manipulations in insulin signaling affect the immune system, we are using the Gene-Switch System to over-express dFOXO in a tissue-specific manner and looking for changes in susceptibility to various bacterial infections. We have chosen to examine dFOXO induction in the adult fat body (the major site of antimicrobial peptide production) and ubiquitously. We are also in the process of analyzing dFOXO-null flies for differences in bacterial susceptibility. POSTERS: Immune System and Cell Death 341

746B Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. Thomas Hauling1, Gawa Bidla1, Mitchell Dushay2, Ulrich Theopold1. 1) Molecular Biology & Functional Genomics, Stockholm University, Stockholm, Sweden; 2) Department of Comparative Physiology, Uppsala University, Norbyvägen 18A, Uppsala, Sweden. How immune responses are activated remains a central question in immunology. Activation of the prophenoloxidase-(PPO) activating cascade is one of the key components of arthropod immunity. In Drosophila, systemic activation of PPO is known to occur in response to microbial infection and involves transcriptional activation triggered by recognition of microbial elicitors. Here we show that local activation of Drosophila PPO after wounding depends on endogenous signals. We provide evidence that PPO activation is locally restricted by the systemic inhibitor Serpin27A. This contrasts with PPO activation in the lepidopteran wax moth Galleria mellonella, where melanization shows a different distribution in hemolymph clots and is more sensitive to regulation by foreign elicitors. We present a modified model of PPO activation which incorporates our findings.

747C Peroxiredoxin 5 modulates immune response in Drosophila melanogaster. William Orr, Svetlana Radyuk, Katarzyna Michalak, Vladimir Klichko, Judith Benes. Dept of Biological Sci, Southern Methodist Univ, Dallas, TX. The peroxiredoxins comprise a ubiquitous family of redox-sensing enzymes with multiple cellular functions. We identified a Drosophila homologue of the mammalian peroxiredoxin 5 (dPrx5) and found that a P element-associated mutant for this gene exhibited enhanced resistance to bacteria, which normally trigger the immune response via the Imd pathway. Molecular analysis showed that production of the Relish-dependent antimicrobial peptides was increased in the dprx5 mutant and occurred in the absence of I?B kinase complex activity, normally required for the activation of Relish-dependent genes, unlike Dredd caspase activity, which was still required. Further analysis revealed that dPrx5 acts at the level of dTak1 and affects both Imd and JNK pathways, which was documented by enhanced phosphorylation of Relish and Basket and increased expression of Relish and Puckered in the dprx5 mutant. Taken together, our studies placed dPrx5 in the dTak1-JNK arm of immune signaling, where it acts as a negative regulator of the immune response.

748A Danger signals in Drosophila Host defence. Jean-Marc Reichhart. UPR 9022 CNRS, IBMC, Strasbourg, France. Drosophila mounts a potent host defence when challenged by various microorganisms. Molecular and genetic analyses of this defence have now provided a global picture of the mechanisms by which insects sense infection, discriminates between various classes of microorganisms and induces the production of effector molecules, among which antimicrobial peptides are prominent. In particular, the finding that the Toll receptor is implicated in the fly anti-fungal response paved the way for the discovery of the mammalian Toll-like receptors. Toll receptor activation differs between vertebrates and invertebrates as the immune response is tightly adaptated to the corresponding anatomy. Recently, exciting data point to the possibilities that Drosophila would be able to sense pathogen-derived danger signals in order to activate defence mechanisms. Recent progress in the field will be discussed. 342 POSTERS: Immune System and Cell Death

749B The effects of accessory gland proteins and sperm on immune response in female Drosophila melanogaster. Sarah M. Short1, Brian P. Lazzaro2. 1) Field of Genetics and Development, Cornell University , Ithaca, NY; 2) Department of Entomology, Cornell University, Ithaca, NY. Previous work in Drosophila melanogaster has shown that mating triggers an upregulation in immunity gene expression in the female reproductive tract and the whole female. Furthermore, male Accessory gland proteins (Acps) and sperm, when transferred to the female reproductive tract, each induce expression of a specific and non-overlapping group of immune response genes. One recent study performed by Fedorka et al. (2007) determined that, despite a rise in antimicrobial peptide (AMP) gene expression in the whole body after mating, females’ ability to fend off an infection decreased. This is intriguing, because it presents a seemingly contradictory situation: sperm and Acps cause the female to produce more antimicrobial compounds, but this does not seem to increase her efficiency at fighting infection. One possible explanation for this is that Acps and sperm upregulate antimicrobial peptide production largely in the reproductive tract, which would likely not translate to an increase in systemic infection resistance. We are testing this hypothesis by studying the individual effects of Acps and sperm on expression of AMP genes in the mated female reproductive tract vs. the whole female by quantitative real-time PCR. We are also studying the specific individual influence of Acps and sperm on resistance to systemic infection by artificially infecting females post-mating and assaying systemic bacterial load.

750C Recognition and Signaling in the Drosophila IMD Pathway. Neal Silverman, Deniz Erturk-Hasdemir, Nicholas Paquette, Kamna Aggarwal. Dept. of Medicine, Division of Infectious Diseases, UMass Medical School, Worcester, MA. The Drosophila immune response is characterized by the rapid induction of a battery of antimicrobial peptides immediately following infection. This response is controlled by the IMD and Toll pathways, which drive antimicrobial peptide gene transcription. The IMD pathway is triggered by DAP-type peptidoglycan, the cell wall material found in all Gram-negative bacteria and in certain Gram- positive microbes. We have shown that distinct immune receptors recognize different forms of DAP-type peptidoglycan. Polymeric DAP-type peptidoglycan is recognized by the cell surface PGRP-LCx. On the other hand, a monomeric form of DAP-type peptidoglycan, known as TCT, is recognized either by a cell surface receptor, consisting of a PGRP-LCx and -LCa heterodimer, or by the intracellular receptor PGRP-LE. Signaling by these receptors likely involves ligand-induced multimerization and a conserved signaling motif found in the N-termini of both PGRP-LC and -LE. Downstream of these receptors, signal transduction culminates in the cleavage, nuclear translocation and activation of the NF-κB homolog Relish, which is essential for antimicrobial peptide gene expression via the IMD pathway . This pathway requires the caspase DREDD, the RING-finger protein dIAP2, and the Drosophila I?B Kinase (IKK) homolog. In fact, dIAP2 is rapidly ubiquitinated, in a DREDD-dependent manner, following immune stimulation. Given the established role for ubc13 (bendless) and uev1a in this pathway, we hypothesize that dIAP2 is K63-polyubiquitinated. Further molecular characterization of this ubiquitination event will be presented. dIAP2 is required for activation of the IKK complex which, in turn, phosphorylates Relish on two specific serines. These serines are required for immune-induced antimicrobial peptide gene expression but do not control Relish cleavage or nuclear translocation. Instead, phosphorylation of these residues appears to be critical for the Relish-mediated recruitment of RNA polymerase II to the promoter of the antimicrobial peptide gene diptericin.

751A Identification of the trafficking receptor of the serpin degradation pathway during the innate immune response. Sandra F Soukup, David Gubb. Functional Genomics, CICBiogune, Derio, Basque Country, Spain. The Necrotic (Nec) protein is a serpin (serine proteinase inhibitor). The innate immune response to fungal and Gram+ bacterial challenge in the fly is mediated through the Toll-receptor. This signalling pathway is controlled via a proteolytic cascade, which is modulated via the Nec inhibitor. Serpins interact with their target proteases via a “suicide” mechanism, which generates an inactive serpin/protease complex. The balance between the production and degradation of serpins and their target proteases controls many physiological responses. In addition, serpin molecules have an inherent tendency to form inactive homopolymers. The accumulation of serpin polymers underlies several human pathological conditions e.g: the liver disease and emphysema associated with Z-variant α1-Antitrypsin. The nec20 mutant carries a Glu→Lys substitution homologous to the human Z α1-Antitrypsin. In flies, the nec20 mutation gives a hypomorphic phenotype as most of the serpin is present as inert polymers. We are studying the mechanisms by which serpins are processed in the fly. In nec20 mutants, Nec accumulates in the mid-gut, specially in ventriculus and gastric caeca and we detect Nec antibody staining in endocytotic vesicles in the Garland and pericardial cells. Infection of wild-type flies with Micrococus luteus gives similar results, with positive antibody staining for both Nec and Spn27. To confirm and extend these observations, we are injecting LDLR ds-RNAs into wild-type flies and monitoring transcription of the anti-microbial peptide, Drosomycin, by qRT-PCR. In mutants of the Low Density Lipoprotein Receptor (LDLR) superfamily, Nec is undetectable in the Garland cells. In addition, these mutants act as allele-specific enhancers, en(nec20), which have no effect on nec (null) mutants. The main route for uptake (and presumably elimination) of serpin complexes and polymers from the hemolymph is via the Garland and pericardial cells. The main site of serpin synthesis is the fat body, but we have strong evidence that the Nec protein detected in the mid-gut is synthesised within the gastric caecae. POSTERS: Immune System and Cell Death 343

752B A serpin that controls the melanization reaction in the tracheal system of Drosophila. Huaping Tang1, Zakaria Kambris2, Bruno Lemaitre2, Carl Hashimoto3. 1) Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; 2) Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France; 3) Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520. The Drosophila immune system features multiple defense reactions to combat infections. Two important defense reactions are the synthesis of antimicrobial peptides (AMPs) by the fat body and the melanization reaction involved in wound healing and encapsulation of pathogens. In addition to these systemic immune responses, epithelial tissues exposed to the external environment mount local immune responses such as the generation of reactive oxygen species and the synthesis of AMPs. Here, we describe a novel role for a protease inhibitor of the serpin family in controlling the melanization reaction that is activated in an epithelial tissue, the respiratory tract. We found that the serpin Spn77Ba is required in larval tracheal cells to prevent melanization of the tracheal epithelium, in addition to being essential for larval viability. Spn77Ba is expressed and secreted by tracheal cells into the extracellular space below the cuticle lining the tracheal lumen, where melanization appears to be triggered. Spn77Ba inhibits the melanization cascade involving the MP1 and MP2 proteases, which in the hemolymph is inhibited by another serpin, Spn27A. We propose that Spn77Ba prevents the melanization cascade in the trachea from being constitutively activated and from spreading beyond the site of infection or injury. Surprisingly, we found that tracheal melanization resulting from loss of Spn77Ba function was associated with expression of the antifungal peptide Drosomycin by the fat body. Our data suggest that a final product of the melanization reaction serves as a diffusible signal to activate the Toll pathway leading to Drosomycin expression. Such signaling could represent a mechanism by which the systemic immune response is prepared for a potential invasion of the hemolymph by a pathogen that evades the local immune response of epithelial tissue.

753C Regulation of Drosophila Inhibitor of Apoptosis Protein1 in Developmental Apoptosis. Hui Li, Kenneth Cadigan. Dept MCDB, Univ Michigan, Ann Arbor, MI. Apoptosis in Drosophila is mainly initiated by Reaper(Rpr), Hid, Grim, Sickle and Jafrac2 which are known collectively as RHG proteins. They promote apoptosis, in part, by direct binding to Drosophila Inhibitor of apoptosis protein 1 (DIAP1), antagonizing its ability to inhibit caspases. RHG proteins are also suggested to stimulate DIAP1 ubiquitination and degradation; however this hypothesis is based on the analysis of overexpression phenotypes, raising the question of whether this regulation is physiologically relevant. Our laboratory has previously shown that Wingless signaling induces apoptosis in perimeter ommatidia of mid-pupal eyes by activating expression of RHG proteins (Lin et al. Development 131:2409). We now show the ommatidia destined to die have a dramatic reduction in DIAP1 levels compared to interior ommatidia. This downregulation is independent of the effector caspase Drice and depends on physiological levels of Hid, Grim and Rpr. This is the first evidence that DIAP1 level is regulated by RHG proteins in developmental apoptosis. Further analysis is carried out to test whether auto-ubiquitination by the E3 ubiquitin ligase activity of DIAP1 is the mechanism that leads to the downregulation. Surprisingly, we find that the Apaf-1 homolog Dark and the initiator caspase Dronc are also required for DIAP1 downregulation. This suggests a two branch model for RHG regulation of DIAP1, one branch involving direct binding to DIAP1 and another through activation of Dark and Dronc. We are currently testing the two-branch model. In particular, we are exploring whether mitochondrial factors are mediators between RHG proteins and caspase activation machinery, which might reveal a missing connection in our current understanding of the apoptosis network.

754A Probing the Diap1-Dronc Interaction in vitro and in vivo. Peter J. Shapiro, Hyung-Don Ryoo. Cell Biology, NYU Medical Center, New York, NY. Drosophila is an excellent model system to study the anti-apoptotic role of IAP (Inhibitor of Apoptosis Protein) family of proteins. Drosophila IAP1 (Diap1, also known as thread) is an ubiquitin-ligase that is required for the survival of most somatic cells in this organism. It has been established that Diap1 physically binds and ubiquitylates many proteins, including the Drosophila initiator caspase (Dronc), as well as other caspases. Due to the complexities of these interactions, mutant alleles that abolish specific Diap1/ caspase interactions have been useful in understanding Diap1’s precise mechanism of action. Previous biochemical and crystallographic studies of the Dronc-Diap1 complex suggest that the interaction is between the second BIR (Baculovirus Inverted Repeat) domain of Diap1, and a region in the N terminal domain of unprocessed Dronc. Here, we show that a previously isolated allele of Diap1, with a missence mutation in the second BIR domain, completely disrupts the interaction between Diap1 and Dronc in vitro. This suggests that Dronc’s Diap1 interaction domain is lost after its proteolytic cleavage and activation. This led us to test the idea that Diap1 interacts primarily with unprocessed Dronc for ubiquitylation. We took advantage of mutant apaf1 (also known as dark, or hac1), which encodes an adaptor protein required for Dronc’s proteolytic cleavage and activation. Under this condition, Diap1 was still able to reduce Dronc immunostaining. This supports the idea that Diap1 ubiquitylates unprocessed Dronc, as part of its anti-apoptotic mechanism. 344 POSTERS: Immune System and Cell Death

755B Dicer-2 mediated inducible antiviral response in drosophila. Safia Deddouche1, Delphine Galiana-Arnoux2, Stefanie Mueller1, Bassam Berry3, Christophe Antoniewski3, Anette Schneeman4, Jules Hoffmann1, Jean-Luc Imler1. 1) UPR9022 CNRS, Institut de Biologie Moléculaire et Cellulaire, 67000 Strasbourg, France; 2) UMR5242 CNRS Institut de Genomique Fonctionnelle de Lyon ENS, 69000 Lyon, France; 3) URA2578 CNRS, Institut Pasteur, 75015 Paris, France; 4) Department of Molecular Biology, CB262 The Scripps Research Institute, La Jolla, CA 92037. Insects have been known for more than a century to be strongly resistant to microbial infections, and Drosophila has proved to be a good model to unravel innate mechanisms of host-defence. We are using three RNA viruses, the Dicistrovirus Drosophila C virus (DCV), the Nodavirus Flock house virus (FHV) and the Alphavirus Sindbis virus (SINV) to study antiviral responses in flies. Our data indicate that resistance to viral infection involves two types of mechanisms: on one hand, infection triggers a transcriptional response that depends in part on the JAK-STAT pathway, and leads to the production of antiviral molecules that remain to be identified; on the other hand, viral double stranded RNAs are recognized by the RNaseIII enzyme Dicer-2, and degraded into siRNAs. These siRNAs are incorporated into the RISC complex that will target and degrade viral RNAs. We have undertaken the characterization of the gene Vago, which is induced by infection with DCV in a JAK-STAT independent manner. Vago is induced in the fat body, which serves as a major replication site for the three viruses. Expression of Vago is also upregulated following infection by SINV, but not by FHV. The FHV viral suppressor of RNAi (VSR) B2, which binds to double-stranded RNAs, interferes with the upregulation of Vago in DCV infected flies, thus explaining why FHV does not induce this gene. We furthermore show that Dicer-2 is involved in the induction of Vago. These data establish a link between RNAi and the inducible antiviral response in Drosophila.

756C Trancriptional architecture of age-specific variation in immune function. T. M. Felix1, G. Wu2, J. Leips1. 1) Dept Biological Sciences, UMBC, Baltimore, MD; 2) Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland. Senescence is reflected in the age-related functional decline of many physiological traits. One trait that shows age-related decline across a broad range of taxa is the immune response. While extensive work on mammalian systems has begun to reveal the cellular and physiological hallmarks of age-related declines in immunity, we currently know very little about the genetic basis of this decline. Two independent studies in our lab using Drosophila lines derived from a natural population have demonstrated genetic variation in the age-specific ability to clear an artificially induced infection. Here we report the results of a microarray study using 12 of these lines that identified those genes associated with age-specific clearance ability. We also explore the extent to which the transcriptional co-expression networks are preserved when individuals are infected at different ages and relate these expression networks with age-specific immune function. Our results elucidate the complexity of the genetic basis of age-specific immune function and suggest that the genetic/transcriptional architecture of this trait changes with age.

757A Identification of host factors and pathways essential for West Nile Virus replication. Sheri Hanna, Robert Doms, Sara Cherry. Dept Microbiology, University of Pennsylvania, Philadelphia, PA. The emergence of epidemic arboviral diseases infecting both humans and domestic animals has led to significant world-wide morbidity and mortality. Little is known about the host factors required for the replication cycles of these viruses in either their insect or vertebrate host, and less about the innate immune pathways that restrict pathogenesis. This has impeded the development of antiviral treatments. The identification of cellular factors involved in viral replication and pathogenesis has been difficult due to the lack of virus-host systems amenable to genetic screening. Therefore, we developed novel high-throughput approaches to identify such factors in Drosophila cells. West Nile virus (WNV) has a worldwide distribution and has been of significant concern in the United States since its introduction in 1999. WNV is a member of the flavivirus genus, which includes other important arboviruses, such as Dengue and Yellow Fever virus. By developing RNAi high-throughput assays for this important class of mosquito-borne emerging pathogens, we anticipate identifying genes that are important in the viruses’ life-cycles, as well as genes required for antiviral innate immunity. Moreover, we are also screening small molecule libraries for inhibitors of West Nile virus replication both in insect and human cells. These complementary approaches will yield new insights into the host factor requirements of this virus, similarities and differences between their lifecycles in the vertebrate versus arthropod hosts, and will hopefully uncover conserved targets for antiviral intervention. The results from our WNV RNAi and chemical screens will be presented. POSTERS: Immune System and Cell Death 345

758B Immunity and Maternal Effects in D. melanogaster. Jodell E. Linder, Daniel E.L. Promislow. Dept Genetics, University of Georgia, Athens, GA. Maternal effects describe how a female’s condition influences the quality of her offspring independent of the offspring genotype. One way that maternal effects can arise is from trade-offs between a female’s investment in her life history traits versus her developing offspring. For example, costs to the female of activating her immune system may lead to trade-offs with investment in offspring. Thus, costs of immunity may lead to a reduction in offspring quality via maternal effects. At the same time, immune-related mRNA or protein expressed in the mother may be passed directly on to the female’s eggs, thereby providing benefits to her offspring. Despite enormous research effort on both maternal effects and immunity in D. melanogaster, little work has been carried out on the effect that mounting an immune response has on reproductive quality through maternal effects. There is some recent evidence that infection in the parental generation may cause a beneficial ‘priming’ response in offspring in some insects. This project examines both the costs and benefits to offspring when mothers are immune challenged. We examine several fitness components of the offspring from both immune challenged and naïve mothers (egg size and viability, larval competitiveness, offspring longevity and survival of offspring after immune challenge). This allows us to determine if there are costs to the offspring when females are immune challenged, and if these costs are compensated for by increased immune response in offspring.

759C Innate immune response activity correlates to diversity of Drosophila susceptibility to bacterial infection. Kiyoshi Okado, Naoaki Shinzawa, Hiroka Aonuma, Shinya Fukumoto, Shin-ichiro Kawazu, Kanuka Hirotaka. NRCPD, Obihiro University of Agri. and Vet. Med., Obihiro, Hokkaido, Japan. Drosophila melanogaster provides an interesting model to study genetic variation of resistance to infection because of the relative ease in assessing functional differences between various wild type lines. Some pathogens are able to infect both Homo sapiens and Drosophila; one such bacterial pathogen is Listeria monocytogenes, a gram-positive, intracellular bacterial pathogen that causes serious food-borne infections and is capable of establishing lethal infections in adult fruit flies. In this report, nine wild type lines of Drosophila were examined for their susceptibility to Listeria infection. Canton-S was the most resistant line while white was especially susceptible to infection with Listeria. When the number of Listeria in these lines were counted it was apparent that compared to white, Canton-S was able to suppress pathogen growth. In order to determine the nature of this pathogen growth suppression we examined the activity of the innate immunity through analysis of expression of antimicrobial peptides (AMPs), activity of phenoloxidase (PO) and phagocytosis. All examined AMPs were induced in both Canton-S and white in response to Listeria infection, however, Canton-S showed increased AMP expression at early timepoints. The melanization reaction is one of the most immediate immune responses against pathogens and is required for wound healing. Phenoloxidase (PO) was monitored as an indicator of the melanization reaction and found to be higher in Canton-S compared to white. Phagocytosis is another key process required to fight pathogens and was measured in adult males through injection of fluorescently-label heat-killed Listeria. Consistent with both AMP production and melanization activity, phagocytosis activity was greater in Canton-S. These results suggest that wild type lines display variation to susceptibility to infection and that this variation is a result of differences in immune response to infection with Listeria.

760A Deciphering the interactions between Drosophila melanogaster and a yeast Candida glabrata. Jessica Quintin, Joelle Asmar, Dominique Ferrandon. UPR9022 IBMC, CNRS, Strasbourg, Alsace, France. Candida glabrata is an opportunistic pathogen of humans that asymptomatically colonizes a wide variety of body locations such as our gastrointestinal and genitourinary tracts. However, C. glabrata causes systemic infections (candidiasis) in immunocompromised individuals. It accounts for approximately 15% of all symptomatic mucosal and bloodstream Candida infections worldwide. This pathogen is naturally resistant to some antifungal drugs and therefore represents a major problem for the coming decades. Although both innate and acquired immunity play important roles in the resistance of mammalians to Candida infections, the innate immune response is the first line of defence against C. glabrata systemic infections. Research into the molecular mechanisms of Candida virulence is hindered by the lack of good animal models. Moreover, the immune system of mammals is complex and it is difficult to determine the relative contributions of the different mechanisms. Drosophila melanogaster represents an interesting alternative for the study of host-pathogen interactions. Drosophila, which is devoided of a mammalian-like adaptive immune system, harbors an innate immune response with striking similarities to plant and mammalian defence mechanisms. This model has been effectively used to study both microbial virulence factors and host immune-defences. We have implemented the study of the interactions between C. glabrata and Drosophila melanogaster in a septic injury model. We have analysed the host immune response of the fly to this infection. We identified that the cellular immune response does not appear to play an essential role in host defense. In contrast, some Toll pathway mutants are susceptible to C. glabrata. The Drosophila system is reminiscent of the situation found in neutropenic patients. We have established the proof of concept that Drosophila can be used to screen mutant libraries of C. glabrata for attenuated virulence. 346 POSTERS: Immune System and Cell Death

761B Identification of Host Factors Involved in Flock House Virus Infection. Leah R. Sabin1, Paul Ahlquist2, Sara Cherry1. 1) Microbiology, University of Pennsylvania, Philadelphia, PA; 2) Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI. Virus-host interactions exist as a delicate balance between opposing forces; as a virus subverts cellular machinery to aid in replication, the host mounts an immune response to eliminate the infection. Historically, most virus research has focused on the virus side of the détente, dissecting virally encoded proteins and their functions. However, an important facet that has been more difficult to study is the cellular contribution to replication and pathogenesis. Using the model organism Drosophila melanogaster, our goal is to identify and study the host requirements for viral infection. To identify the host genes or pathways involved in pathogenesis, we use a cell-based high-throughput screening approach, which includes genome-wide RNAi screening and small molecule screens. We are interested in applying these novel approaches to the study of Flock House Virus (FHV). FHV is a small, positive-sense, non-enveloped RNA virus that has traditionally been used as a model virus to study basic mechanisms in molecular virology. We have completed a small molecule screen and plan to perform a genome-wide RNAi screen, with an emphasis on the identification of factors involved in innate immunity. In our pilot FHV RNAi experiments, we identified a previously uncharacterized gene, oldlace, as having an antiviral function. Knockdown of oldlace allows increased infection by FHV and several other RNA viruses in tissue culture, as measured by percent infection. We are now further characterizing the function of oldlace on a cellular and organismal level. Using forward genetics to identify cellular factors important in viral infection, we hope to uncover new insights into host-pathogen interactions and increase our understanding of the normal cellular roles of the identified proteins and pathways.

762C Black dots and inflammation: the role of p38 MAPK signalling in a chronic inflammation-like phenotype in Drosophila. Gerhard Seisenbacher, Ernst Hafen, Hugo Stocker. Institute of Molecular Systems Biology, ETH Zurich, Switzerland. The Mitogen-activated protein kinase family of is involved in the response to a large variety of external signals implicated in cellular responses, including growth, differentiation, inflammation and apoptosis. Among this family the Stress-activated protein kinase (SAPK) or p38 module is of special biological and medical interest, because of its implication in multiple diseases such as cancer and inflammation. MAPK-activated protein kinases (MAPKAPKs) form an important group of targets of p38 signalling, and studies in cells and mice have shown that these MAPKAPKs are essential for the p38 signalling output. In order to get a more detailed insight into the biological role of one of the MAPKAPKs, we generated deletion mutants of the MAPKAPK-2 gene (MK2) in Drosophila melanogaster. MK2-mutant flies are homozygous viable, fertile and show no obvious phenotypes when reared under standard conditions. However, when reared on “stress food” mutant larvae show melanisation in the hindgut. Upregulation of JNK pathway activity and of antimicrobial peptides indicate an activated inflammation/immune response. Using genetics and biochemistry we identified and characterized a stress-activated MAPK pathway that is involved in this phenotype. We propose Drosophila MK2 mutants as a fly model for a chronic inflammation-like process. We are using these mutants to identify new components of this process.

763A Mitochondrial Morphology and Programmed Cell Death in the Drosophila Ovary. Elizabeth Tanner, Todd Blute, Kim McCall. Biology, Boston University, Boston, MA. Programmed cell death in the Drosophila ovary occurs normally as part of egg chamber maturation. Nurse cells provide essential components for development of the embryo by transferring their cytoplasmic contents to the oocyte and then die through a process which is largely caspase independent. Cell death can also occur during mid-oogenesis in response to nutrient deprivation. This form of PCD leads to the degeneration of entire egg chambers and is caspase dependent. Mitochondrial fragmentation occurs in association with programmed cell death in mammals and C. elegans. In Drosophila, recent findings have shown mitochondrial changes during developmental cell death of larval tissues and in S2 cells in response to reaper and hid expression. However, the Drosophila ovary is a unique system in which cell death occurs independently of the main cell death regulators reaper, hid, and grim. To investigate whether mitochondrial fission and fusion events are involved in either developmental nurse cell death or starvation induced cell death, we are analyzing the mitochondrial morphology of wild-type egg chambers as well as cell death mutants. We have found that during mid-oogenesis cell death, distinct changes in mitochondrial morphology occur. These changes are blocked by overexpression of diap-1, indicating that mitochondrial events occur downstream of caspases. Experiments are underway to investigate whether mitochondrial fission events occur in late oogenesis and if germline cell death is affected in mitochondrial fission and fusion mutants. Current progress will be presented. POSTERS: Immune System and Cell Death 347

764B Reaper- and Grim-induced cell death is suppressed by Ras/MAPK signaling in Drosophila developing indirect flight muscles. Hidenobu Tsujimura, Tomohiro Yoneda, Hiroka Aonuma, Hideaki Arai, Shinobu Hirai. Dept Developmental Biol, Tokyo Univ Agric & Technology, Tokyo, Japan. Cell death is one of the important molecular mechanisms for animal development. It works for morphogenesis and regulating organ size in the embryogenesis. In Drosophila, many cell death-related genes have been identified using tissues that consist of undifferentiated cells. But there were few studies using cells on terminal differentiation. It may be possible to elucidate a new mechanism if we study the mechanisms in cells on terminal differentiation. Here we use Actin88F-GAL4 to study cell death, because GAL4 is expressed in the myotubes but not in the undifferentiated myoblasts in this line. We first checked whether the cell death cascade established using undifferentiated cells work in the same way. When pro-apoptotic genes, reaper, hid, or grim, were ectopically expressed using Actin88F-GAL4, muscles failed to develop. We could trace developing flight muscles until middle pupal stages. But, at 3/4 pupal stage, they began to collapse and disappeared completely or mostly by the eclosion. This process is apoptosis controlled by dIAP1 and caspases. Ectopic expression of dIAP1, p35 or dominant negative form Dronc suppressed the breakdown. In this system, we tested the effect of Ras/MAPK signaling on reaper- and grim- induced cell death. Activation of Ras/ MAPK signaling suppressed muscle breakdown in each cell death. We expect a mechanism for suppressing cell death that is common to reaper- and grim- induced cell death.

765C Spiroplasma infection in natural populations of Drosophila species. Thomas Watts, Sergio Castrezana, Therese Markow, Nancy Moran. Ecology & Evolutionary Biol, Univ Arizona, Tucson, AZ. Male-killing and non male-killing endosymbiotic Spiroplasma infections are found to occur in a number of Drosophila species. The incidence of infection in natural populations of these species, however, is unknown. We collected over 50 females and 50 males of each of 12 different species directly from the wild and screened them for infection with Spiroplasma. The highest incidence of infection was found in D. hydei, where approximately 30% of flies carried Spiroplasma. Both sexes had similar infection rates. Drosophila aldrichi had the lowest infection incidence; less than 5% of females and males had the endosymbiont. No species were found in which only females were infected, although half of the species contained no evidence of infection at all. We are currently examining fitness effects of non male killing Spiroplasma in order to determine factors maintaining infection in natural populations.

766A JAK/STAT signaling controls prohemocyte fates in a dose dependent manner in Drosophila larval hematopoiesis. Soichi Tanda, Ying Shen, Catherine Dominguez, William Bell. Dept Biological Sci, Ohio Univ, Athens, OH. The JAK/STAT pathway is important for controlling larval hematopoiesis in Drosophila melanogaster. A dominant mutation of hopscotch (Drosophila JAK), hopTum, produces many hemocytes, although this effect is temperature-dependent. At a low culture temperature such as 17oC, larvae with this allele produce 30 times as many hemocytes as wild type. However, hopTum larvae raised at a high temperature such as 28oC produced hemocytes to a level similar to those of wild type larvae, but the majority of hemocytes were lamellocytes. This is quite different from what we observed in larvae raised at a low temperature. These larvae mostly produced prohemocytes. Since it is known that the hopTum protein becomes more active at a higher temperature, these observations imply that higher JAK/STAT activity does not necessarily stimulate prohemocyte proliferation. To better understand how JAK/STAT signaling controls hematopoiesis, we examined whether the hemocyte population changes by increasing JAK/STAT activity using a UAS- hopTum transgene in the hopTum mutant background. Additional JAK/STAT activity provided by a UAS-hopTum transgene at 22oC suppressed hemocyte proliferation, but stimulated lamellocyte differentiation. These observations strongly suggest that prohemocytes change their fates depending on a level of JAK/STAT signaling. We also found that Toll signaling enhances the effect of the hopTum allele. The hopTum; Toll10B double mutant larvae showed a reduction in the total hemocyte number, but an increase in the lamellocyte population. The effect of the Toll10B allele on hopTum is likely mediated by Dorsal-responsive genes. These observations were further supported by immunohistochemical and molecular methods. This study indicates that JAK/STAT signaling is a key pathway to maintain the stem cell state of prohemocytes, and that its activity is also modulated by Toll signaling. 348 POSTERS: Techniques and Genomics

767B Genome-Wide Mapping of Chromosomal Proteins in Drosophila. Akiko Minoda1, Art Alekseyenko2, Nicole Riddle3, Yuri Schwartz4, Cameron Kennedy1, Sarah Elgin3, Peter Kharchenko5, Mitzi Kuroda2, Peter Park5, Vincenzo Pirrotta4, Gary Karpen1. 1) Dept. of Genome Biology, Lawrence Berkeley National Lab, Berkeley, CA; 2) Dept. of Genetics, Harvard-Partners Center for Genetics & Genomics, Boston, MA; 3) Department of Biology, Washington University, St. Louis, MO; 4) Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ; 5) HPCGG, Harvard Medical School, Boston, MA. The packaging of DNA into chromatin and chromosomes has major implications for understanding how genome sequences function in eukaryotic cells. We will be determining the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We are performing Chromatin ImmunoPrecipitation (ChIP) with antibodies obtained commercially and generated and validated by this project, isolate and label the precipitated DNA, and apply the probes to genomic tiling arrays. Data generated by scanning the hybridized arrays are analyzed by statistical methods, and the array data are validated by independent analyses in cells and animals. We are initially assaying localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. We will perform a variety of bioinformatic comparisons between protein “landscape” data sets, including analyses of combinatorial patterns of modifications and chromosomal proteins, tissue-specific differences, and interactions among proteins involved in the same epigenetic pathways. Successful completion of this project will provide basic information about the distributions of chromatin components across the Drosophila genome, which will serve as a foundation for future functional studies. The data and analysis are very likely to provide information critical to understanding the roles of chromatin packaging in human cells, and the mispackaging that can be associated with disease.

768C Adaptive image segmentation methods applied to the analysis of Snail repressor dosage impact on nascent vnd, sog, rho and brk transcription. William Beaver1, Adam Paré2, David Kosman2, Ethan Bier2, William McGinnis2, Yoav Freund1. 1) Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA; 2) Cell and Developmental Biology Department, University of California, San Diego, La Jolla, CA. Despite our ability to acquire image stacks containing activity patterns for multiple transcription units in individual nuclei, transforming such image stacks into quantifiable entities is far from commonplace. We have developed adaptive segmentation methods for high resolution microscopy that can be tuned using machine learning methods. They allow experimentalists to teach programs to automatically classify objects in images, for example: “these 100 objects are nuclei” or “these objects are active transcription units”, and relate such datasets to each other. This is in contrast to the arduous task of tuning multiple computer vision parameters in a trial and error fashion, attempting to optimize object recognition. We apply these adaptive segmentation methods to the quantization of nascent transcripts from the vnd, sog, rho, and brk genes, all of which are repressed in ventral regions of the Drosophila embryo by the Snail transcriptional repressor. Transcription from these genes is quite distinct when Sna repressor dosage is altered, and these dose dependent behaviors correlate with the affinities of Snail protein for regulatory regions of these four target genes.

769A Mining embryonic expression images reveals novel developmental pathway components. Erwin Frise, Amy Beaton, Richard Weiszmann, Ann Hammonds, Susan E. Celniker. Berkeley Drosophila Genome Project, Lawrence Berkeley Nat Lab, Berkeley, CA. Analyzing both temporal and spatial gene expression is essential for understanding the developmental and regulatory networks of multicellular organisms. We have hybridized Drosophila mRNAs to embryos and collected over 80,000 2D images for over 6,000 genes. We developed a novel computational image processing approach to explore the relationships between gene expression patterns by converting expression patterns from the images into virtual representations. Using an automated pipeline we segmented the image files such that the primary embryo was separated from nearby or touching embryos, using texture segmentation, morphological image processing and refinement by active contours. Each embryo was aligned to an elliptical mesh comprised of 311 small triangular regions each defining a unique location within the embryo. We created a score to identify similar patterns by comparing corresponding triangles. To identify subtle expression pattern overlaps, we developed a novel probabilistic Markov Random Field (MRF) based algorithm. The MRF can detect shared expression boundaries, generate similarity measurements and is able to discriminate even faint/uncertain patterns, if they appear in both embryos. We conducted a comprehensive analysis of the expression landscape. We filtered the expression patterns and automatically compacted and temporally sorted multiple images in each stage range using a novel algorithm of clustering and graph theoretic approaches. At stage 4-6 we reduced the initial set of about 5,800 patterns to 553 representative ones containing 364 genes. Temporal sorting showed 99 genes having highly dynamic patterns. To further organize the patterns we generated 39 clusters that contain known and previously unidentified genes using affinity propagation clustering and dissected cluster members for similarities and differentiations with the MRF algorithm. For example, using the cluster that contains sna, we identified numerous genes with known involvement in mesoderm development, the previously known hkb which restricts sna expression, and zfh1, which interacts with tin. POSTERS: Techniques and Genomics 349

770B Drosophila genome resources at NCBI. Terence Murphy, Pavel Bolotov, Stacy Ciufo, Karen Clark, Wratko Hlavina, Yuri Kapustin, Boris Kiryutin, Michael Ovetsky, Sergey Resenchuk, Sasha Souvorov, Igor Tolstoy, Paul Kitts, Donna Maglott, Ilene Mizrachi, Tatiana Tatusova, Kim Pruitt. NCBI/NLM/NIH/DHHS, Bethesda, MD. The National Center for Biotechnology Information (NCBI) integrates map and genome data from multiple sources and makes it available to the scientific public as interactive web resources, through programming utilities, and for FTP. Genomic data is available from several resources including Entrez Gene, the reference sequence (RefSeq) collection, the Map Viewer genome browser, and the Genome Projects database. Additional resources include organism-specific BLAST pages, pre-computed BLASTp results (Blink), computed homologous clusters (HomoloGene), and organism-specific web pages. These resources are extensively cross-linked to facilitate navigation across a broad spectrum of biological information and to provide access to model organism databases such as FlyBase. These resources are available for several insects, including Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Nasonia vitripennis, and Tribolium castaneum. NCBI collaborates with FlyBase to represent 1) Drosophila loci in Entrez Gene, and 2) the genome assembly, transcripts, and proteins as RefSeq accessions. This supports the insect community by integrating FlyBase curated data into NCBI genome-oriented resources and providing convenient access to unique NCBI tools. NCBI supports FlyBase annotation and curation efforts by providing transcript and protein alignments, and gene models calculated by NCBI’s Gnomon gene prediction tool were used to aid annotation of the 12 sequenced Drosophila species. Further information is available at the NCBI web site (http://www.ncbi.nlm.nih.gov/), or the Drosophila Genome Resources page (http://www.ncbi.nlm.nih.gov/projects/genome/guide/fly/). Questions and suggestions for additional resources are welcome and can be directed to the “Drosophila Genome Champion” ([email protected]).

771C QBLAST; a so far missing but indispensable on-line tool. Erwin Seinen, Ody CM Sibon. SSCB, UMCG, Groningen, Groningen, Netherlands. BLAST is a well known on-line application to find and align sequences against genomic resources. Although BLAST is a powerful tool, one should realize that the on-line BLAST version is not appropriate for short alignments due to its low sensitivity. However, new discoveries and applications such as RNA interference technology do require the ability to make short alignments against the genome. In case the on-line version of BLAST is used to design siRNA constructs, numerous off-targets may be missed, resulting in non-specific or false-positive results. Alternative applications with a higher accuracy do exist, however these tools require specialized bioinformatics, high demanding hardware and tailored applications to analyze the output. We now present QBLAST (Quick Basic Local Alignment Search Tool), a new user friendly and on-line available alignment tool with 100% accuracy, which overcomes the mentioned limitations of classic BLAST. QBLAST takes any sequence up to 23 nucleotides and aligns this sequence against the whole genome with a user defined number of mismatches within milliseconds. The output shows a detailed presentation of alignments in a way that heuristically based search tools so far are unable to accomplish. QBLAST has the additional ability to search for consensus sequences to search for possible response elements or other protein-DNA interaction hot-spots. The output of these searches are presented in an interactive and intuitive genome browser which reveals possible patterns on a genome-wide level by simple visual inspection. Using QBLAST, we have analyzed 1.5 million sequences of 21 bp originating from Drosophila melanogaster exonal gene sequences. All of these sequences show many alignments against the genome that remained undetectable when a classical BLAST search was used.

772A FlyExpress: Computational Biology and Bioinformatics for spatial gene expression patterns from Drosophila embryogenesis. Bernard Van Emden1, Christopher Busick1, Hector Ramos1, Kailah Davis1, Sethuraman Panchanathan1,2, Stuart J. Newfeld1,3, Sudhir Kumar1,3. 1) Biodesign Inst, Arizona State Univ, Tempe, AZ; 2) School of Computing and Informatics, Arizona State Univ, Tempe, AZ.C; 3) School of Life Sciences, Arizona State Univ, Tempe, AZ. A vast collection of spatial patterns of expression, from a large number of genes, is now available through the efforts of individual and high throughput laboratories. These data are the key to the discovery of previously unknown links and components of developmental networks. In order to accelerate such discovery, we have constructed the largest digital library of standardized fruit fly embryonic expression images and developed novel computational biology methods and bioinformatics tools (www.flyexpress.net). The standardization and alignment of expression patterns is required for meaningful comparison and synthesis of developmental patterns at a genomic scale. This large-scale image standardization capability facilitates the building of Gene-Expression-Maps (GEMs) that capture the spatial distribution of all genes in a given stages and views. We find that GEMs show exceptional correspondence with developmental fate maps. FlyExpress also makes available a pattern-matching discovery tool that generates a list of genes with varying degrees of spatial expression overlap to a query expression by comparing image contents. FlyExpress now connects this gene list (or a selected subset) for further analysis in the Query Builder (FlyBase) and Lists feature of FlyMine. This would allow users to investigate enrichment of the genes identified in terms of their chromosomal distribution, adult expression, and gene ontology. We now invite investigators with large number of expression patterns to submit their data for standardization and inclusion in the FlyExpress resource (e-mail: [email protected]). 350 POSTERS: Techniques and Genomics

773B Automatically determining the developmental stage of embryos captured in the Gene Expression Pattern Images. Jieping Ye 1,3, Jianhui Chen1,3, Sudhir Kumar2,3. 1) Department of Computer Science and Engineering, Arizona State University, Tempe, AZ; 2) School of Life Sciences, Arizona State University, Tempe, AZ; 3) Center for Evolutionary Functional Genomics, The Biodesign Institute, Arizona State University, Tempe, AZ. Knowledge of the developmental stage of the embryo is a prerequisite for large-scale computational analysis of image expression data, because the determination of the extent of pattern overlap is the most biologically meaningful for images that contain spatial patterns from the same stage of development. Annotation of the stage of the embryo contained in the image is currently done manually, which becomes time-consuming and prone to error when collecting data from high-throughput experiments producing tens of thousands of images. Therefore, to automate this task, we are developing computational methods based on a novel formulation of Linear Discriminant Analysis (LDA). Our method employs a multivariate linear regression with the L1-norm penalty controlled by a regularization parameter for feature extraction and visualization. It computes an entire solution path for all values of regularization parameters with essentially the same computational cost as fitting one linear discriminant model, which facilitates efficient model selection. Our method is based upon the equivalence relationship between LDA and the least squares method for multi-class classifications, with the equivalence relationship established under a mild condition. We show that the equivalence relationships hold for many high-dimensional data, such as expression pattern images. Our experiments on a collection of 2705 BDGP expression patterns have shown the effectiveness of the proposed algorithm. When about 10% of the data is used for training, the proposed system achieves about 87% accuracy in recognition. We found that part of the misclassification was due to mislabeling during manual image annotation. We are now developing methods that can help assign images to individual stages, rather than stage ranges (e.g., 4-6) to facilitate more precise analyses of existing data.

774C Identification of novel transcription initiation and polyadenylation sites in the Hdc gene. Daniel E. Boozer1, Sara Smolinski2, Martin G. Burg1,2. 1) Cell and Molecular Biology Program, Grand Valley State University, Allendale, MI; 2) Department of Biomedical Sciences, Grand Valley State University, Allendale, MI. Histamine is a biogenic amine that is used as a neurotransmitter in a variety of cell types in Drosophila melanogaster. Histidine decarboxylase (HDC) is the enzyme that synthesizes histamine, using histidine as a substrate. Mutants in the Hdc gene have been previously identified that have no detectable levels of histamine (Burg et.al., 1993). Further analysis of Hdc expression using Hdc transgenes in this mutant background has indicated that elimination of a genomic fragment 5’ to the coding region, disrupts expression of Hdc specifically in the centrally located neurons, but not photoreceptors (Burg and Pak, 1995). This suggested that either the region 5’ to the Hdc transcription unit functioned as a transcriptional enhancer element or a transcriptional promoter element for Hdc. To help elucidate the manner by which this cell-specific regulation occurs, 5’ and 3’ RACE was performed on the Hdc transcript. Sequence analysis of cDNA ends obtained through 5’ and 3’ RACE indicates alternative 5’ and 3’ UTRs for the Hdc transcription unit. One unique alternative 5’ UTR maps to the region earlier identified to be required for central brain Hdc expression, while a second 5’ UTR extends the currently identified 5’ UTR another 70 bp. RT-PCR was conducted, using the new cDNA sequence, to identify alternative Hdc transcripts. This analysis has revealed 2 additional splicing sites in the Hdc transcription unit. These alternative mRNA’s do not change the coding region for HDC, but could be involved in the regulation of tissue-specific expression. Analysis of the relative levels of Hdc transcripts by Q-PCR will provide further insights into Hdc regulation. Burg, MG et. al. (1993) EMBO J. 12(3): 911-919. Burg, MG and Pak, WL. (1995) Invest. Ophthal. and Vis. Sci. 36(4): 1979.

775A Genome-Wide Expression-Based Lineage Analysis. John M. Olson1, Cory J. Evans1, Eunha Kim1, Kathy Ngo1, Noemi E. Lee1, Edward Kuoy1, Alexander N Patananan1, Daniel Sitz1, PhuongThao Tran1, Minh-Tu Do1, Kevin Yackle1, Albert Cespedes1, Gerald B. Call2, The UCLA URCFG1, Volker Hartenstein1, Utpal Banerjee1. 1) Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA; 2) Midwestern University, Glendale, AZ. The UCLA Undergraduate Research Consortium for Functional Genomics has undertaken the development of a comprehensive genome based analysis of lineage in several tissues of Drosophila. We have created a Drosophila chromosome that has the following genotype: UAS-FLP, UAS-RFP, Ubiquitin promoter-FRT-STOP CASSETTE-FRT-GFP. Students assigned various Gal4 lines from the NP consortium GET DB project, cross into the above background as follows:Random enhancer-Gal4 X UAS-FLP, UAS-RFP, ubi- FRT-STOP-FRT-GFP. In the progeny, the Gal4 is expressed in a pattern that is characteristic of the enhancer. This causes expression of Flp in these cells, excision of the stop cassette and permanent marking of these cells and all their progeny with GFP under the control of the ubiquitously expressed promoter. additionally, the RFP reporter continues to monitor the expression pattern due to the enhancer in real time. By comparing the RFP and GFP patterns the students are providing a comprehensive gene-expression based lineage map for the genes in the developing brain, blood, and imaginal discs. POSTERS: Techniques and Genomics 351

776B An improved Tet-On expression system. Carlos Flores, Jerry C-P Yin, William R Engels. Dept Genetics, Univ Wisconsin, Madison, WI. We are developing an improved system for chemically-induced expression based on the “Tet-On” system. By altering both the Tet- responsive promoter and the transactivator components, the improvements should provide very low background and high-level inducibility. We are combining individual improvements that have been previously published including optimized spacing between the Tet-responsive elements in the promoter and amino acid changes in the “rtTA” transactivator that enhance sensitivity to the tetracycline analog doxycycline. The effect of a strong nuclear localization signal attached to the transactivator is also being analyzed. Currently induction of gene expression by heat-shock is the most widely used method in flies. Unfortunately heat-shock alters the expression of many genes and has broad physiological consequences that are detrimental to many experimental procedures. We will use the improved Tet-On system to drive expression of I-SceI endonuclease, which will create the first heat-shock independent, inducible DNA break generating system in Drosophila. The improved Tet-On system is being tested in Schneider cells and transgenic Drosophila, and is likely to be useful more widely in other model organisms.

777C Optimizing Ends-Out Gene Targeting In Drosophila. Yang Hong, Juan Huang, Wenke Zhou, Annie Watson. Cell Biology and Physiology, Univ of Pittsburgh Medical School, Pittsburgh, PA. The recently developed ends-out (replacement type) gene targeting in Drosophila provides an essential tool for directed modifications of a chosen target gene. However, a significant obstacle in applying the current ends-out targeting routine is its low efficiency that results in a steep requirement of time and labor. Here we developed several measures that significantly improve the efficiency of ends-out targeting. We first synthesized new vectors that facilitate the molecular cloning of targeting constructs. We also constructed new targeting fly stocks that eliminate a major labor-intensive step in targeting, making it possible to set up targeting experiments at much larger scales. Finally, we introduced a conditionally-expressed cell death gene Reaper (“UAS-Rpr”) as a negative selection marker into the targeting routine. By selecting against UAS-Rpr, the number of non-specific targeting candidates can be reduced by nearly thirty-fold, drastically increasing the throughput of actual targeting experiments. Together these new reagents make ends-out targeting in Drosophila a much more optimized and efficient tool for generating both knock-out and knock-in alleles.

778A Development of a “Split GeneSwitch” System for Refined Spatial and Temporal Control of Transgene Expression in Drosophila. Haojiang Luan, Andrew Vreede, Benjamin White. Lab Molecular Biol, NIMH, Bethesda, MD. Targeting of gene expression in Drosophila is commonly accomplished using binary systems, for example the non-inducible Gal4- UAS and the RU486 inducible GeneSwitch-UAS system. Although these techniques have proven enormously powerful, under many circumstances it would useful to be able to further restrict expression patterns to selected subsets of cells. We recently introduced a combinatorial method (Split Gal4) for restricting transgene expression to subsets of cells within the expression pattern of a given promoter (Luan et al. Neuron 2006; 52: 425-36). While this method permits the spatial refinement of gene targeting it does not offer concomitant temporal control. To develop a system that would permit both conditional and spatially-refined targeting we have experimented with several modifications of our original Split Gal4 system. We describe here the development of a new “split” system which uses, instead of Gal4, the inducible GeneSwitch transcription factor. GeneSwitch is compatible with existing UAS lines in Drosophila, but unlike Gal4 can be regulated by the chemical inducer RU486. Our Split GeneSwitch system takes advantage of the protein trans-splicing ability of a hybrid split intein. By fusing N-intein and C- intein moieties to the inert halves of an artificially split GeneSwitch molecule, we have been able to demonstrate the reconstitution of GeneSwitch function in cells expressing both constructs. In transfected SL-2 cells, we find that co-expression of the Split GeneSwitch constructs promotes expression of an EGFP reporter gene at levels close to intact GeneSwitch only in the presence of RU486. We are currently testing transgenic flies that express each of the two split GeneSwitch components in overlapping patterns to determine the efficacy of this system in vivo. 352 POSTERS: Techniques and Genomics

779B A Temperature-Sensitive Protein Switch. Guihong Tan, Ming Chen, Change Tan. Department of Biological Sciences, Bond Life Sciences Center, University of Missouri-Columbia, U.S.A. A special vector has been constructed that allows turning on or off fly genes by a temperature shift. This vector contains the neomycin phosphotransferase (neo) gene without termination signal for in-gene insertion selection, mCherry(RFP) gene for in- intron and in-frame insertion isolation, a temperature sensitive intein to switch on or off gene by temperature shift and the mini- white(w+) gene as a transgenic marker. We used p-element transposon to obtain initial transgenic flies, and piggyBac tansposon to make RFP, neo and intein cassette jumping. Flp/FRT system is used to delete the RFP and neo cassette to get the final temperature sensitive switch, so that the genes were turned off at high temperature and on at low temperature. We used cre/loxP system to delete the whole cassette and recovery the genes. Furthermore, we will use this switch to perform a genome-wide screen in Drosophila melanogaster.

780C ProStack, the image analysis software to process and quantify patterns of gene expression in the Drosophila blastoderm. Konstantin N. Kozlov1, Pisarev Andrei1, Samsonova Maria1, Reinitz John2. 1) St.Petersburg State Polytechnical University, St.Petersburg, Russian Federation; 2) Stony Brook University, Stony Brook, USA. Information on where and when a gene is expressed is important to understand its function. Modern sophisticated microscope techniques allow to monitor gene expression in real time, with a single cell resolution, and can easily produce thousands of images in a single day. However a bottleneck exists at the step of image analysis. Due to large number and frequent need for extraction of fine details any visual analysis of images is impractical. Image analysis packages available today are either expensive commercial or do not support an automatic analysis of images and require strong programming skills to adapt programs to a new project. We present a new software package ProStack developed to process and quantify patterns of gene expression in the Drosophila blastoderm. ProStack is capable to process 2D and 3D digital images of gene expression patterns acquired with confocal microscope. It implements more than 50 operations that include both domain specific and domain-independent methods. Each image processing procedure is implemented as a separate module. Several modules can be joined in a complex image processing scenario, and the intermediate results can be visualized during the workflow enactment. The processing operations afford tuning to ensure customization and flexibility without the loss of efficiency. The designed workflow can be saved as a complex program module and re-used in other workflows. The Prostack package was successfully applied to correctly orient, rotate and crop the embryo images, identify and count objects, remove background and extract quantitative information on gene expression from nuclei and cytoplasm.

781A Multiphoton Investigation of Myosin-Based Drosophila Muscle Degeneration Induced by Proteasome Inhibition. Chiao- Ying Lin1, Chen-Yuan Dong2, June-Tai Wu3, Chii-Wann Lin1,5, Jyh-Horng Chen1, Sung-Jan Lin4,5. 1) Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan; 2) Department of Physics, National Taiwan University, Taipei, Taiwan; 3) Department of Medical Research, National Taiwan University Hospital , Taiwan; 4) Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan; 5) Institute of Biomedical Engineering, College of Engineering and College of Medicine, National Taiwan University, Taipei, Taiwan. Multiphoton laser scanning microscopy is a powerful tool for investigating living specimens in three-dimensions. Several biologically important protein structures, such as collagen and well organized myoglobulins in muscles, give rise to strong second-harmonic generation (SHG) signals in their native form. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information. In this study, we utilize the advantages of multiphoton fluorescence and SHG laser scanning microscopy to image and study muscle degeneration in Drosophila. In this study, we built a chamber that integrates larvae anesthesia capabilities into a multiphoton imaging system to facilitate the in-vivo imaging of Drosophila larvae, To induce muscle degeneration in third instar lavae, a temperature sensitive dominant negative Pros26 allele, DTS5 is over expressed in muscles to conditionally inhibit proteasomal activities by shifting to restrictive temperature 29?. After 12 hours of proteasome inhibition, there is a marked reduction of SHG in the motionless but otherwise living larvae. The reduction of SHG in the larvae correlates well to the gross disorganization of muscle architecture and prominent muscle atrophy observed in the fixed specimens. Our data indicate that the minimally invasive imaging technique of SHG microscopy is a sensitive tool to study muscle degeneration in living specimens. In addition, the high sensitivity and minimally invasive nature of this technique renders SHG to be a promising imaging modality for diagnosing muscle degeneration in patients. POSTERS: Techniques and Genomics 353

782B A novel epitope tag for purification of macromolecular complexes from invertebrates. Matthias Kroiss1, Katharina Apfel1, Julia Wiesner2, Matthias Grimmler1, Albert Sickmann2, Utz Fischer1. 1) Department of Biochemistry, Theodor-Boveri-Institute, Würzburg, Germany; 2) Rudolf-Virchow-Center, Würzburg, Germany. Genetic studies in Drosophila melanogaster have been a fundament of cell and developmental biology for one century. However, functional analyses of gene products by biochemical means have remained scarce for this model organism. One major obstacle has been the lack of an efficient purification strategy for macromolecular complexes. Here, we present the novel TagIt-epitope specifically designed for Drosophila and other invertebrates. By applying this tag in conjunction with a monoclonal antibody in stably transfected Schneider2 cells, we demonstrate efficient purification of target proteins and their complex partners under native conditions. As a proof of principle, we chose to investigate one processive enzyme, a ribonucleoprotein particle (RNP) and an RNP chaperone. First, we have analyzed the protein composition of isolated complexes by using protein mass spectrometry. Second, functional assays have been performed to validate their biochemical activity in vitro. In addition, we demonstrate that RNAs are co-purified in a specific manner together with their protein binding partners. Taken together, we provide the Drosophila community with a powerful tool to perform purification of macromolecular complexes under native conditions with high yield and purity. The TagIt-technique will boost biochemical investigations in this and other invertebrate model organisms.

783C New Drosophila GS-TAP vectors for protein complex purification and proteome exploration. Alexey Veraksa, Phillip Kyriakakis, Marla Tipping, Louka Abed. Department of Biology, University of Massachusetts Boston, Boston, MA. We created a set of vectors for expressing tagged proteins in Drosophila using a recently developed GS-TAP cassette. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by the TEV protease cleavage site. pMK33-based GS-TAP vectors allow for generation of stable cell lines using hygromycin selection and inducible expression from metallothionein promoter, while pUAST-based vectors can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done side-by-side purifications of proteins tagged with the new GS-TAP tag or with conventional TAP tag (containing Protein A and calmodulin binding peptide tags) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. Overall, the new GS-TAP vectors offer an improvement in current protein complex purification approaches by providing a superior signal-to-noise ratio in purified material.

784A Expanding the coverage and versatility of the Gene Disruption Project collection using Minos mutators with swappable cassettes. Robert W. Levis1, Koen J.T. Venken2,3, Yuchun He2,3, Joseph W. Carlson4, Martha Evans-Holm4, Karen L. Schulze2,3, Roger A. Hoskins4, Allan C. Spradling1,3, Hugo J. Bellen2,3. 1) Dept Embryology, Carnegie Inst of Washington, Baltimore, MD; 2) Dept Molecular and Human Genetics, Dept Neuroscience, Baylor College of Medicine, Houston, TX; 3) Howard Hughes Medical Institute; 4) Dept Genome & Computational Biology, Lawrence Berkeley National Laboratory, Berkeley, CA. The Gene Disruption Project (GDP) is using a non-targeted transposon mutagenesis strategy to create a collection of insertion mutants (see Bellen et al (2004) Genetics 167: 761-781). The collection can be searched online (http://flypush.imgen.bcm.tmc.edu/ pscreen/) and new mutants may be requested while they are being balanced and rechecked. Balanced stocks of the mutants are available from the Bloomington Stock Center (BSC). Previous screens with P-element and piggyBac mutators have generated insertions in over half of the known genes. To mutate the remaining genes, we have switched to a Minos mutator. We found that Minos generates five times more new gene hits than P elements. As of November 1, 2007, we have generated > 7,500 mutants in the ongoing Minos MB screen, using an enhancer-trap vector, and have sent 1,478 of these to the BSC. In order to enhance the versatility of insertion mutants, we have engineered a new Minos mutator that incorporates two inverted φC31 attP target sites flanking a standard gene trap cassette. The attP sites allow the DNA located between them to be exchanged heritably in germ cells at the site of integration with any DNA cassette flanked by attB sites that is microinjected into embryos, a process known as Recombination Mediated Cassette Exchange (RMCE). For any insertion allele with a swappable cassette, users may use RMCE to rapidly and efficiently derive additional alleles with a broad array of customized properties. 354 POSTERS: Techniques and Genomics

785B Generation of FRT lines to enable germ line clonal analysis of pre-existing FRT collections. Ernesto Lujan1, Douglas Bornemann1, Carmen Rottig2, Ernst Hafen2, Rahul Warrior1. 1) Dept of Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Zoologisches Institut, Universitat Zurich, SWITZERLAND. The FLP/FRT system permits rapid screening and analysis of lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD-bearing dominant female-sterile transgenes enables efficient screening in germline clone (GLC) embryos as well. Two sets of FRT lines are in common use. One set, frequently used for somatic screens, lacks matching ovoD chromosomes for arms 2R (FRT42D) and 3L (FRT80B). Completing this set enables re-use of pre-existing FRT-mutant collections for comprehensive maternal-effect screens. Because meiotic recombination in Drosophila is female-specific and ovoD causes female sterility, we produced the 2R and 3L FRT-ovoD lines by inducing recombination in males. To demonstrate functionality, we created GLC mutant embryos for alleles of brother of tout velu (botv) and sugarless (sgl) with known GLC phenotypes. These embryos displayed the expected phenotypes. GLCs generated for additional uncharacterized botv alleles identified in an FRT eye screen demonstrate the utility of the FRT-ovoD lines.

786C Innovations in a one-pot system of genome walking and mutational mapping. Kyl Myrick, William Gelbart. Dept Molecular & Cellular Biology, Harvard University, Cambridge, MA. We developed an exceptionally fast molecular screen, based on the coordinated actions of thermophilic/mesophilic polymerases and specialized exonuclease, for expediting the determination of novel mutations and genomic structures [Myrick, K.V. and Gelbart, W.M. Nature Protocols 2, 1556-1563 (2007)]. Our process — which in this laboratory has superseded the traditional methods for these purposes — utilizes controlled strand conversion, polymerase-mediated “ends-jumping” and looping that repositions the target interval so as to lie within a known origin segment, thus permitting rapid, definitive acquisition of new coding sequence, transposon boundaries and genome gap information. Per-reaction walk-lengths are conceivably unbounded in design. Most recently, specific properties of polymerases, relating to the enzymes’ behavior with partly abasic oligonucleotide analogs and graduated melting, have been exploited in this system, resulting in blockade of undersized intermediates, reduced reliance on heterologous exonuclease in the reaction mechanism and hence further robustness and streamlining. Various schemes (“single-tube transposon mapping”, L-UFW, UFW) based on the core reaction sequence have now been successfully deployed by ourselves and others in a multitude of contexts, such as long-range direct genome walking, mutational element discovery, deletion analysis, mRNA ends retrieval, TE distribution studies, DNA fingerprinting, as well as dependable “one-pot”, half-day recovery of flanks from this laboratory’s mutation collections, including the P{wHy} compound elements.

787A Systematic characterization of Drosophila transcription factor specificities via a bacterial one-hybrid system. Michael Brodsky1, Marcus Noyes1, Xiangdong Meng1, Atsuya Wakabayashi1, Saurabh Sinha2, Scot Wolfe1. 1) Program in Gene Function & Expression, University of Massachusetts Medical School, Worcester, MA; 2) Department of Computer Science, University of Illinois at Urbana-Champaign, Urbana, IL. A combination of experimental and computational approaches is required to understand how specific genomic sequences regulate spatial and temporal patterns of transcription. Chromatin immunoprecipitation (ChIP) data can be used to identify genomic sites occupied by transcription factors (TFs) in vivo, but cannot be used for all possible cell types and may not distinguish between functional and “neutral” binding sites. In a complementary approach, DNA binding specificity data for TFs can be used to computationally identify evolutionarily-conserved binding sites for one or more TFs,. The primary limitation for computational methods is the paucity of DNA-binding specificity data for most TFs. We describe a modified bacterial one-hybrid system that provides a rapid method for characterizing TF specificities. Using this system, we have characterized over 100 Drosophila factors, approximately 14% of all predicted TFs in the genome. These factors include examples of all of the major classes of DNA-binding domains. Our specificity data matches and often improves existing DNase I or SELEX data, when available, and can be used to accurately predict known target genes and cis-regulatory modules for many TFs. To maximize the utility of this dataset for the Drosophila community, we have developed web-based tools to identify cis-regulatory modules throughout the fly genome using TF specificities coupled with phylogenetic comparisons. We have implemented a new computational strategy that allows users to rapidly examine individual genomic regions (using Gbrowse) or perform genome-wide searches for DNA segments with a statistical overrepresentation of binding sites for any combination of factors. Our specificity dataset coupled with these search tools provides a powerful resource for the identification of cis-regulatory modules within fly genomes. POSTERS: Techniques and Genomics 355

788B Incorporation of piggyBac-based insertional mutagenesis and GAL4 system vectors in ten species of Drosophila with sequenced genomes. Stacy Holtzman1, David F.B. Miller1, Teruyuki Niimi2, Thomas C. Kaufman1. 1) Biology Department, Indiana University, Bloomington, IN; 2) Lab of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan. The sequencing of the genomes of 12 Drosophila species has created an opportunity for much in the way of comparative molecular analyses amongst these species. To aid that endeavor, we have made several transformation vectors based on the piggyBac transposon that will be useful for mutagenesis and establishing the GAL4-UAS system in these species. We have tested the piggyBac- based insertional mutagenesis system in ten species including D. melanogaster, D. simulans, D. sechellia, D. erecta, D. yakuba, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, and D. virilis. The mutagenesis system is composed of a source of piggyBac transposase (the “mobilizer” component), and either a binary 3XP3-Ds-Red/hsp70-EGFP enhancer trap or a binary w+mC/ 3XP3-EGFP marker as “mutator” components. Additionally, we have tested the ability of D. melanogaster Ubiquitin, nanos and β2- tubulin enhancer elements to drive expression of GAL4 in D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni and D. virilis. The efficacy of the enhancer::GAL4 transgenes was determined by measuring the response of UASP::Ds-Red and UASP::α- Tubulin::GFP transgenes in all five species, and a UAS::Centrosomin-RNAi transgene in D. melanogaster only. To eliminate an absolute requirement for epiflourescence microscopy, the D. melanogaster mini-white gene has been incorporated into almost all of the transformation vectors and characterized in several species based on eye color. Our addition of white-eyed D. erecta and D. yakuba strains to the extant w- strains makes it possible to use white rescue in all the sequenced Drosophila species except D. persimilis and D. grimshawi. In summary, we hope to provide a basic toolkit for both insertional mutagenesis and GAL4/UAS system- based experiments in multiple species of Drosophila.

789C Genetic information in FlyBase: what goes in must come out. Peter McQuilton, The FlyBase Consortium. FlyBase-Cambridge, Dept Genetics, University of Cambridge, Cambridge, United Kingdom. FlyBase (www.flybase.org) is the central database for information concerning the genes and genomes of Drosophila species, with a particular focus on Drosophila melanogaster. The FlyBase project involves a consortium of curators and computer scientists based at three distinct sites: Harvard University (USA), Indiana University (USA), and the University of Cambridge (UK). At FlyBase, we curate genetic data from a variety of sources, including primary research papers, reviews, conference abstracts and personal communications. We collate these data using controlled vocabulary terms. To do this, we employ numerous controlled vocabularies, including Gene Ontology (GO), Sequence Ontology (SO), and our own anatomy, and developmental stage controlled vocabularies. These terms capture information at the gene and allele level, detailing molecular function, biological process, morphology changes, phenotypic class, allele type, and molecular data, among other characteristics. This allows the grouping and display of genetic and functional data in a concise and easily understood manner on our website. This also enables the use of complex search tools, such as QueryBuilder and TermLink, to mine the data and combine queries across datasets. Here, we detail the process of curation and how this has implications on the display and querying of genetic data in FlyBase.

790A Expression profiling by massively parallel sequencing. Christian Schloetterer1, Tatiana Torres1, Muralidhar Metta1, Birgit Ottenwaelder2. 1) Inst Tierzucht, VMU Wien, Wien, Austria; 2) Eurofins, Medigenomics GmbH, Martinsried, Germany. Massive parallel sequencing holds great promise for expression profiling, as it combines the high throughput of SAGE with the accuracy of EST sequencing. Nevertheless, until now only very limited information is available on the suitability of the current technology to meet the requirements. Here, we evaluate the potential of the 454 sequencing technology for expression profiling. We show that short (<~80bp) and long (<~300-400bp) cDNA fragments are under-represented in 454 sequence reads. Nevertheless, sequencing of 3’ cDNA fragments generated by nebulization could be used to overcome the length bias of the 454 sequencing technology. Gene expression measurements generated by restriction analysis and nebulization were highly correlated (r>0.83). Similar correlations were reported for replicated microarray experiments. 97% of the cDNA fragments could be unambiguously mapped to the genomic DNA demonstrating the advantage of longer sequence reads. Our analyses suggest that the 454 technology has a large potential for expression profiling and the high mapping accuracy indicates that it should be possible to compare expression profiles across species. 356 POSTERS: Techniques and Genomics

791B Efficient generation of continuous cell lines from Drosophila embryos by expression of oncogenic Ras. Amanda Simcox, Ting Chen, Jon Buthchar, Steven Justiniano. Dept Molecular Genetics, Ohio State Univ, Columbus, OH. Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. We found expression of the conserved oncogene, RasV12 (a constitutively activated form of Ras), profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was silenced. We successfully created several cell lines and found these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines. This offers an opportunity to create cell lines of specific genotypes and potentially of a given cell type.

792C Drosophila melanogaster: a GO reference genome. Susan Tweedie, The FlyBase Consortium and The Gene Ontology Consortium. Department of Genetics, University of Cambridge, Cambridge, United Kingdom. FlyBase uses gene ontology (GO) terms to describe the molecular function of gene products, the processes they play a role in and where they are located within a cell. One aspect of our GO annotation work is participation in ‘The Reference Genome Annotation Project’ organised by the Gene Ontology Consortium. The aim of this project is to provide comprehensive high quality GO annotation based on experimental evidence for the human genome and a set of 11 well-characterised model organisms including Drosophila melanogaster. In the process, we aim to improve the quality of our existing GO term assignments, for instance by removing terms that are predictions based upon other predictions or author statements that cannot be traced to an experiment. These GO data will be a useful resource for rapid annotation of newly sequenced genomes, including other insect species. Another outcome of the project will be tools, such as a graphical viewer that will allow GO terms to be compared easily for related genes across the set of reference genomes. To date, our priority curation targets have been orthologs/homologs of human disease genes. Each database works from an agreed list of 20 genes/month, identifying the related genes in their species and aiming to capture all possible GO terms from the available experimental literature. We describe how FlyBase assigns GO terms, summarise our progress with the reference genome annotation project, and show the tools we have for viewing the data.

793A Phosphoproteome analysis of Drosophila melanogaster embryos. Bo Zhai, Judit Villén, Sean A. Beausoleil, Julian Mintseris, Steven P. Gygi. Department of Cell Biology, Harvard Medical School, Boston, MA. Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography and high accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13,720 different phosphorylation sites were discovered from 2,702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Owing to the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events. POSTERS: Techniques and Genomics 357

794B A Genome-Wide RNAi Screen to Identify New Components of the RAS/MAPK Pathway. Dariel Ashton-Beaucage1, Anne- Sophie Guenier1, Jean Duchaine1, Patrick Gendron1, Marc Therrien1,2. 1) Institue for Research in Immunology and Cancer, Montreal, Canada; 2) Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Canada. The RAS/MAPK pathway participates in many essential cellular processes. Moreover, mutations affecting pathway activity are found in many forms of cancer. The principal components of the pathway, Ras (Ras85D), Raf (Phl), MEK (DSor) and MAPK (rolled), form an evolutionarily conserved signalling unit called the MAPK module. This module has been extensively studied but, the specific means by which the pathway is regulated are still poorly understood. In order to identify new pathway components, we have developed a high-content screening method in drosophila S2 cells to measure pathway activity. We have completed a genome-wide RNAi screen and identified 315 primary hits, representing genes that act on MAPK signalling. The results from this screen should provide us with a better understanding of the network of regulators involved in the intracellular portion of MAPK signalling. 358 POSTERS: Drosophila Models of Human Diseases

795C Function of Dorsal and Dif in multistep hematopoietic microtumor formation. Marta Kalamarz, Indira Paddibhatla, Christina Nadar, Shubha Govind. Department of Biology, The City College of New York, CUNY, New York 10031, USA. Many hematopoietic mutants exhibit overproliferation and differentiation of hemocytes and develop melanotic tumors. The details of how tumor formation is initiated and how it progresses remain largely unexplored. We are using genetic backgrounds (dUbc9-/lwr- or cactus-) where Toll signaling is unrestrained, to study the discrete steps and mechanisms governing hematopoietic microtumor formation. It was previously established (Chiu et al., 2005) that the hematopoietic defects observed in lwr- mutants are suppressed in the presence of a neomorphic allele of cactus (cactE10) or in the absence of Toll pathway transcription factors Dif and dorsal. We characterized the size and morphology of a large number of lwr- and lwr- Dif- dl- tumorous masses and have found that (1) there are many more aggregates (less than ~ 0.5 x 10-3 mm3) in the larval hemolymph than there are structures greater than 0.5 x 10-3 mm3. Therefore, we called these structures microtumors (0.5 x 10-3 mm3 - 1 mm3). (2) Not all microtumors are melanized. (3) Microtumors are complex structures with distinct morphological areas and gene expression patterns. (4) In contrast to single loss-of-function mutants of lwr or cact, triple mutants of lwr- Dif- dl- do not contain any microtumor structures. (5) Confocal and electron microscopy studies of cellular composition and morphology of microtumors reveal that while most tumors are made up of hemocytes, some of them also contain fragments of the fat body tissue. The presence of fat body in association with hematopoietic microtumors suggests that factors from this tissue may play a role in the development and growth of the microtumor. Based on these results from single and triple mutants, we hypothesize that dorsal and Dif play a critical role in the early stages of microtumor formation. We are examining whether Toll signaling also contributes to other steps in microtumor formation such as proliferation, aggregation and differentiation of hemocytes. The morphological characterization of microtumors and description of discrete steps of their formation will be presented.

796A Enthoprotin regulates hematopoiesis in Drosophila. Wei-Ru Li1,2, Yung-Heng Chang1,3, Y. Henry Sun1,2. 1) Inst Molecular Biology, Taipei, Taiwan; 2) Inst of Genomic Science, National Yang Ming University, Taipei, Taiwan; 3) Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan. The blood cell development in Drosophila shares several interesting features with hematopoiesis in vertebrates, including spatiotemporal regulation as well as the use of similar transcriptional regulators and signaling pathways. In a misexpression screen, we found that the misexpression of the human enthoprotin and its fly homolog caused the formation of melanotic masses, suggestive of an effect on hematopoiesis. The same phenotype was obtained by expressing enthroprotin either ubiquitously or specifically in hemocytes. To analyze the endogenous function of enthoprotin in fly, we used RNAi and also generated mutation by P-element excision. Surprisingly, melanotic mass can also be observed in the loss-of-function mutants. We checked to see if the phenotype is caused by overproliferation of hemocytes and found that the number of hemocytes increased significantly. There were also more proliferating cells in the lymph gland, the hematopoietic organ during larval stages. These results suggested that a critical level of enthoprotin expression is important for maintaining proper level of hematopoiesis in Drosophila. Enthoprotin has been shown to be a clathrin adaptor protein involved in the endocytosis pathway, especially from TGN to endosome. We are currently studying the mechanism by which enthoprotin influences hematopoiesis.

797B Inhibition of RAS-Mediated Transformation and Tumorigenesis by Targeting the Downstream E3 Ubiquitin Ligase SIAH. Amy Tang1,2, Rebecca Schmidt2, Cheol Park1, Atique Ahmed1, Justin Gundelach1, Nanette Reed1, Shen Cheng1, Bruce Knudsen1, Amy Tang1,2. 1) Department of Surgery, Mayo Clinic College of Medicine, Rochester, MN; 2) Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN. Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How best to contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signal might be a suitable anti-RAS target. The E3 ubiquitin ligase SEVEN-IN-ABSENTIA (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into the altered proteolysis in the RAS pathway in tumor initiation, progression and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer. POSTERS: Drosophila Models of Human Diseases 359

798C The effects of Bcr-Abl on Ena-associated proteins in Drosophila melanogaster . Kaitlyn M Vernier, Traci L Stevens. Biology, Randolph-Macon College, Ashland , VA. The mutant protein Bcr-Abl, which is the result of a translocation between chromosomes 9 and 22 that fuses sequences of the bcr gene to the abl gene, causes leukemia in humans. Bcr-Abl has activated tyrosine kinase activity relative to cellular Abl, and studies in cell culture and Drosophila have shown that expression of this fusion protein alters the structure of actin projections during cell migration. These effects on cell migration are thought to contribute to the oncogenic nature of Bcr-Abl. The overall goal of our research is to define the molecular pathways by which Bcr-Abl alters the actin cytoskeleton during cell migration. One target of Bcr- Abl kinase activity is the actin regulator, Enabled (Ena). In Drosophila embryos that express Bcr-Abl, increased levels of Ena tyrosine phosphorylation are observed, and Ena is mis-localized from the leading edge of migrating epithelial sheets during dorsal closure. Ena is known to interact with many proteins throughout development, and the objective of this study was to characterize the proteins that interact with Ena in the presence of Bcr-Abl. Using co-immunoprecipitation assays (co-IPs), we found that several tyrosine phosphorylated proteins associate with Ena in the presence of Bcr-Abl, but not in wild-type embryos. In addition, we examined the effects of Bcr-Abl on a specific Ena-associated protein, zyxin, during embryonic development in Drosophila. In cell culture, zyxin, an adhesion protein that also regulates actin assembly, interacts with Ena. Western analysis showed that expression of Bcr-Abl did not affect the overall levels of zyxin protein in Drosophila embryos, and co-IPs were used to examine whether Ena and zyxin interact in embryos that express Bcr-Abl. These studies will lead to a clearer understanding of the molecular pathways that regulate actin dynamics and cell migration in the presence of Bcr-Abl and how these pathways are altered during the onset of leukemia.

799A The Helicobacter pylori virulence factor CagA can function as an adaptor protein in receptor tyrosine kinase pathways. Anica M. Wandler1, Crystal M. Botham2, Karen Guillemin1. 1) Institute of Molecular Biology, University of Oregon, Eugene, OR; 2) Department of Pediatrics, Stanford University, Stanford, CA. During Helicobacter pylori infection of the human stomach, the CagA effector protein is translocated into host cells and its presence is associated with gastric cancer formation. In tissue culture cells CagA interacts with several host cell proteins including SHP-2, an important component of receptor tyrosine kinase (RTK) pathways whose misregulation has been liked to cancer formation. However, the effects of this interaction on intact epithelial tissues and the mechanisms by which it promotes carcinogenesis have not been determined. Using Drosophila melanogaster as a model organism we hope to determine CagA’s activities through analysis of photoreceptor development, an RTK-dependent process. Our data show that CagA can functionally substitute for the Drosophila Gab adaptor protein, Daughter of Sevenless, to specify photoreceptors. This process requires Corkscrew, the Drosophila homolog of SHP-2, further indicating that CagA can function as an adaptor protein in RTK pathways. CagA’s activation of this pathway is unique because it results in excessive RTK signaling that leads to the formation of extra R7 photoreceptors. Unlike other mutations that up-regulate RTK signaling through constitutive activation of downstream components, CagA’s effect on this pathway requires the presence of both the receptor and ligand. These data lead to two possible models: one predicting that CagA is a more potent activator of SHP-2, and another in which CagA allows extra cells to undergo RTK signaling through disruption of cell-cell contacts. CagA’s ability to interfere with cell junctions in tissue culture, and our observation that expression of CagA causes disorganization of the retinal epithelium provide support for the second model. By combining biochemical analysis of RTK signaling in cell lines with genetic and cell biological examinations of CagA’s effect on RTK pathways in Drosophila tissues we aim to gain further insight into CagA’s mechanisms and oncogenic activities.

800B Drosophila model for Kif1A related hypoplastic left heart syndrome. Takeshi Akasaka1, Grant Hogg1, Karen Ocorr1, Rolf Bodmer1, Paul Grossfeld2. 1) Burnham Inst, La Jolla, CA; 2) Department of Pediatrics, UCSD, La Jolla, CA. Advancements in therapeutic strategies have successfully improved the prognosis of many forms of congenital heart diseases (CHD). However, hypoplastic left heart syndrome (HLHS) - characterized by severe left ventricular hypoplasia and thickening of the myocardium (due to an abundance of fibrous tissue) - is still a disease that is refractory to effective treatment and can require heart transplantation. Identification of the underlying mechanism of HLHS is an important prerequisite that should lead to earlier diagnosis, improved management, and ultimately, prevention of HLHS. There are multiple lines of evidence supporting a genetic etiology for HLHS, however, to date the genetic mechanisms underlying HLHS are largely unknown. We recently found a family with a balanced translocation [t(2,8;q37,p11)], whose breakpoints are in close proximity to the Kif1A gene at 2q37. Interestingly, left-sided heart defects and the translocation are well correlated in this family. To test whether Kif1A overespression can produce cardiomyocyte defects, we generated transgenic fly lines that overexpress Kif1A in mesodermal tissue or heart muscles using the Gal4-UAS system. We found that Kif1A overexpression results in cardiac myofibers that are disorganized and the heart is hypoplastic. These findings are consistent with the histological abnormalities seen in HLHS patients, which include myofibrillar disarray and scant cytoplasm in cardiomyocytes. Physiological analysis of fly hearts also revealed that Kif1A overexpression flies show impaired fractional shortening compared to the outcrossed controls. These findings suggest that Kif1A affects not only morphology but also cardiac performance in vivo. We also examined the effect of Kif1A in C2C12 myoblasts and found that Kif1A overexpression affects actin but not microtubule organization of the cytoskeleton. These data suggest that overexpression of Kif1A may be an important factor in the pathogenesis of HLHS, and they further underline the utility of Drosophila as a useful model to address the mechanisms underlying human cardiac disease such as HLHS. 360 POSTERS: Drosophila Models of Human Diseases

801C A wildtype Drosophila model of age-dependent heart failure. Rolf Bodmer1, Karen Ocorr1, Tim Crawley1, Greg Gibson2. 1) Burnham Inst, La Jolla, CA; 2) University of Queensland, Brisbane, Australia. Cardiac function in Drosophila melanogaster decays in an age-related manner, providing a suitable model for genetic dissection of heart disease. We have characterized variation among 50 wild-type nearly isogenic lines for electrical pacing-induced heart failure at 1 and 5 weeks of age, and describe 7 lines with extreme age-dependent arrhythmicity. High speed video imaging of the adult heart beat in a subset of these lines reveals a variety of defects that resemble the consequences of loss of genetic functions involved in insulin signaling, cardiac potassium channel activity, and heart morphogenesis. Measurement of defects in F2 or Backcross progeny suggests that the defects are nearly Mendelian in some cases, and led us to rapidly map one naturally occurring variant to the 97C interval on Chromosome 3 using a combination of bulked segregant and single feature polymorphism (SFP) mapping by genomic DNA hybridization to Affymetrix Gene Chips. Our hypothesis is that heart failure in natural populations of flies occurs at a similar rate as is observed in humans, and is largely due to combinations of rare alleles of large effect. Experiments are under way to map and clone such alleles and precisely determine the mechanisms by which they cause age-dependent heart disease.

802A Deletion screen in adult Drosophila identifies candidate genes for dilated cardiomyopathy. Michelle E. Casad1, Il-Man Kim2, Matthew J. Wolf2, Howard A. Rockman1,2,3. 1) Department of Cell Biology, Duke University Medical Center, Durham, NC; 2) Department of Medicine, Duke University Medical Center, Durham, NC; 3) Department of Molecular Genetics, Duke University Medical Center, Durham, NC. Traditional genetic screens in Drosophila have focused on early patterning and heart development, however adult heart phenotypes have not been well-characterized due to the difficulty in accurately measuring cardiac function in adult Drosophila. Using optical coherence tomography (OCT), we recently developed an innovative method to phenotype cardiac function in awake adult Drosophila, and we initiated a genome-wide screen for deletion mutants with abnormal cardiac function in the adult. We have identified two deletion strains on the X chromosome with a dilated cardiomyopathy phenotype. These cover 125kb and 92kb, corresponding to deletions of 10 and 16 genes, respectively. Next we engineered smaller genomic deletions within the regions to narrow the interval responsible for the phenotype. For each genomic deletion, we identified a smaller deletion that maintained the abnormal cardiac phenotype, narrowing the potential candidate genes to and 4 and 5, respectively. Interestingly, these candidate genes do not include any sarcomeric proteins, nor any proteins previously implicated in heart function. Furthermore, we have identified a transposon insertion mutant which phenocopies one of the deletion mutants. The insertion lies between the four candidate genes for that deletion, but outside any coding region. We have obtained stocks from the Vienna Drosophila RNAi Center in order to knock down candidate genes in a temporal or tissue specific manner, and we are evaluating their effects on cardiac function. Additionally we are creating transgenic Drosophila that express candidate genes to evaluate the rescue of cardiac function. In conclusion, we show that we can readily identify adult Drosophila with dilated cardiomyopathy and genetic strategies can lead to discovery of novel genes that are potentially responsible for this disease.

803B Drosophila model of muscular dystrophy. Jeffery Goldstein1, Michael Allikian2, Gira Bhabha2, Patrick Dospoy2, Ahlke Heydemann2, Pearl Ryder2, Judy Earley2, Matthew Wolf4, Howard Rockman5, Elizabeth McNally2,3. 1) Departments of Pathology; 2) Medicine; 3) and Human Genetics, University of Chicago, Chicago, IL; 4) Departments of Medicine; 5) and Cell Biology, Duke University, Durham, NC. The dystrophin-sarcoglycan complex is a key element in maintaining muscle cell integrity. Diverse mutations in members of this complex are associated with muscular dystrophy in humans and in murine models. Where mammals have three of the 35 KDa class of sarcoglycans (γ, δ and ζ), Drosophila have only one (Sgcδ). Concordantly, deletions of Drosophila Sgcδ described here result in a complex phenotype with many parallels to the human disease. Using imprecise P-element excision, we generated a deletion of the unique amino-terminal tail region (Line 28), as well as more complete Sgcδ deletions (Line 169, Line 840). Compared to the parental KG5430 line, Sgcδ deletion mutants show an age dependent decrease in climbing ability as adults, corresponding to the muscle weakness that defines muscular dystrophies. Optical coherence tomography demonstrated a dilated and poorly contractile heart tube in the complete deletion Lines 840 and 169. This is reminiscent of the dilated cardiomyopathy seen in muscular dystrophies associated with dystrophin and the sarcoglycans. The partial deletion, Line 28, resulted in an increased end systolic diameter with a normal end diastolic diameter compared to the control w1118 stock. The phenotype is also characterized by muscle tears evident on routine histology and sarcomeric distruption revealed by electron microscopy. We will report on the genetic interaction between Sgcδ and its two closest homologues in mouse, γ- and δ-sarcoglycan (mSgcγ, mSgcδ). Specifically we will investigate the ability of Drosophila Sgcδ to rescue formation of the dystrophin-sarcoglycan complex in mice lacking Sgcδ, and the ability of tissue-specific expression of mSgcγ or mSgcδ to rescue tissue-specific phenotypes in Drosophila. POSTERS: Drosophila Models of Human Diseases 361

804C Using Drosophila heart to uncover the genes contributing to Down syndrome congenital heart disease. Tamar R. Grossman1, Amir Gamliel2, Robert J. Wessells3, Ouarda Taghli-Lamallem4, Kristen Jepsen2, Julie R. Korenberg5, Rolf Bodmer4, Ethan Bier1. 1) Div. Biol. Sci; 2) HHMI, Dept. Med., UCSD, La Jolla, CA; 3) Univ Michigan, Ann Arbor, MI; 4) Burnham Institute Med Res, La Jolla, CA; 5) Cedars-Sinai Medical Center, Los Angeles, CA. Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects. It has been suggested that DS-CHD is a multigenic disease that results from elevated expression of several genes from the DS-CHD candidate region, which include SH3BGR, DSCAM, WRB, Collagen VI A1 and A2, Collagen XVIII and HES1 genes. In attempt to identify which gene(s) are responsible for DS-CHD we used the Drosophila heart as an in vivo Drosophila assay system where we mis-expressed human and fly DS-CHD candidate genes using the UAS/GAL4 system and tested the effect on the heart’s physiology. In order to identify possible gene interactions we also mis-expressed all pairwise combinations. The effect on heart physiology was analyzed using sensitive heart function assays such as electrical pacing and high-speed video recordings from adult fly hearts. Our results show that mis-expression of certain DS-CHD candidate genes results in significant heart defects, including increased rate of pacing stress-induced heart failure. We found that out of all double gene combinations tested only two showed a synergistic aggravation of the heart phenotypes when simultaneously mis-expressed in the fly heart. In order to confirm our results in a mammalian system, we generated double transgenic mice with the two genes that we found to have the strongest interaction in the fly heart. Our preliminary data show cardiac specific overexpression of two DS-CHD genes results in heart malformations. Taken together, our data suggest that specific genes from the DS-CHD region on chromosome 21 may be responsible for the heart defects in Down syndrome.

805A Characterizing how mGluR-activated G-coupled pathways are misregulated in the absence of dFMR1. Balpreet Bhogal, Thomas Jongens. Dept Genetics, University of Pennsylvania, Philadelphia, PA. Fragile X syndrome is the most common heritable form of mental retardation. Patients with this disease display mild to severe mental retardation, behavioral traits such as developmental delay, autistic-like behavior, sleep disorders and attention deficit hyperactivity disorder, as well as prominent physical features. This disease is caused by loss of function of the Fragile X mental retardation (FMR1) gene, which encodes an RNA binding protein. Although previous studies suggest that FMR1 functions as a translational regulator, a molecular function, as well as its regulatory targets, remains to be elucidated. Previous studies have implicated metabotropic glutamate receptor (mGluR) signaling as a pathway that is hyperactivated in the absence of FMR1. In mammals, mGluR signaling has been shown to primarily activate Gq- and Gi-coupled signaling cascades, yet how the mGluR pathway functions in flies has not been very well characterized. Our lab uses Drosophila melanogaster to study the molecular basis of Fragile X syndrome. In Drosophila, there is a single gene, Drosophila fragile x related (dfmr1), that is orthologous to FMR1. Loss of dFMR1 results in defects in circadian rhythm and naive courtship, learning and memory defects, and β-lobe crossover of the mushroom body, and treatment of dfmr1 flies with mGluR antagonists rescues most of these phenotypes. We are interested in determining how misregulation of mGluR signaling and downstream pathway members give rise to the dfmr1 mutant phenotype. We are currently performing courtship assays on Gq- or Gi- coupled pathway mutants to test how naive courtship is affected. We are also treating dfmr1 mutants with pharmacological antagonists to either Gq- or Gi-coupled pathway members to determine if naive courtship is rescued. We will also examine how immediate recall, short-term memory and mushroom body morphology are affected to determine if misregulation of Gq- and Gi-coupled pathways give rise to specific phenotypes.

806B A role for the steroid hormone ecdsyone in embryonic tracheal development. Tina Chavoshi, Anne Uv. Department of Medical and Clinical genetics, Biomedicine, Gothenburg, Gothenburg, Sweden. Epithelial tubular networks are fundamental to life. We breathe through them, our blood circulates in them and they form the basic units of our kidneys and glands. Tubular organ formation involves several steps, including tubular network pattering and growth to attain correct tubular diameters and lengths. These activities occur in controlled sequential steps during development to ensure functional organs. Here, we have investigated the role for the steroid hormone ecdysone during development of the embryonic drosophila trachea, a network of branched epithelial tubes. The level of ecdysone rises during a few hours at mid-embryogenesis, coinciding with early tracheal organ formation. We show that embryos deficient for zygotic ecdysone biosynthesis develop narrow and short tracheal tubes with irregular lumen diameter. These defects are associated with impaired deposition of apical/luminal matrix components and terminal branch differentiation as well as a gradual loss of expression of key tracheal genes. The results indicate that the mid-embryonic ecdysone pulse is needed for the tracheal cells to proceed through their genetic programme of tube formation. 362 POSTERS: Drosophila Models of Human Diseases

807C Requirement for O-mannosylation in Drosophila development. Dmitry Lyalin, Naosuke Nakamura, Stacey Whitman, Kate Koles, Vlad Panin. Dept Biochemistry & Biophysics, Texas A&M Univ, College Station, TX. O-mannosylation is a mucin-type glycosylation of proteins with O-linked glycans attached via mannose to serine or threonine residues of protein backbones. This posttranslational modification is presumably important for several aspects of cell interactions, including cell adhesion, migration, and interaction with extracellular matrix. POMT1 and POMT2 are the two O-mannosyltransferase genes that have been described in mammalian genomes to date. Notably, mutations in these genes have been linked to the Walker- Warburg syndrome - a severe form of human muscular dystrophy. One of few known targets of O-mannosylation in humans is Dystroglycan (Dg), and its abnormal glycosylation underlies developmental and physiological defects in a number of muscular dystrophies. Drosophila rt and tw genes encode homologs of mammalian POMT1 and POMT2 O-mannosyltransferases, respectively. Mutations in these genes result in “rotated abdomen” phenotype, a clockwise rotation of abdominal segments. It is not known what causes the rotation of the abdomen in Drosophilaa. We studied temporal and spatial requirements of tw for the abdomen development. We found that abdomen rotation phenotype could be rescued by tw expression during an unusually wide “window” of developmental stages. Current experiments are aimed at investigating possible mechanisms that result in abdomen defects in O-mannosyltransferase mutants.

808A Binding partners of dFMRP suggest a regulatory role in translational initiation. Ophelia Papoulas1, Kate Monzo1, Greg Cantin2, Cristian Ruse2, John Yates III2, John C. Sisson1. 1) The Section of MCD Biology and The I.C.M.B., The University of Texas at Austin, Austin, TX; 2) Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA. In humans, loss of Fragile X mental retardation protein (FMRP) activity in brain neurons is thought to result in the aberrant translation of potentially hundreds of mRNAs within dendrites and cause the cognitive symptoms of Fragile X syndrome, the most common form of heritable mental retardation. Although FMRP is clearly involved in translational regulation, its exact mechanism of action is unknown. We have previously shown that the Drosophila ortholog of FMRP is required in cleavage stage embryos for cellular morphogenesis prior to nervous system formation, helping to establish the early Drosophila embryo as a promising new system for elucidating the mechanism of dFMRP function. Here we describe the identification of two proteins that specifically coimmunoprecipitate with dFMRP from early Drosophila embryo extracts that directly implicate dFMRP in the regulation of translational initiation. The vertebrate homolog of one of these proteins, dFMRP-associated protein (dFAP), encodes an RNA-binding protein implicated in transcript-specific translational repression in dendrites. In order to study the functional significance of the dFMRP/ dFAP interaction we have generated alleles of dfap and raised an antibody. Similar to dfmr1 mutants we find that dfap mutants are viable and females are semi-sterile, producing embryos with defects in cellularization. Immunofluorescence analysis reveals extensive colocalization of dFMRP and dFAP to cytoplasmic RNP bodies of cleavage stage embryos and complementary biochemistry shows extensive cosedimentation of dFAP and dFMRP in sucrose gradients away from polyribosomes. Both proteins are also extensively coexpressed in the embryonic nervous system and primordial germline cells in older embryos. Together our results suggest that dFAP and dFMRP function together to regulate translational initiation of specific mRNAs required for normal cellular morphogenesis of cleavage stage embryos.

809B Lamin processing and Progeria: studies in Drosophila. Sandra R Schulze1, Beatrice Curio-Penny2, Melissa Petersen1, Lori L Wallrath2. 1) Dept Biol, Western Washington Univ, Bellingham, WA; 2) Dept Biochemistry, University of Iowa, Iowa City, IA. Hutchinson Gilford Progeria Syndrome (HGPS) is a premature ageing disorder in humans arising from improper processing of Lamin A, a component of the nuclear envelope. Lamins are type V intermediate filament proteins that exist as two types: A-types (Lamin A and C) and B-types (Lamin B1 and B2). All lamins except Lamin C are prenylated on a C-terminal moiety to aid in membrane anchorage. However, Lamin A undergoes an additional processing step to remove this anchor, presumably carried out by the prenyl protease Zmpste24 (FACE1). The reasons for this transient prenylation are unclear. HGPS correlates with an absence of Lamin A processing and the persistence of the prenylated form. We are interested in developing a fly model to study A-type lamin biology, particularly in the context of processing. Human lamins incorporate into the fly nuclear envelope and interact with both

Drosophila A and B type lamins (Lamin C and Lamin Dm0 respectively). It is not clear whether transgenically expressed human Lamin A is processed in Drosophila, but there are three potential homologs for the prenyl protease Zmpste 24, (CG9000, CG9001 and CG9002). A preliminary characterization of these genes will be presented, together with data describing conserved interactions between Drosophila and human nuclear envelope components. POSTERS: Drosophila Models of Human Diseases 363

810C The role of alg10 in regulating N-glycosylation during Drosophila development: a novel model for Congenital Disorders of Glycosylation. Erica M. Selva, Carly Dominica, Evan Lebois. Dept Biological Sci, Univ Delaware, Newark, DE. Proteins involved in extracellular signaling must first traverse the secretory pathway where they are posttranslational modified before the mature protein is able to execute its function. Congenital disorders of glycosylation are a group of human genetic diseases characterized by disruption of glycan attachment to substrates that require this modification for function. In this study, Drosophila harboring mutations in alg10 are characterized. alg10 encodes a glycosyltransferase that catalyzes addition of the terminal glucose residue to the growing dolichol-linked oligosaccharide just prior to its en masse transfer to nascent polypeptides. This terminal glucose is removed from the oligosaccharide even before nascent glycoproteins exit the endoplasmic reticulum and alg10 is not expected to be required in tissues that express oligosaccharyltransferase isoforms that transfer under glycosylated substrates. The study of alg10 in Drosophila thus provides a unique model to explore N-glycosylation defects at a cellular level in a genetically tractable organism. Our study of P-element excision alleles of alg10 demonstrates that this gene product is of surprisingly critical importance during Drosophila development. Removal of both zygotic- and maternal-derived alg10 results in severe and pleiotropic deficits in embryos, demonstrating the importance of regulated N-glycosylation during the initial phases of Drosophila development. During larval development, removal of alg10 from the eye imaginal disc leads to a disordered eye of reduced size. These effects might be mediated through the Sevenless activated MAP kinase cascade, as we find alg10 mutant eye discs display pathway gain- of-function phenotypes. These data suggest that regulated N-glycosylation of a component of the Sevenless receptor tyrosine kinase pathway, perhaps the receptor itself, is one important target of Alg10 function. Together, our data suggest that tissue specific addition of terminal glucose to the dolichol-linked oligosaccharide is an essential regulatory event in executing the Drosophila developmental program.

811A Dube3a expression in neurons increases synaptic bouton number through regulation of Pbl and downstream Rac targets. Kyle Summers1, Hemachand Tummala2, Priti Azad1, Lawrence Reiter1. 1) Neurology, University of Tennessee Health Science Center, Memphis, TN; 2) Biology, University of Memphis, Memphis, TN. Maternal deficiency for the human UBE3A gene results in the severe autism spectrum disorder Angelman syndrome while maternally inherited duplications for the UBE3A gene region are the most common cytogenetic abnormality found in autism. We recently identified protein targets of the UBE3A ubiquitin ligase through proteomic analysis in Drosophila head extracts. One protein found to be regulated by Dube3a in both fly and mouse models is the Drosophila Pebble (Pbl) protein (Reiter et al. 2006. Human Molecular Genetics. 15:2825-35). Pbl is a Rho-GEF involved in actin cytoskeletal rearrangements through the regulation of both the Rho and Rac pathways. Here we describe a phenotype in synaptic boutons that results from Dube3a expression under the panneuronal c155-GAL4 driver. In Dube3a over-expressing neurons, boutons were not only small, but significantly more numerous than in wild type larvae. Furthermore, we demonstrate that this phenotype may be the result of down-regulation of Pbl protein in boutons. Genetic interaction studies indicate that the bouton morphology phenotypes observed may be the result of downstream effects on Pbl regulated Rac1 and Rac2 proteins. In C. elegans, the Pbl orthologue let-21 interacts with Rho1 during neuronal P-cell cytokenesis, but depends on Rac interactions for the regulation of axon guidance, cell migration and morphology. Here we propose a model for synaptic plasticity based on the pre-synaptic regulation of the Pbl/Rac pathway by Dube3a in synaptic boutons.

812B Characterization of the Drosophila mitochondrial elongation factor iconoclast: a model system for Combined Oxidative Phosphorylation Deficiency. Catherine Trivigno, Theodor E. Haerry. Dept. of Biology, Florida Atlantic Univ, Boca Raton, FL. Mutations in the human mitochondrial elongation factor GFM1 gene have recently been shown to cause Combined Oxidative Phosphorylation Deficiency (COX1) Syndrome. Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities, including early-onset Leigh’s disease. Here, we describe the identification and characterization of iconoclast (ico), the Drosophila homologue of GFM1. Interestingly, while point mutations in this gene result in developmental defects and death during embryogenesis, animals null for ico survive until the second instar larval stage. Similarly, somatic eye clones of point mutations in ico are mostly cell lethal, while clones of null mutations are not. These observations suggest that point mutations in ico produce toxic proteins. Consistent with this hypothesis, we find that expression of a mutant ICO protein inhibits growth and can be lethal. Currently, we are testing whether the human homologue can compensate for the lack of the Drosophila gene and whether expression of human mutant alleles exhibits toxic effects as well. Since all eukaryotic organisms have a second mitochondrial elongation factor, GFM2, we are also examining the role of this protein. These experiments will contribute to the elucidation of the mechanisms of the underlying pathology caused by GFM1 mutations in an animal model system. 364 POSTERS: Drosophila Models of Human Diseases

813C Characterization of a new tracheal gene required in tube elongation. Erika Tång Hallbäck, Anne Uv. Biomedicine, Medical genetics, Göteborg, Göteborg, Sweden. Epithelial tubular organs are vital to multicellular organisms. The ability of such tubes to transport gases and liquids in an efficient manner depends on correct tube size and shape, which in turn requires precise rearrangements and shapes of the cell within the tube wall. We are using the Drosophila trachea (respiratory organ) as a model system to identify mechanisms that coordinate these events across the tubular epithelium to ensure correctly formed tubes. In previous work we have found that formation of uniform tubes in the trachea requires the correct assembly of an intraluminal chitinous matrix, which appears to coordinate tracheal cell shape changes during tube growth. Without a functional chitin filament the tracheal tubes develop local dilations and constrictions during tube diameter expansion and later in development the tubes also become too long. I will present our recent analysis of a new tracheal tube size mutant that develop too short tubes at the end of embryogenesis. The mutation disrupts a chloride channel and is expressed after the initial luminal matrix has formed and performed its role in tube diameter dilation. We speculate that loss of this chloride channel alters the lumen environment and thereby prevents elongation of the luminal matrix and subsequently tube elongation. Thus, a genetic program may modulate the luminal tracheal matrix during development to accommodate sequential changes in tube dimensions.

814A Identification and characterisation of a new class of proteins required for epithelial barrier formation. Fariba Zare, Anna Tonning, Anne Uv. Department of Clinical and Medical Genetics, Institution of Biomedicine, Gothenburg, Sweden. Epithelial tubes are fundamental to life, and for the functional units in most of our vital organs. An impaired tube wall integrity breaks the barrier between the luminal environment and the surrounding body tissues leading to passage of pathogenic organisms, toxic molecules and immune cells across the wall. We use the well characterized Drosophila model system, namely the respiratory organ (trachea) for formation of branched tubular organ. By screening collections of Drosophila genes for a potential role in tracheal tube formation I have found four new genes required for formation of the transepithelial barrier and encoding apical surface proteins. All are non-characterized genes and one has a vertebrate ortholog with unknown function. I propose to combine genetic, molecular and biochemical methods to reveal the cellular and molecular functions of these genes in epithelial tube formation. As fundamental biological mechanisms are shared across species, our findings are expected to apply to higher organisms and to have impact on the development of treatments and regeneration of damaged human tubular organs.

815B Functional characterization of dPGC, a member of the PPARγ Coactivator 1 family. Gretchen V. Gee, Jennifer T. Paul, Marc Tatar. Brown University, Providence, RI. Department of Ecology and Evolutionary Biology. Drosophila gene CG9809 (dPGC) is a homolog of the mammalian PPARγ Coactivator 1 (PGC-1) family of transcriptional coactivators that serve as key regulators of metabolism. In mammals, perturbations in PGC-1 expression have been linked to type 2 diabetes, cardiovascular disease and neurodegenerative diseases. Our experiments, in both cell culture and whole flies, indicate that dPGC exhibits characteristics of both PGC-1α and PGC-1β; including transcriptional induction following cold exposure, insulin stimulation and nutrient uptake. We show that homozygous P-element insertion flies with very low transcription levels of dPGC are developmentally delayed and have reduced fertility compared to wild-type flies. Notably, these flies have low basal expression levels of multiple genes involved in both fatty acid β-oxidation and lipid storage. Taken together, our data indicate that dPGC can be induced in Drosophila under conditions that are known to up-regulate mammalian PGC-1 genes, and that the basal transcriptional role of dPGC in Drosophila is to maintain lipid homeostasis. POSTERS: Drosophila Models of Human Diseases 365

816C Drosophila as a model for metabolic syndrome. Laura Reed, Stephanie Williams, Mastafa Springston, Julie Brown, Greg Gibson. Dept Genetics, North Carolina State Univ, Raleigh, NC. We are developing Drosophila as a model system for the genetic dissection of Metabolic Syndrome (MetS), which is the most prevalent disease affecting modern humans. Our hypothesis is that altered diet and other environmental factors expose cryptic genetic variation, and that dietary perturbation in Drosophila will similarly disrupt metabolism resulting in disease-like symptoms. In this study we characterize the standing genetic variation for MetS-like symptoms in Drosophila melanogaster when experiencing the environmental perturbation of high fat and high sugar diets. We measured pupal weight, larval lipid content, larval hemolymph sugar concentration, and larval survival of 150 natural genetic isolates raised across four dietary treatments, normal lab food, low-fat low- sugar, high-fat, high-sugar. We found significant genotype by environment interactions for all four phenotypes, with the greatest variance in phenotype occurring on the high fat diet. Out of these 150 lines, 10 phenotypically extreme and 10 randomly selected lines have been characterized by gas chromatography-mass spectrometry metabolite profiling on two diets. We will describe the diversity, heritability, and genetic covariance of metabolites across genotypes and diets, and their relation to genetic variance for weight gain and the other MetS-like symptoms.

817A Regulation of energy homeostasis and obesity in Drosophila melanogaster. Tânia Reis, Iswar Hariharan. MCB, Univ California, Berkeley, Berkeley, CA. We are increasingly aware that obesity is not simply an effect of excessive food intake. Maintaining energy balance requires coordination of the nervous system (sensing satiety and regulating feeding behavior) and energy storage tissues (storing energy as fat/sugar or mobilizing it). The bulk of current obesity research is focused on a small number of pathways regulating fat storage and energy homeostasis, yet many more are likely to exist. We turned to the fruit fly to identify new genes controlling organismal fat levels. Drosophila melanogaster features a complex physiology, including a dedicated fat storage organ called the fat body, and a brain that discriminates among tastes and odors and directs complex feeding behaviors. We used a density-based flotation assay to identify mutant larvae with increased fat levels, confirming by gas chromatography/mass spectrometry that all of the mutants tested have elevated triacylglycerides. Encouragingly, we isolated mutations in several genes implicated in the regulation of mammalian adipogenesis. Some genes mutated in fat flies are normally expressed in the brain. To further investigate the role of the brain in regulating fat storage, we also screened for fat flies in a collection of lines in which the function of specific neurons or groups of neurons was conditionally ablated. Certain regions of the brain are overrepresented in the fat lines compared to the overall collection, and some affect feeding behavior when inhibited. Interestingly, both overeaters and undereaters were identified, suggesting that the brain can regulate fat storage directly as well as by altering food consumption. With this work we aim to define a regulatory network for energy homeostasis, including which organs sense and process physiological changes in energy levels and food sources. These studies will identify potential targets for the treatment of human obesity and associated diseases.

818B STRESS PROTECTION PATHWAY - NEW WAYS OF TREATING DISEASE. Qiong Wang, Wesley Dobbs, Adriana Villella, Gerasimos Sykiotis, Dan Garza, Marc Hild. DMP / Model Organisms, NIBRI, Cambridge, MA. Stress, disease and aging are causally linked. Genetically or pharmacological induced activation of stress response pathways leads to lifespan extension in animal models and has been shown to have beneficial effects in multiple models of neurodegenerative disease. A deeper understanding of the role of theses pathways in aging, stress and disease should open up new roads for drug discovery and ultimately result in new therapeutic approaches. Due to its excellent RNAi system and advantages as a genetic model system, we have begun using Drosophila to study stress response pathways and their role in disease, beginning with the establishment of Drosophila cells as an in vitro system to identify novel modulators of the HSF1 pathway (heat shock response). We have performed genome-wide RNAi screens under multiple stimuli conditions as well as using unstimulated cells and will present the results from these screens as well as further validation experiments in Drosophila cells and whole flies. 366 POSTERS: Drosophila Models of Human Diseases

819C Genetic suppression of neurodegeneration and neurotransmitter release abnormalities caused by expanded full-length huntingtin accumulating in the cytoplasm. Guang-Ho Cha1, Romero Eliana1, Patrik Verstreken1,2,6, Cindy Ly3, Robert Hughes4, Hugo Bellen1,2,3,5, Juan Botas1,7. 1) Dept Molecular Human Gen, Baylor Col Medicine, Houston, TX 77030; 2) Howard Hughes Medical Institute; 3) Department of Neuroscience, Baylor Col Medicine, Houston, TX 77030; 4) Buck Institute 8001 Redwood Blvd, Novato, CA 94945; 5) Program in Developmental Biology, Baylor Col Medicine, Houston, TX 77030; 6) VIB Department of Molecular and Developmental Genetics K.U.Leuven Department of Human Genetics Herestraat 49, bus 602 B3000 Leuven, Belgium; 7) Department of Molecular and Cellular Biology, Baylor Col Medicine, Houston, TX 77030. Huntington’s Disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N-terminus of the huntingtin protein. Here we describe the generation and characterization of a novel full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded huntingtin is cleaved and imported into the nucleus in detectable amounts. We find that expanded full-length huntingtin (128QhttFL) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128QhttFL animals. Partial loss of function in synaptic transmission (Syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length huntingtin during early stages of pathogenesis.

820A Protein nuclear transport and polyglutamine toxicity. Wing Man Chan1, Frankie Ho Tsoi2, Pang Chui Shaw1,2, Ho Yin Edwin Chan1,2. 1) Molecular Biotechnology Programme, CUHK, HKSAR; 2) Department of Biochemistry, CUHK, HKSAR. Polyglutamine (polyQ) disease is a group of genetic disorders caused by abnormal elongation of an existing polyQ domain in disease proteins. Those proteins that harbor the expanded mutant polyQ domain cause progressive dysfunction and death of neurons. The nucleocytoplasmic localization of polyQ disease proteins is closely correlated with toxicity. Classical nuclear localization signals (NLSs) and nuclear export signals (NESs) are found on polyQ disease proteins. However, the activities of these transport signals are compromised by polyQ expansion, which subsequently alters the nuclear transport properties of disease proteins. In addition to the classical nuclear transport signals found on polyQ disease proteins, our preliminary data demonstrated that the mutant polyQ domain also possesses NLS and NES activities. We used RNA interference to further study nuclear transport properties of mutant polyQ domain, and identified several members from the karyopherin nuclear transport receptor family that are involved in mutant polyQ-mediated nuclear transport. Understanding the molecular mechanisms that govern the nuclear translocation of the mutant polyQ protein will provide new insights into polyQ pathogenesis.

821B Regulation of Tau Phosphorylation and Toxicity : Insights from a Drosophila Model. Shreyasi Chatterjee1, Tzu Kang Sang2, George Jackson1. 1) Department of Neurology and Center for Neurobehavioral Genetics. David Geffen School of Medicine at University of California, Los Angeles. CA; 2) Department of Neurology,National Tsing Hua University, Taiwan, Republic of China. Although a number of kinases have been implicated in tau phosphorylation, including GSK-beta and MARK, the relationship between tau phosphorylation and toxicity has not been fully understood. We have used a Drosophila model expressing the longest isoform of human tau to explore the relationship between tau phosphorylation and toxicity.The fly homologue of MARK is PAR-1, and the phosphorylation of tau by MARK is thought to promote subsequent phosphorylation by other proline directed kinases.Mutations of sites phosphorylated by MARK (S262 and S356) abolishes PAR-1 induced phosphorylation and diminishes tau toxicity but does not impair tau phosphorylation by GSK-3beta/Shaggy.Coexpression of human GSK-3beta or it’s fly homologue Shaggy,increases tau phosphorylation and toxicity.Mutation of eleven important Ser/Thr phosphorylation sites abolishes the Shaggy induced increase in tau phosphorylation ; however this S11A form of tau retains it’s toxicity. Comparison of toxicity of wild type tau, S2A and S11A revealed a corelation between the affinity of each construct for microtubules and its toxicity. Our work suggests that although phosphorylation of tau is an important determinant of its neurotoxicity, other factors are likely to play additional roles in the development of tauopathy. POSTERS: Drosophila Models of Human Diseases 367

822C Characterizing the function of the Drosophila LRRK2 homolog, lrrk. Mark W. Dodson1, Changan Jiang2, Ming Guo1,2. 1) Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA; 2) Departments of Neurology and Pharmacology, David Geffen School of Medicine, UCLA. Although the pathogenesis of Parkinson’s disease (PD) remains unclear, significant insights have come in recent years from the study of genes mediating inherited forms of the disease. Our lab and others have shown that the fly homologs of the PINK1 and PARKIN genes, which mediate recessive forms of PD, function in a common genetic pathway regulating mitochondrial function. Leucine Rich Repeat Kinase-2 (LRRK2) is the single most common genetic determinant of PD, and mediates a dominant late onset form of the disease. The cellular processes in which LRRK2 normally functions, and the mechanisms mediating the pathogenecity of the mutant alleles, are unknown. It has been reported that human LRRK2 localizes to the outer mitochondrial membrane, and physically interacts with Parkin in vitro, suggesting that LRRK2 may also play a role in mitochondrial function. We have generated loss-of-function mutations in lrrk, the single fly LRRK2 homolog, and have found that these flies are viable and show no gross external defects, but have a marked reduction in female fertility. We have also generated transgenic flies overexpressing Drosophila lrrk bearing a mutation analogous to the pathogenic G2019S substitution in human LRRK2 (referred to as lrrkGS). Strong ubiquitous overexpression of lrrkGS results in pupal lethality, while equivalent overexpression of wildtype lrrk does not, demonstrating increased toxicity of the mutant protein in vivo. Interestingly, neither lrrk loss-of-function nor overexpression of lrrkGS disrupts mitochondrial structure in muscle or testes, tissues in which mitochondrial defects are readily apparent in pink1 and parkin mutant flies. Furthermore, overexpression of wildtype lrrk, unlike parkin overexpression, does not suppress pink1 loss-of-function phenotypes. Together, these findings suggest that lrrk functions in a distinct pathway from pink1 and parkin, and does not play a major role in the maintenance of mitochondrial structure.

823A Cell and Organism Toxicity by Atrophins. Manolis Fanto1, Bernard Charroux2, Ilaria Nisoli1, Elise Peyre2, Francesco Napoletano1. 1) Dulbecco Telethon Institute, DIBIT-HSR, Milan, Italy; 2) IBDML, Campus de Luminy Case 907, F-13288 Marseille Cedex 9, France. Polyglutamine (polyQ) diseases are neurodegenerative syndromes caused by the expansion of CAG repeats resulting in long polyQ stretches in the protein. The molecular mechanisms of polyQ proteins’ toxicity are not fully characterised yet and many key aspects are under intense scrutiny. Dentatorubropallidoluysian Atrophy (DRPLA) is one of these disorders caused by mutations in the Atrophin-1 protein. Since Atrophins represent the polyQ proteins that are best conserved between Drosophila and humans, we have set up a thorough approach that merges gain of function and classic loss of function analysis to unravel the mechanisms of cellular and organism toxicity by polyQ Atrophins. We are focusing on neuronal photoreceptors to analyse cell degeneration and on lifespan to analyse organism-level toxicity. Our genetic and ultrastructural analysis of neurodegeneration in the retinal neurones suggests a critical role for autophagy and Tor signalling, whereas there is little evidence that apoptotic cell death is induced. At the organism level, polyQ Atrophins display a glial specific toxicity which leads to a sharp decrease in lifespan. Similar intracellular autophagic modifications to those found in photoreceptor neurones are displayed and we are currently investigating whether this mechanism is responsible for the reduction in viability. Comparison with other polyQ Drosophila models underline similarities and differences in cell specific toxicity and draw an intriguing picture, which suggests that the complexity and variegation of polyQ pathologies may be due to a combination of common polyQ mechanisms and specific loss of function effects and that these diseases may affect a larger part of the nervous system than the degenerated areas, perhaps through glial malfunction.

824B Soluble Oligomers of α-Synuclein in the Neurodegeneration of Parkinson’s Disease. Madhu Gajula Balija1, DP Karpinar1, F Opazo3, B Falkenburger3, D Riedel2, A Herzig1, H Jaeckle1, S Eimer2, JB Schulz3, C Griesinger1, M Zweckstetter1. 1) Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; 2) European Neuroscience Institute, Goettingen, Germany; 3) Center for Molecular Physiology of Brain, Goettingen, Germany. Soluble aggregation intermediates and insoluble aggregates of the different aggregation-prone proteins that characterize neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and prion diseases share some common structural features. Accumulating evidence indicates that soluble oligomers of the misfolded proteins in the aggregation pathway can diffuse and interfere with synaptic structure, plasticity, vesicle trafficking and electrical properties of the neuronal membranes as exemplified by the Aβ-42 oligomers in the AD.In an attempt to investigate whether this emerging concept also holds true for the soluble α- Synculein oligomers in Parkinson’s disease, we have used a rational approach based on structure to generate a mutant form of α- Synuclein, which exclusively forms oligomeric species but does not fibrillate in the biophysical and biochemical assays. Expression of this mutant form of α-Synclein genetically in different model organisms, like Drosophila, C.elegans, and in cell culture, indicates that it is potentially toxic. In our Drosophila model for the Parkinson’s disease, we have used phiC31 site-specific integration system to control position effects, and permit precise comparisons between different transgenic constructs. We are also studying circadian rhythms and sleep behavior as sensitive readouts for the cytotoxicity by specifically expressing the various mutant forms of α- Synuclein in the Dopaminergic neurons. Altogether our results from different model organisms support that diffusible oligomers of α- Synuclein are apparently the most toxic species in the neurodegeneration of Parkinson’s disease. This realization not only indicates that soluble oligomers produced by different amyloid proteins might initiate similar cytotoxic mechanisms, but also raises the possibility of targeting them for therapy. 368 POSTERS: Drosophila Models of Human Diseases

825C Genetic screen of human neurological disease genes for defects in synaptic function and development. Dominik M. Haddad1,2, Maarten Leyssen1,2, Patrik Verstreken1,2. 1) Laboratory of Neuronal Communication, K.U.Leuven, Center for Human Genetics, Belgium; 2) VIB, Department of Molecular and Developmental Genetics, Belgium. While many genes are implicated human hereditary neurological disease, their putative role in neuronal communication often remains poorly defined. Using database analyses we have identified the Drosophila orthologues of 187 such genes. We have knocked down these genes using nervous system specific RNAi and screened the flies for neuronal dysfunction with a variety of assays. We used behavioral tests including survival, geotaxis, bang sensitivity and flying ability as these assays frequently show defects in fly disease models. Interestingly, 28 out of 152 analyzed genes show neurological defects when inactivated in neurons. To scrutinize the phenotypes in these positives, we also investigated neuronal development including assessment of the projection pattern of the LNv neurons that orchestrate circadian rhythms and determination of the neuronal morphology of the third instar motor neuron endplates that innervate the larval muscles. Finally, we tested synaptic function of photoreceptors and of motor neurons by recording electroretinograms from the surviving flies and by testing the ability of mutant neurons to internalize the fluorescent dye FM 1-43 during stimulation, a measure of synaptic vesicle cycling efficiency. Our screen identified several known genes, including imac and gars, but also numerous genes whose role in nervous system function and development is not well defined. We will present our progress on the analyses of these neurological disease genes.

826A Circadian Rhythms as Model Systems to Study the Effects of Transcriptional Dysregulation in MJD-afflicted Drosophila. Amy B. Hart, John M. Warrick. Department of Biology, University of Richmond, Richmond, VA. Machado Joseph disease (MJD) is a member of the family of human polyglutamine diseases that is caused by an extended number of CAG repeats that occur in the DNA sequence and encode an expanded polyglutamine repeat. Transcriptional dysregulation due to the inactivation of transcriptional regulators has been proposed as a mechanism of pathology in human polyglutamine disease. Circadian rhythms drive the daily locomotor activity of humans and Drosophila. These rhythms are regulated by tightly controlled transcriptional feedback loops. Analysis of daily locomotor rhythm in Drosophila may serve as a model system in which to study the role of transcriptional dysregulation in the disease pathology. Expression of full-length mutant protein Ataxin-3 (ATX-3) causes disruption in circadian activity, while the normal ATX-3 has no effect on circadian activity. A substantial rescue of circadian behavior was observed in behavioral assays of flies expressing human heat shock protein, a molecular chaperone. Co-localization of the circadian proteins PER and CLK with mutant protein was observed in the lateral ventral neurons, revealing a possible role of transcriptional dysregulation in disease pathology. Presently, the role of CLK as a histone acetyltransferase is being explored, as histone acetylation is important for rhythmic transcription. In addition, studies of the function of CREB binding protein in the disease pathway and its effect on transcription are being conducted.

827B A Drosophila Model for neuroinflammatory response in neurodegenerative disease. Arati Inamdar1, Anathbandhu Chaudhuri2, Hakeem Lawal1, Faiza Ferdousy1, Janis O’Donnell1. 1) Dept Biological Sci, Univ Alabama, Tuscaloosa, AL, 35487; 2) Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198. The pathogenic mechanisms of various chronic neurodegenerative diseases including Parkinson’s disease are proposed to be defective mitochondrial function, increased oxidative stress, apoptosis and exitotoxicity. In addition, various studies have elucidated the pathogenic role of activated microglia in initiating neuroinflammatory responses in these chronic neurodegenerative diseases. Most studies of the mechanisms of neuroinflammatory responses have been performed in mammalian cell cultures. We have established a Drosophila Parkinson’s disease model using the herbicide, paraquat. Using this model, we have tested dopamine regulatory mutant genes and found that they are differentially sensitive to paraquat (Chaudhuri et al., 2007). We now report that paraquat also causes activation of an inflammatory response in adult brain by activating nitric oxide synthase in glial cells. We tested whether minocycline, a tetracycline derivative known to inhibit the activation of microglia in various mammalian models, in our paraquat-induced neuroinflammatory model and confirm that minocycline modifies the glial response in adult brains. We also tested those genes with increased sensitivity to paraquat and found that a subset of these is also involved in glial cell activation. Thus, we report an in vivo Drosophila model to understand the mechanisms by which activated microglia cause activation of neuroinflammatory response in Parkinson’s disease. We will further report results of mutagenesis screens for potential modifiers of oxidative stress susceptibility and glial function using this model. POSTERS: Drosophila Models of Human Diseases 369

828C Overexpression and RNAi silencing the Drosophila ortholog of UBQLN1, dUbqln, decreases anoxia tolerance. A Li1, KM McKay1, Y Dong1,2, Z Xie1,2, RE Tanzi1. 1) Genetics & Aging Research Unit, Department of Neurology; 2) Department of Anesthesia and Critical Care. Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline accompanied by neuronal and synapse loss, neurofibrillary tangles (NFTs), and senile plaques. UBQLN1 variants are associated with increased risk for late- onset AD. UBQLN1 interacts with presenilins and has been detected in NFTs and Lewy bodies in brain tissue from AD and PD patients. Severe hypoxia can trigger apoptosis in the brain, which may contribute to the neuronal loss and memory impairment seen in AD patients. To further characterize the molecular function of UBQLN1 in vivo, we isolated the Drosophila ortholog of UBQLN1, dUbqln, and created dUbqln transgenic flies. We overexpressed and silenced dUbqln by RNAi (Li, et al. Hum Mol Genet 2007) in the central nervous system (CNS) with ELAV-GAL4 and ubiquitously with Actin-GAL4 and assessed hypoxia tolerance in dUbqln transgenic flies. We exposed flies at age 3-6 days old (200 flies in each group) in a specially designed chamber with anoxic conditions (O2 concentration = 0% with administration of 100% N2) for 20 minutes before allowing recovery in room air (O2 = 20.8%) and examined recovery time from anoxia. Compared with control flies (Elav-GAL4/+), flies in which dUbqln was silenced in the CNS (UAS-dUbqln- RNAi; Elav-GAL4) showed a significantly prolonged recovery time from anoxia (p=0.04 by student t test), while flies overexpressing dUbqln in the CNS (UAS-dUbqln; ELav-GAL4) also exhibited a trend toward prolonged recovery time though the differences did not reach statistical significance (p=0.07). Flies in which dUbqln was silenced ubiquitously were lethal. In flies overexpressing dUbqln ubiquitously (UAS-dUbqln; Actin-GAL4), a significantly prolonged recovery time was observed as compared with control flies (Actin- GAL4/+) or flies overexpressing dUbqln in the CNS (p=0.01). Collectively, these data demonstrated that altered expression of dUbqln decreases tolerance to O2 deprivation in Drosophila.

829A modeling the role of human ApoD in neuropathologies. Julien Muffat, Seymour Benzer. Dept Biol, CalTech, Pasadena, CA. Apolipoprotein D expression increases in several neurological disorders, and in spinal cord injury. We provide evidence of a physiological role for human ApoD: flies overexpressing hApoD are long-lived, and protected against stress conditions associated with aging and neurodegeneration, including hyperoxia, dietary paraquat, and heat. We have shown that the fly orthologs of hApoD are strongly upregulated by these extrinsic stresses, and also can protect in vitro cultured cells in situations modeling Alzheimer’s disease (AD) and Parkinson’s disease (PD). In adult flies, hApoD overexpression reduced age-associated lipid peroxide accumulation, suggesting a proximal mechanism of action. These data suggest that ApoD and its homologs play an evolutionarily-conserved role in response to stress, possibly managing lipid peroxidation in degenerating cells. We are now testing the ability of these proteins to rescue fly models of neurodegenerative diseases such as AD or PD, as well as models of Wallerian degeneration in peripheral nerve. To that end we are using various techniques, including laser resection of wing or leg nerves, antennal extirpation, and crush injury to the legs. We are also studying, in Drosophila, the recruitment of the proteins to puncture wounds to the thoracic flight muscle, characterized by massive apoptosis, in an attempt to model the known recruitment of hApoD to lesions, in particular lesions of the nervous system.

830B NEUROTROPHIC FACTORS GDNF AND BDNF AS APPROACH FOR GENOTHERAPY OF NEURODEGENERATIVE DISORDERS. Ekatherina A Nikitina, Elena V. Savvateeva-Popova. Neurogenetics, Pavlov Institute of Physiology, St-Petersburg, St-Petersburg, Russian Federation. Neurodegenerative diseases, such as Huntington’s (HD), Parkinson’s (PD) are characterized by a late onset disturbance of memory, synaptic and glial pathology, neurodegeneration of neurons of the substantia nigra and striatum. Strategies to rescue or to protect injured neurons usually involve promoting of neuronal growth and function. A large number of therapeutic approaches have been explored for their potential in the treatment of PD and HD using animal models which mimic these disorders. Neurotrophic factor therapy is a promising approach in that it addresses the basic mechanism underlying neurodegeneration that occurs in PD and HD. Neurotrophic factors are difficult to deliver to the brain in humans in the clinical setting. However, human growth factors such as the glial cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) can be expressed in animal model systems,such as Drosophila, to study their effects. We investigated learning ability and memory formation in transgenic flies which carry GDNF under HS promoter. HS leads to improvement of memory in this mutant. Moreover, we investigated learning acquisition and memory retention in transgenic flies which carry BDNF under HS promoter using conditioned courtship suppression paradigm following HS. However, we did not observe such improvement of memory in BDNF mutant, as in GDNF transgenic flies. It may be related to different effects of these neurothrofic factors on memory formation. Supported by grant of St-Petersburg Government. 370 POSTERS: Drosophila Models of Human Diseases

831C Degradation of functional TPI protein underlies sugarkill pathology. Michael Palladino1,2, Jacquelyn Seigle1,2, Alicia Celotto1,2. 1) Pharmacology Dept, Univ Pittsburgh, School of Medicine Pittsburgh, PA. 15261; 2) Pittsburgh Institute for Neurodegenerative Diseases University of Pittsburgh School of Medicine Pittsburgh, PA 15260. TPI deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The condition is caused only by specific missense mutations affecting the triose phosphate isomerase protein (TPI) and is characterized clinically by anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehydes 3 phosphate (G3P), however, little is known about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPIsugarkill mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, is capable of forming a homodimer and retains function, but the mutant protein is efficiently degraded by the major 20S proteosome leading to loss-of-function pathogenesis.

832A A Drosophila model for motor neuron disease caused by a mutation in VAPB/DVAP33A: Evidence for a dominant negative mechanism. Anuradha Ratnaparkhi, George Lawless, Felix Schweizer, Peyman Golshani, George Jackson. University of California, Los Angeles, Los Angeles, CA. A dominant missense mutation (P56S)in the evolutionarily conserved protein, VAPB (VAMP associated membrane protein B/ ALS8) has been shown to cause ALS and spinal muscular atrophy Nishimura et al., 2004, Am J Hum Genet 75: 822-31. Although mutations in VAPB are a rare cause of ALS, insights gained from study of rare familial forms of disease may provide powerful insights into the pathogenesis of more common sporadic forms. We have established a fly model of ALS8 using a corresponding mutation(P58S) in fly VAPB/dVAP33A. Previous study has shown that dVAP33A regulates bouton size and microtubule organization at the 3rd instar larval neuromuscular junction (Pennetta et al., 2002, Neuron, 35:291-306). We find that expression of P58S-VAP affects synaptic morphology in a manner similar to dVAP mutant. Expression of the mutant protein also interferes with the function of the wild type, consistent with a dominant negative effect of the ALS8 mutation. This model provides a platform with which to begin to identify genetic modifiers of VAPB that may provide powerful insights into the mechanisms of neuronal dysfunction and death that play a role in ALS8 and more generally in sporadic ALS.

833B Developmental functions of two acyl-CoA synthetases, Bubblegum and Double Bubble , in Drosophila. Anna Sivatchenko, Anthea Letsou. Dept Human Genetics, Univ Utah, Salt Lake City, UT. The Drosophila melanogaster homologous genes bubblegum (bgm) and double bubble (dbb) code for very-long-chain fatty-acid (VLCFA) acyl-CoA synthetases that are required for degradation of VLCFAs. Sequence similarity of the bgm and dbb genes and their close proximity in the genome led us to the hypothesis that they are duplicated genes playing redundant developmental roles. Consistent with this idea is our demonstration that dbb and bgm exhibit overlapping expression profiles and are co-regulated by the Dorsal pathway that is essential for specification of ventral cell fates during dorsoventral patterning in Drosophila embryos. Whereas an amorphic bgm allele was pre-existing, we generated dbb and bgm dbb mutant animals by homologous recombination. Amorphic mutations in bgm , dbb, or bgm dbb are homozygous viable, but exhibit neurological defects. Loss of both bgm and dbb leads to disruption of fenestrated membrane, and this phenotype is enhanced in double mutant, revealing their important overlapping functions in the retina. It has been previously suggested that neurodegeneration and VLCFA accumulation in Drosophila mutant bubblegum point to a link between bgm phenotype in flies and human peroxisomal disorder X-ALD. We are currently testing if bgm, dbb and bgm dbb mutants recapitulate other aspects of the peroxisomal disorders. POSTERS: Drosophila Models of Human Diseases 371

834C A Genetic Screen in Drosophila for Modifiers of Ab42-induced lifespan. Ho-Juhn Song1, Neda Hoseinovic1, Thomas Neufeld2, Mary Konsolaki3, Daniel Curtis1, Dan Garza1. 1) Dept Dev Molec Pathway, Novartis, Cambridge, MA; 2) Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 3) Department of Genetics, Rutgers University, Piscataway, NJ. Alzheimer’s disease (AD) is a human elderly dementia known to be caused by the accumulation of amyloid peptides, produced from proteolytic cleavage of the amyloid precursor protein (APP). While one of the amyloid protein species, Ab42, is known to be the major constituent of amyloid plaques, it still remains unclear how it becomes toxic to neurons. To understand the pathology of Ab42 we have been using a Drosophila AD model in which Ab42 peptide is expressed in the fly central nervous system. We present here the results of a genetic screen to identify modifiers of a shorted lifespan phenotype induced by Ab42 toxicity in the CNS. A total of 1757 P element insertions were screened in the heterozygous condition (+/-) against the lifespan phenotype, leading to the identification of a total of 38 suppressors and 28 enhancers. Both lifespan phenotype and locomotor deficits are suppressed by heterozygosity for dTor loss-of-function mutations, and this suppression can also be achieved by pharmacological treatment of Ab42-expressing flies with the Tor inhibitor RAD001. In contrast, reduced gene dosage for Atg1 enhanced both phenotypes. Total Ab42 was reduced in Ab42 flies heterozygous for dTor but increased in flies heterozygous for Atg1, suggesting that the observed suppression of Ab42- mediated neurotoxicity occurs at least in part through increased clearance of Ab42 by autophagy-associated processes. Other genetic modifiers identified using this neurotoxicity model will be presented, and reveal potential insights into the mechanisms of Ab-mediated neurotoxicity.

835A Modified co-activator levels in Spinocerbellar Ataxia 3 drosophila model. Naoum Tavantzis, John Warrick. University of Richmond, Richmond, VA. Spinocerebellar Ataxia 3, also known as Machado-Joseph Disease (MJD) is a hereditary human disease that causes neurodegeneration due to polyglutamine residues in the Ataxin 3 protein. The disease is characterized by the creation of insoluble aggregates in the cell nuclei. Currently we are using a transgenic Drosophila model to study the normal Ataxin 3 protein and the mutant protein, and the effects of modifiers, such as drosophila CREB binding protein (dCBP) in the disease development. dCBP is a transcription co-activator and has histone acetyltransferase activity, and has been implicated in polyglutamine disease pathology, and specific levels of dCBP are required during development of the fly eye; overexpression and underexpression will disrupt eye morphology. To combat this, we are using the Rhodopsin-1 Gal4 driver, which begins to be expressed late in development, so the dCBP will be expressed in adult photoreceptors to avoid the morphology problems caused when the levels of dCBP are altered in the fly eye. Doublestranded RNA-mediated interference (RNAi) against dCBP will be used to lower the level of CBP in the adult fly eye. EP 1179 encodes for the Drosophila dCBP, and in the presence of the GAL4 transcription factor will overexpress the dCBP and we will demonstrate how raised and lowered levels of CBP influence adult eye degeneration.

836B Genetic evidence for the involvement of the 26S proteasome in age-related neurodegenerative diseases. Ayako Tonoki1, Erina Kuranaga1,2, Takeyasu Tomioka1, Jun Hamazaki3, Shigeo Murata3, Keiji Tanaka3, Masayuki Miura1,2. 1) Department of Genetics, Grad. Sch. Pharm. Sci., University of Tokyo; 2) JST, CREST; 3) Laboratory of Frontier Science, Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science. Aberrant protein aggregation and late-onset of symptoms are common features of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease. The ubiquitin-proteasome system (UPS), which contributes to protein quality control by degrading unfolded or misfolded proteins, is closely related to the etiology of neurodegenerative disorders. Although they generally do not appear or progress until late in life, it has not been clear why the risk of contracting neurodegenerative diseases should increase with age or what factors influence this increased risk. Here we show that attenuation of the 26S proteasome during aging underlies the progression of polyglutamine-induced toxicity in vivo. The 26S proteasome is composed of one, proteolytically active, core particle (alias 20S proteasome) and one or two 19S regulatory particles (RPs) subdivided into lid and base subcomplexes. In a genetic gain-of-function screen, we identified one of the lid subunits as a suppressor of expanded polyglutamine-induced neurodegenerative phenotypes in a Drosophila model. The overexpression of the lid subunit prevented the age-dependent reduction of proteasome activity that was associated with impaired assembly of the 19S and 20S proteasome and suppressed the accumulation of ubiquitinated proteins. Our results demonstrate that an age-related reduction in 26S proteasome activity is the key factor of the increased risk for late-onset of neurodegeneration, and implicate that improving the amount of the 26S proteasome by providing an overexpressed lid subunit or an unidentified lid assembler will provide a way to inhibit the onset of neurodegeneration in vivo. 372 POSTERS: Drosophila Models of Human Diseases

837C Roles of E3 ubiquitin ligases in polyglutamine diseases. Azaria K.Y. Wong1,2, Alan S.L. Wong1,2, C.M. Chan1,3, H.Y. Edwin Chan1,2,3. 1) Laboratory of Drosophila Research; 2) Molecular Biotechnology Programme; 3) Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong SAR, China. Polyglutamine diseases define a group of late-onset, progressive neurodegenerative disorders caused by CAG trinucleotide repeat expansion in the coding region of the disease genes. Such expansion results in the translation of an expanded polyglutamine tract which confers a gain-of-function toxicity to the disease proteins and causes neuronal dysfunction. Misfolding and aggregation of the disease proteins are common features of polyglutamine diseases. Several lines of evidence suggest that dysregulation of protein degradation is involved in polyglutamine pathogenesis. Ubiquitin-proteasome system (UPS) is responsible for proper protein degradation in the cells, which involves the conjugation of ubiquitin to cellular protein substrates. E3 ubiquitin ligases play essential roles in substrate recognition. Some components of UPS including E3 ubiquitin ligases are known to be recruited to the polyglutamine protein aggregates and alteration of their activities associates with polyglutamine-induced toxicity. These suggest that E3 ubiquitin ligases and polyglutamine pathogenesis are linked. Using a Drosophila polyglutamine disease model, we further explore the functions of E3 ubiquitin ligases in polyglutamine-induced toxicity.

838A Analysis of the de novo purine synthesis gene CG3590 encoding adenylosuccinate lyase. Denise V. Clark, Bethany R. Herrmann. Dept Biol, Univ New Brunswick, Fredericton, NB, Canada. The de novo purine synthesis pathway produces inosine monophosphate (IMP) in 10 steps and then branches to produce AMP and GMP, each in 2 steps. Adenylosuccinate lyase (ADSL) catalyzes 2 steps in this pathway, one in the production of IMP and the other in the production of AMP. In humans, mutations in the ADSL gene have been associated with accumulation of enzyme substrates and various neurodevelopmental disorders including autistic features and epilepsy. In Drosophila melanogaster, there is a single ADSL ortholog, CG3590, on chromosome 3R. CG3590 is intronless and lies within the 11 kb intron 1 of Pxd, a peroxidase gene. So far, loss of function mutations in other de novo purine synthesis genes have a pupal lethal phenotype. In contrast, a lethal piggyback insertion CG3590 c02781, 6 bp upstream from the transcription start, causes arrest at the 2nd larval instar. However, we could not detect either CG3590 or Pxd transcripts in this mutant by RT-PCR. Thus, in an effort to obtain independent alleles of CG3590 and Pxd, we used a viable P element insertion 2 kb upstream of CG3590, but still within Pxd intron 1, to generate directional deletions by male recombination, either extending toward CG3590 or Pxd. We will present an update on our phenotypic and molecular analysis of these mutations, as part of our effort to determine whether the Drosophila ADSL gene could serve as a model for human ADSL deficiency.

839B LOW CHLOROPHYLLIN CONCENTRATIONS INCREASE GENETIC DAMAGE INDUCED BY GAMMA RAYS IN DROSOPHILA MELANOGASTER. Martha P. Cruces1,2, Emilio Pimentel1. 1) Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Mexico, Mexico; 2) Posgrado en Ciencias Biologicas, UNAM. Chlorophyllin (CHLN) possesses a potent activity, reducing or inhibiting the genetic damage induced by physical and chemical agents. However, there are data demonstrating that in certain circumstances CHLN has an inverse effect increasing the genetic damage of certain agents. In our previous work using the wing spot test, we obtained clear evidence of this effect: in pretreatment with a feeding solution of 69 mM of CHLN to 48h-old Drosophila larvae for 24 h and after a delay of 0,1,2,3 days, treatment with chromium (VI) oxide (CrO3) had a protective effect on days 0 and 1, and an enhancer effect after 2 and 3 days of delay following the exposure to CrO3. In the present study we tested the hypothesis that the low dose of CHLN can cause an increase in the DNA damage induced by some agents. Eight groups of larvae 48 h old were pretreated for 24 h, each one with differing low concentrations of CHLN (0-69 mM), after that were irradiated with 10 Gy of gamma rays. The results confirmed the radioprotective effect of 69mM of CHLN and provided evidence that low CHLN concentrations (1.1 and 2.2 mM) increased the mutation frequency (P < 0.02) compared with the 10Gy control and higher concentrations of CHLN + 10 Gy. The increase in genetic damage induced by the gamma rays promoted by low doses of CHLN seems to be the general effect since this was also observed in other systems. The promoter effect of low doses of CHLN could be related with its intercalation capacity. POSTERS: Drosophila Models of Human Diseases 373

840C Effects of tissue-specific expression of mutant forms of Lamin C: Drosophila as a model for Emery-Dreifuss muscular dystrophy. Diane E. Cryderman1, George Dialynas1, Sandra R. Schulze2, Beatrice Curio-Penny1, Pamela K. Geyer1, Lori L. Wallrath1. 1) Biochemistry, University of Iowa, Iowa City, IA; 2) Biology, Western Washington University, Bellingham, WA. A network of intermediate filament proteins called lamins line the inner side of the nuclear envelope. Lamins possess an N- terminal globular domain, central rod and C-terminal globular domain. Lamins are ubiquitously expressed, yet mutations in human genes encoding lamins give rise to tissue-specific diseases called laminopathies, such as Emery-Dreifuss muscular dystrophy. We are using Drosophila as a model to determine the tissue-specific functions of lamins in development, with implications for understanding the human disease mechanisms. Transgenic flies were generated that express wild type or mutant forms of Lamin C; the mutant forms include domain truncations and amino acid substitutions that mimic human disease forms. Ubiquitous expression of wild type Lamin C did not affect nuclear architecture or viability. Ubiquitous expression of mutant forms containing amino acid substitutions in the rod domain resulted in nuclear defects similar to those observed with human disease and did not affect viability. In contrast, ubiquitous expression of an N-terminal deletion or a mutant form possessing an amino acid substitution within the C-terminal globular domain caused lethality at the pupal stage, yet larvae exhibited no nuclear defects. These data demonstrate that nuclear defects are not responsible for lethality. To address tissue-specific functions of Lamin C, mutant forms were expressed using the Gal4-UAS system. The N-terminal deletion and a specific amino acid substitution within the C-terminal globular domain caused lethality when expressed in muscle, but not non-muscle tissues. Lethality was observed upon muscle-specific expression in the embryo or larvae, but not adult. In one case, semi-lethality resulted in a few adult escapers possessing leg defects similar to those caused by defects in ecdysone signaling. Collectively, these data demonstrate a temporal and tissue-specific role for Lamin C in Drosophila development.

841A Genetic screens uncover new genes potentially involved in the pathogenesis of Myotonic Dystrophy Type 1. Maria de Haro1, Ismael Al-Ramahi1, Thomas Cooper2, Juan Botas1. 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) Dept Pathology, Baylor Col Medicine, Houston, TX. Myotonic Dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG expansion in the 3’ UTR of the DMPK gene. Most experimental observations indicate that pathogenesis in DM1 is triggered by a toxic gain of function of the expanded DMPK RNA. We have generated a Drosophila DM1 model expressing a non-coding mRNA containing 480 interrupted CUG repeats. This (iCUG) 480 transcript accumulates in nuclear foci and its expression leads to muscle wasting and degeneration in Drosophila as well splicing pattern alterations in several genes. We found that altering the levels of two RNA-binding proteins known to be involved in DM1 pathogenesis, MBNL1 and CUGBP1, modify the (iCUG) 480 degenerative phenotypes. Expanded CUG-induced toxicity in Drosophila is suppressed by overexpression of human MBNL1, and enhanced when MBNL1 endogenous levels are reduced. In contrast, increasing the levels of CUGBP1 worsens (iCUG) 480-induced degeneration. Based on these data we sought to find novel genes able to modify the expanded CUG-induced phenotypes when altering their levels, in both an unbiased and a biased screen. We have identified several genes able to modify the CUG-induced degeneration. These genes are involved in new pathways not known to be involved in DM1 pathogenesis such as cell cycle control and nervous system maturation.

842B The H. pylori virulence factor, CagA, interacts with Rho signaling to disrupt epithelia. Jonathan B Muyskens, Crystal Botham, JT Neal, Anica Wandler, Lucy Cho, David Reid, Karen Guillemin. Institute of Molecular Biology, University of Oregon, Eugene, OR. Nearly half the world’s population is infected with the gastric bacterial pathogen, Helicobacter pylori. A key virulence factor of H. pylori that has been implicated in a variety of gastrointestinal diseases, such as stomach cancer, ulcer and gastritis, is CagA, a protein that shares homology with no known protein. We would like to understand how CagA induces pathologies associated with H. pylori infection, and specifically how CagA disrupts epithelia, such as the the epithelial lining of the gastrointestinal tract. To this end, we are expressing CagA in epithelial tissues within the fly. These Drosophila epithelial tissues share key features with the epithelium of the human stomach, such as junctional complexes and cell polarity, however, in contrast to the human stomach, they are amenable to genetic manipulation and imaging studies. Our initial studies have involved expressing CagA in the developing eye epithelium using transgenic flies containing a UAS-CagA transgene under the control of the GMR driver, which drives expression throughout the developing eye. The eye was a particularly attractive tissue to initiate these studies because it is easily accessible and thus amenable to screening, and has been well characterized developmentally. Our work reveals that CagA expression precisely phenocopies the disruption of the larval eye disc epithelium observed in larvae with perturbed Rho1 activity. Rho1 is a small GTPase required for epithelial maintenance and cell movement in a variety of contexts. In addition, we show that CagA activates myosin regulatory light chain, a target of Rho, when expressed in eye discs, and that CagA can disrupt the polarized localization of myosin regulatory light chain in the border cells of the fly ovary, which have been used as a model for invasive cell migration. Thus, our studies provide phenotypic evidence that CagA acts through Rho1 to disrupt epithelia, and thus, uncover a novel target for therapy for H. pylori infection. 374 POSTERS: Drosophila Models of Human Diseases

843C Stimulation of the mitochondrial replicase by deletion mutants of mitochondrial single-stranded DNA-binding protein. Marcos T Oliveira, Laurie S Kaguni. Graduate Program in Genetics and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI. Biochemical studies of the human mitochondrial replisome demonstrate that the mitochondrial replicase and the mtDNA helicase Twinkle are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSBs share similar physical and biochemical properties with Escherichia coli SSB, and a high degree of conservation in the central region of their amino acid sequences; however, the roles of their variable N- and C-termini are not understood. The aim of this work is to evaluate the stimulation of the human mitochondrial replicase (Hspol γ) by its cognate mtSSB (HsmtSSBwt) and mutants lacking the N (HsmtSSBΔN), the C (HsmtSSBΔC), and both termini (HsmtSSBΔNΔC), and to compare it to the functional interactions between Drosophila pol γ (Dmpol γ) and its cognate mtSSB (DmmtSSBwt). Comparative gel mobility shift analyses indicate that the DNA binding affinity of the untagged DmmtSSBwt is ~ 60 fold higher than an N-terminally His-tagged version of HsmtSSBwt. However, in a reaction that mimics mitochondrial lagging DNA strand synthesis, we show that the N-terminally His-tagged version of HsmtSSBwt and the untagged HsmtSSBΔN stimulate Hspol γ ~ 10 fold, in a manner similar to Dmpol γ stimulation by its cognate mtSSB. Interestingly, both human proteins show stimulatory effects that are ~ 2 fold higher than that of the untagged HsmtSSBwt. Although differing physical interactions may account for these observations, we are examining these and various biochemical properties of the mutants to understand the mechanism by which mtSSB stimulates DNA synthesis by pol γ. This work is supported by grant GM45295 from the National Institutes of Health.

844A Dominant modifier screens reveal components that interact with the Drosophila Dg-Dys complex. Mario Pantoja1, Mariya Kucherenko1,2, Andriy Yatsenko1,2, Karin Fischer1, Halyna Shcherbata1, Hannele Ruohola-Baker1. 1) Department of Biochemistry, University of Washington, Seattle, WA; 2) Ivan Franko National University in Lviv, Lviv, Ukraine. Many lines of evidence confirm that maintaining the structural link between the extracellular matrix ECM to the actin cytoskeleton is crucial in preventing many forms of Muscular Dystrophy. This conduit is formed by the transmembrane protein Dystroglycan (Dg) binding to laminin of the ECM and to dystrophin (Dys) which binds to cytoskeletal actin in the cytoplasm. Previously, we had initiated a characterization of the Drosophila Dystroglycan-dystrophin (Dg-Dys) complex by determining that mutations in this complex show abnormalities in the fly that are similar to symptoms shown by patients with common forms of Muscular Dystrophy. These abnormalities include age dependent muscle degeneration, defects in eye development and a shorter life span. Recently, other labs have also reported that there are heart defects in the flies mutant for components of the Dg-Dys complex. Since cardiomyopathy is another symptom of patients with common forms of Muscular Dystrophy this is another aspect of the disease that can be modeled in flies. These many similarites make Drosophila an attractive model for further clarification of how the Dg-Dys complex functions. We have used a wing vein phenotype seen in Dg and Dys mutants as a basis for several dominant modifier screens with the idea of finding components that may shed light on the action and regulation of the Dg-Dys complex. We have found interactors of the complex that belong to the TGFβ and EFGR signaling pathways as well as cytoskeletal components and genes involved in cell/neuronal migration.

845B Drosophila melanogaster as model system to study mitochondrial respiratory chain diseases. A. Sanchez-Martinez1, R. Hernández-Sierra1, M. Calleja1, S. Peralta1, V. Domingo1, R. Garesse1, N. Luo2, C. Farr2, Y. Matsushima2, L. S. Kaguni2. 1) Departamento de Bioquimica. Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM. CIBERER ISCIII, Facultad de Medicina, Universidad Autónoma de Madrid, Spain; 2) Department of Biochemistry and Molecular Biology, Michigan State University,East Lansing, Michigan 48824-1319, USA. One of the major challenges in the field of mitochondrial diseases is to generate animal models that reproduce the symptoms associated with the dysfunction of the respiratory chain. Drosophila melanogaster is an excellent model system to study complex biological processes due to its easy manipulation at genetic, biochemical, molecular and physiological levels. The presence in Drosophila of orthologous genes to those involved in human mitochondrial diseases, makes it an excellent model organism for understanding the pathophysiology of this complex group of disorders. To induce an mtDNA mutator phenotype we have generated by homologous recombination transgenic flies that express a catalytic subunit of mitochondrial DNA polymerase (PolG-A) lacking the exonuclease 3'-5' proofreading activity (PolG-Aexo-). In addition, using an RU485 inducible UAS-GAL4 system, we have overexpressed the PolG-Aexo- version specifically in adult flies. This strategy avoids the overexpression of the defective enzyme during embryogenesis and allows studying the effect of the accumulation of mtDNA mutations mostly in postmitotic tissues. We have also established several transgenic lines harboring specific point mutations in the mitochondrial helicase gene associated with adPEO in humans. We are using the UAS-GAL4 system to overexpress the different helicase versions and characterize its molecular impact on mtDNA replication. POSTERS: Drosophila Models of Human Diseases 375

846C Laboratory selection for hyperoxia-tolerance in Drosophila Melanogaster: role of single genes in tolerance. Huiwen W. Zhao1, Dan Zhou1, Gabriel G. Haddad1,2,3. 1) Departments of Pediatrics; 2) Departments of Neuroscience, University of California San Diego, La Jolla, CA 92093; 3) The Rady Children’s Hospital, San Diego, CA 92123. High O2 is deleterious for cells and tissues in vertebrates and invertebrates. In our laboratory, we have developed a Drosophila strain that is extremely resistant to high O2 and can survive perpetually at 90% O2, without overt signs of injury. In order to understand the basis for this tolerance, we utilized microarrays and a mutagenesis screen to identify the genes that contribute to this tolerance. We found that 641 and 272 genes had altered expression in the hyperoxia-selected 3rd instar larvae and adult flies respectively (p<0.0001, 1.5-fold), as compared with the normoxia strain. We also tested 138 P-element Drosophila mutants corresponding to the genes that were changed by at least 1.7- fold in microarrays, using hyperoxia and paraquat. The majority of P- element lines can not tolerate either hyperoxia or paraquat, only seven out of 138 P-element lines were found to significantly tolerate both hyperoxia and paraquat treatments, among the genes that played a role were Glutathione S transferase D2 and CG4666. Precise excision of P-element allele reversed the tolerance to paraquat, indicating that a greater survival after paraquat treatment was related to the P-element insertion in the target gene. Therefore, we conclude that a) single gene modifications can play a role in the tolerance to hyperoxia, and b) hyperoxia may not share the same mechanisms and pathways for inducing injury and survival as paraquat. 376 POSTERS: Physiology and Aging

847A Beneficial role of ad libitum water on lifespan in Drosophila melanogaster. William W. Ja1, Ted Brummel2, Seymour Benzer1. 1) Division of Biology, California Institute of Technology, Pasadena, CA; 2) Department of Biology, Long Island University, Brookville, NY. Dietary restriction experiments in Drosophila are usually performed by dilution of the food medium. However, this has been shown to result in compensatory ingestion and, in some cases, food consumption is largely or fully compensated for, resulting in equivalent nutrient intake on the different media. These experiments fail to account for the differences in fluid intake. We find that providing an ad libitum accessory water supply increases fly lifespan, especially at higher food concentrations, where fluid intake is ordinarily the least due to compensatory feeding. Our results suggest that the longevity presumably produced by dietary restriction may at least be partially attributable to better hydration. In some food medium paradigms, lifespan changes due to food medium dilution can be completely eliminated by providing access to water.

848B Dietary Restriction Increases Lifespan by Targeting Anti-oxidative Defense Systems. Hadise Kabil1,2, Lawrence Harshman1, Scott Pletcher2. 1) School of Biological Sciences,University of Nebraska-incoln, Lincoln, NE; 2) Huffington Ctr on Aging, Baylor Col Medicine, Houston, TX. Dietary restriction (DR) extends lifespan from yeast to mammals. Beneficial readouts of DR at physiological level have been relatively well characterized. However the mechanism translating DR into longer lifespan is not understood. It has been proposed that DR reduces the levels of reactive oxygen species (ROS) and that this reduction in ROS is responsible for the lifespan extending effects of DR. In this study we show that DR induces a robust antioxidative defense system and leads to increased levels of small detoxifying molecules. Inhibition of key biochemical steps supportive for the maintenance of a robust antioxidative defense system results in the loss of lifespan extension by DR. We also show that a diluted anti-oxidative defense system results in significant metabolic shifts affecting both protein and lipid metabolisms.

849C Juvenile hormone as a regulator of the trade-off between reproduction and lifespan in Drosophila. Thomas Flatt1,2, Tadeusz Kawecki2,3. 1) Brown University Division of Biology and Medicine Department of Ecology and Evolutionary Biology Box G-W, 80 Waterman Street Providence, 02912 Rhode Island USA; 2) University of Fribourg Unit of Ecology and Evolution Chemin du Musee 10 CH-1700 Fribourg Switzerland; 3) Department of Ecology and Evolution University of Lausanne Biophore CH-1015 Lausanne Switzerland. Trade-offs between reproduction and lifespan are ubiquitous, but little is known about their mechanisms. Here we combine hormonal manipulation with experimental evolution to investigate the role of juvenile hormone (JH) in mediating such trade-offs. To induce evolutionary changes in JH metabolism or signaling, we selected flies for for resistance to the JH analog (JHa) methoprene in larval medium. We predicted that (1) selected lines become less sensitive than unselected lines to the effects of JHa on lifespan and fecundity and (2) JHa resistance caues lower sensitivity of flies to their own JH. If so, then selected lines should show lower fecundity and longer lifespan than control lines even without JHa treatment. Exposure to JHa in larval medium increased early-life fecundity but reduced lifespan of unselected flies, supporting the role of JH in mediating the trade-off. This effect was much smaller for lifespan, and not detectable for fecundity, in fly lines previously bred for 19 generations on medium containing JHa. Furthermore, selection lines lived longer than unselected controls even in absence of JHa treatment, without a reduction in early-life fecundity. Thus, selection for JHa resistance induced evolutionary changes in JH metabolism or signaling, which led to longer lifespan as a correlated response. Together with previous evidence, these results suggest that JH is a mediator of the trade-off between reproduction and lifespan. POSTERS: Physiology and Aging 377

850A Couch potato aging in Drosophila. Paul Schmidt1, Mariska Batavia1, CT Zhu2, Walter Eanes2. 1) Dept Biol, Univ Pennsylvania, Philadelphia, PA; 2) Department of Ecology and Evolution, SUNY Stony Brook. Reproductive quiescence is often associated with reduced senescence and lifespan extension, as exemplified by the dauer phenotype in Caenorhabditis elegans . An analogous and potentially homologous phenotype, reproductive diapause, occurs in Drosophila melanogaster. This study examined the effects of an identified gene for reproductive diapause, couch potato (cpo), on aging, reproduction, and stress resistance. Expression level of cpo was experimentally manipulated with P-element hypomorphic alleles and wildtype revertants, as well as gene duplications and deletions, in a common genetic background. These manipulations had consistent and predictable effects on patterns of longevity and resistance to multiple stressors: reduced function of cpo was associated with a progressive decrease in longevity and decline in stress resistance, whereas increased expression above normal wildtype resulted in lifespan extension, reduced rates of age specific mortality, and elevated resistance to stress. Furthermore, the functional significance of wild derived cpo alleles on aging was investigated with gene specific quantitative complementation schemes. Allelic variation segregating at cpo in natural populations had significant and differential effects on all assayed traits except resistance to high temperatures; these functional differences also appear to be associated with variation in expression among cpo variants. Our results identify cpo as a novel gene for aging in Drosophila and show that natural allelic variation at cpo has pronounced effects on aging and correlated traits.

851B Sumoylation is necessary for the metamorphosis of Drosophila melanogaster. Ana Talamillo1, Jonatan Sánchez1, Coralia Pérez1, David Martín2, Rafael Cantera3,4, Rosa Barrio1. 1) Functional Genomics, CIC bioGUNE, Derio, Bizkaia, Spain; 2) Institut de Biologia Molecular de Barcelona-CSIC, Barcelona, Spain; 3) Stockholm University, Stockholm, Sweden; 4) IIBCE, Montevideo, Uruguay. To study in vivo the role of the ubiquitin-like protein Smt3 (Sumo) during Drosophila development, we generated transgenic flies carrying the transgene UAS-smt3i to reduce smt3 mRNA levels in specific groups of cells. Low smt3 in the prothoracic gland, the tissue responsible for the synthesis of ecdysteroids, prevents metamorphosis. RNAi knockdown larvae stop their development in their last larval stage and remain alive for up to a month, during which they continue to eat and gain weight. Their prothoracic glands have fewer, but larger cells than normal. These results are in concordance to larvae mutant in lesswright, the homologue of the Sumo conjugating enzyme gene Ubc9. They also have lower ecdysteroid titer than WT. After dietary administration of exogenous ecdysone some of these larvae form pupal cases, but do not proceed further in development and die. However, addition of cholesterol or 7-dehydrocholesterol does not rescue the larval phenotype, indicating that sumoylation must be necessary for later steps in the ecdysteroid synthesis pathway. Interestingly, we observed that, in larvae with lower levels of smt3, as well as in lesswright mutants, the subcellular localization and expression levels of factors involved in the regulation of ecdysteroids synthesis are altered.

852C Neverland, a conserved Rieske-type family of proteins essential for ecdysone synthesis and cholesterol metabolism in the prothoracic gland. Takuji Yoshiyama1, Ryusuke Niwa2, Hiroshi Kataoka1. 1) Department of Integrated Biosciences, The University of Tokyo, Kahiwa, Chiba, Japan; 2) Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT. Conversion of cholesterol to 7-dehydrocholesterol (7-DHC) is the first step of the ecdysone biosynthesis pathway in the prothoracic gland (PG). This step is hypothesized to be catalyzed by a microsomal Cytochrome P450 oxygenase. Previously we have reported that a conserved Rieske-type oxygenase Neverland (Nvd) is essential for 7-DHC formation in the PG and insect development. During embryonic and larval development, nvd is expressed specifically in the PG. Loss of nvd function in the PG causes arrest of both molting and growth. This phenotype is rescued by application of 20-hydroxyecdysone or 7-DHC but not of cholesterol. Therefore, we have postulated that Nvd functions in the conversion of C to 7-DHC in the ecdysteroidogenic pathway. The molecular function of Nvd, however, has not been characterized completely. Here, we report the subcellular location, biochemical functions, and effects of site-directed mutagenesis of Nvd. Our results suggest that Nvd family proteins have a potential role in steroid hormone synthesis and cholesterol metabolism in eukaryotes. 378 POSTERS: Physiology and Aging

853A Characterization of ponchik, a novel gene regulating appetite, adiposity, and lifespan in Drosophila melanogaster. Tammy P. Chan1,2, Sergiy Libert1,3, Emmeline Peng1, Jessica E. Zwiener1, Danielle A. Skorupa1,3, Scott D. Pletcher1,2,3,4. 1) Huffington Center on Aging, Baylor College of Medicine, Houston, TX; 2) Program in Developmental Biology, Baylor College of Medicine, Houston, TX; 3) Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, TX; 4) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX. Systems of energy balance and nutrient storage are evolutionarily conserved, suggesting that mechanistic links among longevity, energy utilization, and health are ancient. Identification of new genes involved in energy balance will lead to a better understanding of the effects of diet on obesity and health span and may provide insight into prevention of age-related functional decline. In a novel screen using Drosophila melanogaster, we identified a gene important for energy balance that we named ponchik. Ponchik mutant animals maintain high levels of triglycerides and exhibit increased fecundity, which are both suggestive of aberrant triglyceride mobilization and utilization. Additionally, these animals have elevated feeding rates, and preliminary data suggests that they are long-lived. Using Lac-Z staining of a ponchik reporter construct, we determined that the gene is expressed in the brain and ventral ganglion. Bioinformatic analysis revealed homology to human neutral sphingomyelinase (N-SMase) activation associated factor, which regulates sphingomyelin, a lipid surrounding nerve cell axons that has been implicated in aging and longevity. Finally, a series of mutant alleles of ponchik has been generated, allowing for detailed characterization of function. Our data suggest that ponchik may play a critical role in coordinating organismal responses to nutrients. Current studies are aimed at elucidating how different nutrients are utilized in ponchik mutants and at molecular characterization of these alleles.

854B Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic and computational systems analysis. Laurence Coquin1, Jacob Feala2, Andrew McCulloch2, Giovanni Paternostro1,2. 1) The Burnham Institute for Medical Research, 10901 N. Torrey Pines road, La Jolla, CA; 2) Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, 0412, La Jolla, CA 92093-0412. The fruit fly Drosophila melanogaster offers promise as a genetically tractable model for studying adaptation to hypoxia at the cellular level, but the metabolic basis for extreme hypoxia tolerance in flies is not well known. Using 1H NMR spectroscopy, metabolomic profiles were collected under hypoxia. Accumulation of lactate, alanine, and acetate suggested that these are the major end products of anaerobic metabolism in the fly.A constraint-based model of ATP-producing pathways was built using the annotated genome, existing models, and the literature. Simulations supported the hypothesis that the ability to flexibly convert pyruvate to these three byproducts might convey hypoxia tolerance by improving the ATP/H+ ratio and the efficiency of, glucose utilization. Traditional enzymatic assay are used to determine the pool of glycogen, trehalose and ATP, in order to refine the model. Metabolomic profile of selected mutants have also been collected, to determine the pathways used to produce alanine and acetate as well as to better understand the biological effects of these metabolites.

855C Metabolic control under hypoxia. Christian Frei1, Nan Chen1, Olivier Rinner2, Matthias Gstaiger2. 1) CC-SPMD and Institute for Cell Biology, ETH Zurich, Switzerland; 2) Institute for Molecular Systems Biology, ETH Zurich, Switzerland. Eukaryotic cells have a conserved response to low oxygen, termed hypoxia: Slowed growth and cell cycle progression and a shift away from oxidative metabolism. Drosophila Sima, the homologue to mammalian Hif-1alpha, is part of a transcription factor that mediates the cellular response to hypoxia. Sima function is regulated by different mechanisms, most importantly by degradation mediated in response to hydroxylation by the Fatiga prolyl hydroxylase, previously called Hph. Since the Fatiga-Sima pathway does not explain all aspects of hypoxia response, we postulated that Fatiga could function in a Sima-independent manner. To identify novel Fatiga-interacting proteins, we performed immunoprecipitation of a tagged Fatiga protein from S2 cells, and mass spectrometry. Among others, we found Sima binding to Fatiga, demonstrating that the assay is sensitive to detect novel substrates. Several other proteins bound Fatiga, and specific binding was reproduced using pull-down of in vitro-translated protein. Furthermore using mutated or truncated Fatiga protein, we found that the novel interactors showed a distinct binding pattern compared to Sima. We will discuss in detail the role of these novel Fatiga binding partners in the cellular control to hypoxia. POSTERS: Physiology and Aging 379

856A Molecular characterization of mutations in the enzymes of the α glycerophosphate cycle. Ross MacIntyre1, Amber Carmon1, Jeff Chien1, David Sullivan2. 1) Dept Molec Biol & Genetics, Cornell Univ, Ithaca, NY; 2) Emeritus Professor, Syracuse University, Syracuse, NY. The two critical enzymes for the operation of the α glycerophosphate cycle in the adult indirect flight muscles are the cytoplasmic the α glycerophosphate dehydrogenase (GPDH) and the mitochondrial the α glycerophosphate oxidase (GPO). Acting in concert, they provide electrons for oxidative phosphorylation and maintain cytosolic levels of NAD+ for pyruvate production. For the past several decades, a number of laboratories have induced and/or recovered mutant GPDH and GPO proteins, meausured their enzymatic activities, and assessed their effects on adult flight ability. In order to learn more about the structure and regulation of GPDH and GPO, we have sequenced the available mutants for each protein. For GPDH, we sequenced RT-PCR products for 14 EMS induced mutants and present summaries of relevant enzymatic activity data and flight abilities. Most are missense mutations, but two nonsense mutants and one mutation affecting a splice site were also detected. For GPO, the genome sequence indicates there are three paralogs- at 34C, 43D, and 52C8. We show by RT-PCR that only the gene in 52C8 is expressed in the adult flight muscles. We propose to call this gene GPO-1. We have directly sequenced 8 mutants, 6 are EMS induced and 2 were isolated from natural populations. As with GPDH, flight activity and enzymatic activities in these mutants are strongly correlated. In order to produce a null mutant of GPO-1, we excised a P-element inserted near the 5’ end of the gene. Two excisions which extended into GPO-1 were recovered and analyzed.

857B Roles for the DHR38 nuclear receptor in carbohydrate metabolism. Anne-Françoise Ruaud, Carl Thummel. Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT. Although nuclear receptors are known to play a central role in controlling vertebrate metabolism, no similar functions have yet been described in Drosophila. Our current studies are focused on DHR38, the only fly homolog of the vertebrate NR4A receptor subclass: Nur77, Nurr1 and NOR-1. Nuclear receptors of the NR4A subgroup are orphan receptors, the activity of which is determined largely by their expression levels. In line with their induction by pleiotropic physiological stimuli, they have been implicated in many processes, including carbohydrate metabolism. Specifically, Nur77, Nurr1 and NOR-1 are transcriptional regulators of several hepatic gluconeogenesis genes and, as such, participate in controlling blood glucose levels both in fasted and diabetic mice (Pei et al., 2006, Nat Med, 12, 1048). A second study indicates that Nur77 regulates the expression of glucose metabolism genes in skeletal muscle (Chao et al., 2007, Mol Endocrinol, 21, 2152). We are taking a genetic approach to investigate roles for DHR38 in controlling fly carbohydrate metabolism. DHR38 null mutants die late during pupal development with cuticular defects. Initial characterization indicates that DHR38 mutant larvae are not starvation sensitive. Their glucose, trehalose and glycogen levels are similar to wild type under fed conditions, but they display reduced levels of glycogen upon starvation. Building on this observation, our ongoing studies aim to further characterize metabolic activity in DHR38 mutants, define the tissue-specificity of DHR38 action, identify DHR38 target genes, and study DHR38 interactions with known metabolic regulators. Taken together, these approaches should provide a better understanding of how NR4A receptors regulate carbohydrate homeostasis.

858C A Drosophila DEG/ENaC gene expressed in the fat body may play a role in lipid metabolism. Yishan Sun1,2, Lei Liu2, Yehuda Ben-Shahar4, Michael J. Welsh1,2,3,4. 1) Neuroscience Graduate Program, Univ of Iowa, Iowa City, IA; 2) Dept of Internal Medicine, Univ of Iowa, Iowa City, IA; 3) Dept of Physiology and Biophysics, Univ of Iowa, Iowa City, IA; 4) HHMI, Iowa City, IA. Drosophila melanogaster has more than 20 genes of the Degenerin/Epithelial Sodium Channel (DEG/ENaC) family, which are collectively referred to as pickpockets (ppks). Loss-of-function studies have implicated ppk genes in salt taste, tracheal liquid clearance, pheromone detection, and developmental timing. These results, together with the conserved presence of DEG/ENaC genes through evolution, suggest that further studies on fly ppk genes may help gain insights to a number of physiological processes. Here we report phenotypic characterization of a null mutant of ppk11. The mutant was generated by FLP-FRT recombination between two Exelixis transponson insertions. With food deprivation, adult ppk11 mutants died more rapidly than wild-type controls. By promoter- driven GFP expression, we found ppk11 expression in the fat body in both larvae and adults. Furthermore, Oil Red O staining revealed that ppk11 mutants had apparently normal triacylglyceride storage in the fat body when raised on normal fly food, but their triacylglyceride mobilization during starvation was much faster than wild types. These results indicate that ppk11 may play a role in lipid mobilization from the fat body in starved flies. 380 POSTERS: Physiology and Aging

859A Mutation in Drosophila adenosine deaminase disturbs the glycogen metabolism. Monika Zuberova, Tomas Dolezal. University of South Bohemia, Faculty of Science, Ceske Budejovice, Czech Republic. Extracellular adenosine is a key stress signaling molecule that is released in most of tissue under various stress conditions. A null mutation in Drosophila adenosine deaminase (adgf-a) leads to dramatically increased levels of adenosine in larval hemolymph. This is accompanied by abnormal proliferation/differentiation of hemocytes and melanotic tumor formation. Mutants are delayed in development and usually die before reaching the adult stage. Overall mass of the larval fat body is greatly reduced and it eventually disintegrates. In genetic screen, we have identified a P-element insertion in the coding region of gamma subunit of phosphorylase kinase (PhKγ). This insertion suppresses the adgf-a phenotype when it significantly increases larval survival and mass of the larval fat body. Phosphorylase kinase is a key regulator of glycogenolysis. We propose that the adgf-a mutation leads to disbalance in the glycogen synthesis/degradation in favor of degradation and the P-element insertion returns it into the balance by decreasing the phosphorylase kinase activity. This is supported by experiments with a diet containing different sugar concentrations. We have found that the survival of the adgf-a larvae strongly depends on the sugar concentration. Higher sugar concentration robustly increases their survival. On the other hand, rearing the larvae on the yeast diet with no sugar supplement leads to rapid death. In agreement with the result of our genetic screen, this sensitivity to low-sugar diet is suppressed by the mutation in PhKγ. Our data suggest that extracellular adenosine is under certain conditions able to regulate the glycogen metabolism and high levels of extracellular adenosine due to the mutation in adenosine deaminase disturb this regulation.

860B Mechanisms of life-span extension in Or83b mutant flies. Peter Poon, Daniel Harmon, Xiaowen Chu, Scott Pletcher. Huffington Center on Aging, Baylor College of Medicine, Houston, TX. The ability to smell provides an organism with a host of environmental cues, including the availability of food, mates and the possibility of disease and predators. The organism interprets and uses the information to properly regulate and modulate behavior, development and aging. Flies with a loss of function in Odorant Receptor 83b (Or83b) has been shown to be broadly anosmic and extend lifespan under a variety of different environmental conditions (Libert et al, 2007). However the mechanisms by which Or83b extends lifespan has not yet been fully elucidated. Therefore, we undertook a series of experiments to determine the physiological changes in Or83b mutant flies. In addition we also performed genetic epistasis experiments with known lifespan regulators Sir2 and Foxo to determine which lifespan extension pathways are affected by Or83b. Furthermore, we also performed Or83b rescue experiments in different subsets of olfactory neurons to determine which neurons are important for the lifespan extension effects of Or83b Here we present the mechanisms and genetic pathways involved in lifespan extension of Or83b mutant flies.

861C Effects of mutation of lot’s wife and starvation on crop motility in Drosophila. Elizabeth M. Bacon, Edward M. Blumenthal. Biological Sciences, Marquette University, Milwaukee, WI. Mutation of the lot’s wife (lwf) gene results in complete adult lethality during the first week post-eclosion. Our data suggest that mutant flies starve to death (see abstract by Peller and Blumenthal), and there appears to be a defect in the movement of food from the crop (the food storage organ) into the midgut. To investigate the possibility that lwf expression is required for the muscle contractions that normally move food from the crop into the midgut, we compared crop motility in females homozygous and heterozygous for the recessive null mutation lwf1. Crop motility was measured by exposing the crop and observing the contractions of the duct and lobes. The lwf1 homozygotes had twice the rate of crop duct contractions (p<0.002) and three times the rate of crop lobe contractions (p<0.0001) as heterozygotes. The ratio of lobe contractions to duct contractions, which was close to unity in the heterozygotes, was greater than 2 in homozygotes, suggesting a lack of organ-level muscle coordination in the mutants. To determine whether the difference in the motility of exposed crops was an artifact of the dissection or was representative of the activity in vivo, we measured crop duct contractions in intact flies that had been fed colored food to allow visualization of the crop through the cuticle. Under these conditions, lwf1 homozygotes continued to show a greater than 2-fold faster rate of duct contractions than heterozygotes (p<0.002). Because we know that lwf1 causes starvation, we questioned whether starving heterozygous flies would phenocopy the effect of lwf1 on crop motility. Starving heterozygotes for 48 hours did not cause any significant change in the rate of either crop lobe or crop duct contractions. It appears, therefore, that crop motility is accelerated by the lwf1 mutation, but not directly affected by starvation. Supported by NIH 1R15 GM080682 to E.M.Blumenthal. POSTERS: Physiology and Aging 381

862A Alterations in triglyceride levels and feeding behavior by a mutation in lot’s wife. Cassandra R. Peller, Edward M. Blumenthal. Biological Sciences, Marquette University, Milwaukee, WI. Mutations in the lot’s wife (lwf) gene result in flies with an extremely short adult lifespan; males hemizygous for the null mutation lwf1 show significant lethality beginning on the second day post-eclosion (P2) and are completely dead by the end of the first week. We have previously reported that mutant flies show retention of food in their crops and reduced defecation, suggesting that their short lifespan is due to starvation. To test this hypothesis, we measured triglyceride levels in Canton S (CS) and lwf1 males throughout the first week of adult life. While triglyceride levels (normalized to total protein) did not differ between wild-type and mutant flies during P0-P2, beginning on P3 the mutant flies had significantly smaller triglyceride stores. Indeed, triglyceride levels in P3 lwf1 males resembled those of CS males that had been starved for 48 hours. The biochemical evidence for starvation in the mutant flies was mirrored in their behavior. We used the proboscis print assay to quantify the flies’ drive to feed (Edgecomb et al., 1994). Both P3 mutants and 48-hour starved CS flies displayed a high level of printing, while fed P3 wild-type flies did not print. Thus at the stage when they are beginning to die in large numbers, lwf1 males show a strong behavioral and biochemical resemblance to starved CS males. Experiments are now in progress to investigate whether the timing of this transition reflects a cessation in feeding, an exhaustion of larval fat stores (Aguila et al., 2007), or some other physiological event. Supported by NIH 1R15 GM080682 to EMB.

863B Effect of different temperature in the spontaneous somatic mutation accumulation in Drosophila. Anamaria Garcia1, Rita Busuttil2, Cody Ginn1, Brent Calder2, Jan Vijg2, Martha Lundell1. 1) Dept Biol, Univ Texas, San Antonio, San Antonio, TX; 2) Buck Institute for Age Research, Novato, CA, USA. Progress in understanding the mechanistic basis of in vivo mutagenesis and its phenotypic consequences has been hampered by difficulties in quantifying and characterizing mutations in somatic tissues of multicellular organisms. Using P-element transformation we generated a mutation reporter system in Drosophila melanogaster based on the recovery of chromosomally integrated plasmids harboring the lacZ gene. Using this system, we have previously reported that the spontaneous somatic mutation frequency in the fly appeared to be significantly higher than observed for various mammalian tissues, and characterized by a very high fraction of genome rearrangements. In addition, the mutation frequency in female flies was slightly, but significantly higher than in males. In order to investigate the effects of the temperature on the spontaneous mutation frequency, our lacZ-fly line was allowed to complete development at 25 °C and was then transferred to three different temperatures: 18, 25 and 29 °C, 1-2 days after hatching. Samples were collected at 5 days, 2 weeks, 4 weeks and 6 weeks of age. The results revealed that the spontaneous mutation frequency increases with an increase of the temperature and aging. These results indicate that our lacZ-fly system is able to detect mutations under different environmental conditions and this is the first report on somatic mutation and aging in Drosophila.

864C Changes in gene expression profiles related to mortality over the life span of large caged populations of Drosophila melanogaster. Kylee M Gardner1, Kimberly Carlson1, Anjeza Pashaj1, Darby Carlson1, Lawrence Harshman2. 1) Biology, University of Nebraska at Kearney, Kearney, NE; 2) School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588. Aging is characterized by a decline in many aspects organism function. Genomic technologies, such as microarrays, allow for a global analysis of gene expression during the aging process. The objective of this study was to perform transcriptome analysis using cDNA microarrays to determine the differentially regulated genes in large caged populations of genetically related Drosophila melanogaster. It addresses the question of whether there is consistency in the gene expression patterns in replicate populations. Moreover, it investigates gene expression in very old individuals made possible by the large starting size of each cohort. Two cages of 10,000 flies (5,000 females and 5,000 males) were established and samples collected at 0%, 10%, 30%, 60%, and 90% population mortality. This microarray study includes very old flies; it is the first D. melanogaster microarray study of aging to include this class of flies. Total RNA was extracted from females and cDNA microarray analyses performed. Two hundred and ninety-five genes exhibited a statistically significant change in gene expression from samples compared to the 0% sample. Of these, 205 were found to be significantly differentially expressed across all time points. Cluster analysis was applied to these genes to identify sets of genes with a similar pattern of altered gene expression. Gene ontology analysis was used to identify the probable function of the 205 genes. 382 POSTERS: Physiology and Aging

865A Identification of Genes that Impact Age-Related Locomotor Impairment. Melanie Jones, Michael Grotewiel. Dept Human Genetics, VCU, Richmond, VA. Normal aging in humans results in a number of age-related functional declines that have grave health consequences. Age-related locomotor impairment is particularly problematic since it is associated with an increased risk of institutionalization, falls, and death. We are using Drosophila as a model to investigate the molecular-genetic basis of age-related locomotor decline. Toward identifying genes and mechanisms involved in this behavioral consequence of aging, we performed a forward genetic screen by assessing senescence of negative geotaxis, a locomotor behavior, on ~1000 transposon insertion lines. Transposon insertion lines that had the most robust delayed aging of negative geotaxis were chosen for further study. We found that all transposon lines with delayed aging of negative geotaxis are resistant to various stresses, suggesting that enhanced stress resistance might be mechanistically linked to the delayed locomotor aging in these flies. Additionally, some of the transposon insertion lines have extended life span whereas others do not. One mutant harbors a P element insertion in PDK1, a component of the insulin signaling pathway known to influence lifespan in worms, flies, and mice. Removal of the P element by precise excision resulted in a normal locomotor senescence phenotype, demonstrating that the P element insertion was causing the delayed aging of negative geotaxis in these flies. Real-time PCR analysis revealed that this fly was a loss of function mutant in PDK1 with decreased insulin signaling. Analysis of other P element insertions in other components of this pathway revealed that Dp110, the catalytic subunit of PI3K also influences locomotor senescence. These results highlight the importance of the insulin signaling pathway in regulating senescence of negative geotaxis in Drosophila. Further analysis of additional transposon insertion lines identified through this screen will lead to a better understanding of the molecular-genetic basis of age-related locomotor impairment.

866B The shuttle craft locus controlling motoneuron axon guidance proves to affect Drosophila melanogaster lifespan. Natalia V. Roshina, Alexandr V. Symonenko, Eugenia A. Tcybulko, Eugenia V. Ershova, Elena G. Pasyukova. Institute of Molecular Genetics of RAS, Moscow, Russian Federation. Previously, deficiency complementation tests and complementation tests to mutations demostrated that shuttle craft (stc) is a novel candidate gene affecting lifespan of Drosophila females (Pasyukova et al, 2000;Pasyukova et al, 2004). stc encodes an RNA polymerase II transcription factor and is an attractive candidate gene for the regulation of lifespan because it is required for motoneuron development and is expressed throughout adult lifespan. To prove directly that stc is involved in lifespan control, we used a standard technique based on the analysis of phenotypic changes in a trait of interest due to insertion mutations and their reversions at the gene of interest. stcKG01230, a homozygous viable allele with a P{SUPor-P} insertion in the 5’ untranslated region, was introduced into w1118 line background. Five lines with reversion of the marker phenotype were obtained. In all five lines true excision of the vector was observed, according to the results of PCR amplification and further sequencing of a 5 kb region ajacent to the insertion site, Southern blot analysis and in situ hybridization with polythene chromosomes. Longevity was measured for mated and virgin flies of w1118 and stcKG01230 lines. No difference in male lifespan was revealed. Virgin stcKG01230 females lived significantly longer than virgin control females (P=0.0001), whereas mated stcKG01230 females - significantly shorter than mated control females (P=0.0001). Overall, mutation affects life span and ageing, depending on sex and reproductive status of flies. Longevity was also measured for mated and virgin females of revertant lines. Lifespan of revertant virgin and mated females was undistinguishable from lifespan of the corresponding control females and differed significantly (P=0.0001, P=0.0001) from lifespan of mutant females. Thus, stc proves to be a gene involved in the control of Drosophila lifespan. Analysis of stc expression in control, mutant and revertant lines is in progress.

867C Mitochondrial superoxide dismutase or SOD2 is more essential for adult life span. Renee Forde, Subhas Mukherjee, Atanu Duttaroy. Howard Univ, Washington, DC. Mitochondrial superoxide dismutase or SOD2 catalyzes the conversion of superoxide radicals into hydrogen peroxide. Superoxide radicals are by products of cellular oxygen metabolism. Sod2 null mutant in mice and Drosophila both displays early adult lethality suggesting the essential requirement of this enzyme. Earlier studies claimed the involvement of SOD2 on aging process. Since SOD2 is essential for survival, we wanted to know how exclusively SOD2 function is required during Drosophila development. We first detected SOD2 in bona fide Sod2-null mutant embryos, which suggest that SOD2 is maternally contributed. This maternal SOD2 disappears completely in 3rd instar larval stage as confirmed through Western blotting and SOD2 activity analysis yet lack of SOD2 protection does not affect the subsequent developmental stages. With the help of various cellular markers we find that development proceed quite normally as there was no indication of any excessive cell death. Eclosion ratios confirm that less than 10% Sod2 null pupae failed to eclose as adult. At this point our data suggest that SOD2 action may be necessary during early embryogenesis, but it is certainly not essential during later part of development. So, early adult lethality of Sod2 nulls possibly happen due to its requirement in adult life. POSTERS: Physiology and Aging 383

868A Is the circadian clock system involved in temporal co-ordination of protein homeostasis during oxidative stress in Drosophila melanogaster? Natraj Krishnan, Andrew Davis, Jadwiga Giebultowicz. Department of Zoology, Oregon State University, Corvallis, OR 97331. •- Reactive oxygen species (ROS) are continuously generated from the reduction of molecular oxygen to the superoxide anion (O2 ) by single electrons that have escaped the mitochondrial respiratory chain. ROS accumulation can cause chronic cell damage and has been associated with aging as well as certain physiopathological conditions. Oxidative stress can give rise to protein carbonyl derivatives, via a variety of mechanisms that include fragmentation and amine oxidation either due to metal catalysis or by hypochlorous acid. Since proteasome is directly involved in protein turnover and degradation of oxidized protein, its fate upon oxidative stress has received special attention. We demonstrate that the circadian clock system may be directly or indirectly involved in conferring protection against oxidative stress in Drosophila melanogaster. In general, we found that specific clock mutants were more susceptible to oxidative stress induced by hydrogen peroxide as well as hyperoxia than their wild type counterparts. Moreover, we observed that oxidized proteins (protein carbonyls) are formed and degraded in the heads (brain) of fruitflies in a circadian manner. We investigated if this temporal fluctuation of oxidized protein turnover is correlated with change in activity of specific proteasome subunits during periods of increased carbonyl accumulation and whether these are regulated by the circadian system. Understanding the mechanisms by which this crucial protein homeostasis is achieved and if deficiencies in a functional circadian clock can be a predisposing factor in cellular susceptibility to oxidative stress may open new strategies for combating oxidative stress induced pathologies.

869B Factors influencing aging of male germline stem cells in Drosophila. Matthew Wallenfang, Tarnima Ahamed, Khadeejah Bari, Christine Chang, Saira Siddiqui, Ayelet Spitzer. Dept Biological Sciences, Barnard College, New York, NY 10027. Adult stem cells persist throughout the lifetime of an organism, and are critically important in many tissues to maintain homeostasis. Because of these properties, it is thought that organismal aging maybe be due, at least in part, to a decline in the number and/or function of adult stem cells. Drosophila male germline stem cells (GSCs) are a particularly attractive model in which to study stem cell aging, as stem cells can be easily identified, and much is known about the cellular and molecular makeup of the niche that maintains these stem cells. We have previously shown that GSC number and cell cycle activity decreases during aging, and that this correlates with a decrease in somatic hub cells that contribute to the stem cell niche. Interestingly, decline in GSC function is not observed in long-lived methusalah (mth) mutant flies. We are currently further exploring the causes of age related changes in GSCs, including localizing the requirement for mth function in promoting aging, and exploring the role of other factors that are thought to influence aging.

870C A novel muscle-spike waveform in the giant fiber response in old flies. Jeff Engel. Dept Biological Sciences, Western Illinois Univ, Macomb, IL. We previously showed age-related deficits in the visually-induced startle response in white-eyed mutant Drosophila melanogaster. We also showed age-related reduction of the electroretinogram (ERG), suggesting that changes in visual processing contribute to the behavioral deficit. Now we are using electrical stimulation of the giant fiber pathway to look for changes in later stages of the pathway. White-eyed cn bw flies were reared at 23°. Stimulating electrodes were placed in the eyes to trigger the giant fibers or their afferents in the brain. Recording electrodes were placed in DLM (wing-depressor) and TTM (leg-extensor) muscles, with a reference electrode in the abdomen. The giant fiber response persisted in flies as old as 100 d. DLM muscle spikes were seen at all ages, and both short- and long- latency responses were seen. However, most flies older than 77 d showed an aberrant TTM waveform, either unusually small or negative-going. The negative waveform might be the normal signature of another muscle in the path between the recording and reference electrodes that is usually masked by a large TTM spike which is diminished or absent in old flies. This decrement in the TTM spike could indicate degeneration of the TTM muscle or its innervation, or it could be due to other changes that affect the electrophysiological recording. Loss of the TTM response would be expected to eliminate the jump component of the startle response, although a direct correlation between the electrophysiological and behavioral changes remains to be shown. 384 POSTERS: Physiology and Aging

871A Initiation and formation of adult type 2 peritrophic matrix. Gae E Kovalick. Biology, University of Texas Permian Basin, Odessa, TX. The peritrophic matrix (PM) is a thin, chitinous sheath that is produced by the midgut and that has protective and digestive functions. A type 2 PM is synthesized continuously by the cardia, a specialized organ located at the foregut-midgut junction. The PM is synthesized as a continuous tube that extends from the cardia through the posterior midgut. Food entering the midgut is surrounded by this tube. The synthesis of the PM was examined 0-12 hours following eclosion in Periodic Acid-Schiff (PAS)-stained sections of whole adults. Individuals were examined for the presence of a PM; those that had a PM were scored for how far the PM extended through the midgut. In wild-type adults, the PM was absent at eclosion. A PM was present in all adults by 2 hr posteclosion (PE), and by 8 hr PE, all had a mature PM that extended through the entire midgut. There was no difference in the initiation or kinetics of PM formation between males and females or between those adults that had access to food and those without such access. Adults that were restrained in glass capillary tubes immediately after eclosion showed a delay in PM formation. This delay persisted through 12 hr PE. Since restraint suppresses the release of bursicon after eclosion, PM formation was examined in rk[4] mutants, in which the bursicon receptor is affected, and in pu[1] mutants, in which one of the bursicon subunits is affected. Both mutants showed a similar delay in PM formation. For the first few hours after eclosion, this delay was similar to that observed in restrained adults. However, by 8-12 hr PE, the PM in rk[4] and pu[1] mutants resembled wild-type, whereas restrained adults continued to display a delay in PM formation. These results suggest that bursicon release increases the rate of PM formation, and that additional factors released later in development also affect this process. To identify these factors, PM formation is being examined in other mutations that affect posteclosion events.

872B Fatty Acid Transporters Regulate Obesity, Cardiac Performance and Lifespan in Drosophila. Samantha Morley, Nicole Piazza, Mike Hayes, Robert Wessells. Dept Intnl Med/Geriatrics, Univ Michigan, Ann Arbor, MI. The group of symptoms, known as the Metabolic Syndrome, including insulin resistance, dyslipidemia, obesity and cardiovascular disease, is becoming an increasing health concern to our aging population. These mutually reinforcing symptoms eventually lead to Type-II Diabetes and a higher risk for heart failure. Fatty acid transporters are found in vertebrate hearts, where they affect metabolism and energy usage. Levels of fat in vertebrates contribute to the Metabolic Syndrome and the symptoms can be regulated by fatty acid transporters. Similar transporters in Drosophila melanogaster may serve as a model to examine the role of fatty acid metabolism in the etiology of obesity and heart disease. We report that fatty acid transporters in drosophila are important for regulation of fat storage and long-term cardiac performance. Mutations in two different fatty acid transporters cause opposite phenotypes in cardiac performance, fat storage and lifespan. Flies carrying the first of these mutations have a shorter mean lifespan and reduced amounts of movement due to a significantly lower amount of overall energy production, but have dramatically improved cardiac performance, especially at older ages. Flies with the other mutation have increased lifespans and vigor, but have significantly more stress sensitive hearts than wild-type flies. Potential specificity of fatty acid transporters’ response is being examined by various fatty acids. These experiments will provide information on fatty acids that can affect cardiac function, cardiac performance, fat storage and lifespan. We hope to use these mutants as a model to investigate relationships between fat metabolism and long term cardiac performance.

873C Changes in GST gene expression in large caged populations of Drosophila melanogaster. Anjeza Pashaj1, Kimberly Carlson1, Kylee Gardner1, Darby Carlson1, Lawrence Harshman2. 1) Biology, University of Nebraska at Kearney, Kearney, NE; 2) School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588. Microarray studies have recently identified detoxification genes as an overrepresented class of genes among those differentially expressed as a function of age in model species for genetic studies. One such family of detoxification enzymes are the glutathione S- transferases (GSTs). GSTs are found in all organisms, with orthologs shared by humans and Drosophila melanogaster. In this study, we investigate the abundance of mRNA corresponding to specific GST genes. Large caged populations (15,000) of D. melanogaster were sampled over a period of 89 days and the levels of GST mRNA analyzed. A time course experiment was performed to analyze the expression levels of GST mRNA on days 5, 19, 31, 43, 61, and 75. Total RNA was extracted for each time point and was used for real time qRT-PCR. The precision and cost effectiveness of qRT-PCR allows many samples to be analyzed. The long-term goal is to identify candidate genes for longevity in laboratory and natural populations. POSTERS: Physiology and Aging 385

874A Antigenotoxicity of some traditional medicinal phytoextracts used for the control of diabetes mellitus type 2. Rosario Rodriguez-Arnaiz, America Nitxin Castañeda Sortibrán, Maria Guadalupe Ordaz Tellez. Departamento de Biologia Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico, DF, Distrito Federal, Mexico. Two herbal phytoextracts used for the control of diabetes mellitus type 2 in folk medicine (Cecropia obtusifoliaand Equisetum myriochaetum) were assayed for antigenotoxicity using the Somatic Mutation and Recombination Test in Drosophila melanogaster. The standard (ST) and the high bioactivation (HB) crosses were used. Hydrogen peroxide was used as an oxidative genotoxicant to test the antigenotoxic potency of the medicinal herbs. Water was used as negative control and urethane as positive control. Three- day old larvae, trans-heterozygous for the genetic markers multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) were treated chronically by oral administration with the test compounds or complex mixtures. For antigenotoxicity studies, chronic co-treatments as well as separate pre-treatments with the genotoxicant followed by a chronic treatment with the complex herbal mixture were assayed. None of the phytoextracts showed a significant genotoxicity but they were able to behave as desmutagens, detoxifying the mutagen hydrogen peroxide. The flavonoids content of such herbal medicinal plants is supposed to be the possible scavenger of reactive oxygen species.

875B IRES dependent translational regulation of dFoxo. Eugenia Villa-Cuesta, Brian T. Sage, Marc Tatar. Department of Ecology and Evolutionary Biology, Brown University, Providence, RI. Drosophila dFOXO is a transcription factor that belongs to the FoxO subfamily of Forkhead proteins. dFOXO is homologous to FOXO1, FOXO3a, and FOXO4 in mammals. In Drosophila, as in mammals, dFOXO regulates cell cycle transition, proliferation, apoptosis, oxidative stress resistance, glucose metabolism, and longevity; all of them key factors in the control of development, metabolism and disease. FoxO proteins are highly post-translationally modified, allowing them to sensor the cell status and rapidly change FoxO activity. Here we show that dFOXO is not only post-translationally regulated, but also the initiation of dFOXO translation is regulated by IRES. Internal ribosome entry sites (IRES) are untranslated segments of mRNA transcript needed to initiate protein synthesis in response to environmental stresses that normally prevent canonical 5’cap-dependent translation. The dfoxo gene has three transcripts variants, each with unique 5’UTRs (5’ untranslated regions). Transcript dFoxo A encodes for isoform A of dFOXO and two of the transcripts (dFoxo B and C) encode for isoform B. Our data shows that the 5’UTR of mRNA dFoxo B has the ability to initiate IRES-mediated translation of dFOXO isoform B, when the cap-dependent translation is reduced. To address the biological and cellular implications of dFoxo IRES, we are studying the differential functions and expressions of dFoxo’s isoforms in Drosophila. We have data showing that dFOXO has differential expression in males and females, as well as heads and bodies of adult flies. To examine the differential functions of the isoforms, we are now generating mutations affecting only the IRES region and generating transgenic flies that over-express dFoxo isoform A and B independently. As mentioned before, dFOXO regulates different cellular stresses. The presence of IRES in one of the isoforms of dFOXO may allow, under environmental stress, the translation of dFOXO protein and therefore a better adaptation of the fly to the stress situation.

876C The ribose-5-phosphate isomerase mutant extends lifespan in Drosophila melanogaster. Yi-Yun Wang, Jing-Zi Wang, Hsun Li, Horng-Dar Wang. Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, R.O.C. Long-lived organisms are often associated with better resistance to various stresses. Therefore, multiple-stress analysis has been used to find genes involved in extension of lifespan (Wang et al., 2004). Using multiple-stress in forward genetic screening, we found EP2456, which has an EP-element insertion nearby CG30410 and displays reduced expression of it. CG30410 encodes ribose-5- phosphate isomerase (rpi) involving in pentose phosphate pathway, which generates NADPH to cope with oxidative stress and provides ribose-5-phosphate for the synthesis of nucleotides and nucleic acids. EP2456 reveals lifespan extension and oxidative stress resistance. Knockdown of rpi by double-stranded RNA interference also exhibits similar results. Insulin signaling required for glucose metabolism and lifespan modulation is well conserved among different species. The analysis of the key components of insulin signaling pathway in EP2456 reveals decreased phosphorylation level of Akt and up-regulation in 4EBP, suggesting that mutation in rpi extends lifespan by means of intervening of downstream components of insulin pathway. 386 POSTERS: RNA Biology

877A A genome-wide screen for genes involved in microRNA biogenesis and activity. Caroline Jacquier, Josette Pidoux, Hélène Thomassin, Christophe Antoniewski. Lab of Drosophila Genetics and Epigenetics, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France. Pre-miRNAs are cropped from long primary miRNA transcripts by the Drosha/Pasha microprocessor complex and exported to the cytoplasm through an Exportin 5 dependent process. In Drosophila, pre-miRNAs are then maturated in double-stranded miRNAs by Dicer-1 and loaded into an Argonaute containing RISC complex. miRNA-mediated target silencing occurs in P-bodies both through translation inhibition and mRNA decay. Although the bases of the miRNA silencing pathway have been established, it is most likely that numerous components remain unknown. We have established a reporter system that monitors biogenesis and activity of artificial miRNAs targeted to the GFP. The backbone sequences of endogenous miR genes were mutagenized to perfectly match regions in the GFP coding sequence. These reprogrammed miRNAs were then placed in an intron and fused to a GFP coding gene. As expected, the resulting “AutomiR” construct, in stable cell lines as well as in Drosophila transgenics, was self-silenced by the reprogrammed miRNAs. In addition, GFP transgenes were dramatically silenced in trans, indicating that reprogrammed miRNAs represent a valuable alternative to long hairpin RNAs for targeted gene inactivation. We have used a stable AutomiR S2R+ cell line in a genome-wide RNAi screen for genes whose inactivation cause AutomiR desilencing. A highly significant set of already characterized components of the miRNA pathway were recovered, including Drosha, Pasha, Exportin-5 and Gawki. Interestingly, RNAi of Ago1 as well as Ago2 genes produced significant hits, suggesting that both Argonautes are involved in mediating AutomiR silencing. Validation and characterization of the other candidates should provide new insights on the machinery involved in miRNA biogenesis and activity.

878B Regulation of Drosophila germline stem cells by a Dcr-1 mediated miRNA pathway. Zhigang Jin, Ting Xie. Stowers Inst, Kansas City, MO. Stem cells are capable of self-renewing themselves and producing other types of cells in plants and animals; therefore, it is of great interest to understand how their self-renewal and proliferation is regulated. Extensive studies carried out in various model organisms as well as mammalian tissue culture systems have elucidated many cellular pathways or components that are required for stem cell regulation. We are interested in testing the involvement of the miRNA pathway in the regulating of stem cells in Drosophila females. As in other species, Drosophila Dicer-1 (Dcr-1) is essential for generating mature miRNAs from their corresponding precursors. In addition to cell proliferation defects, we found that dcr-1 mutant germline stem cells (GSCs) and somatic stem cells (SSCs) cannot be maintained and are lost rapidly from the niche without discernable features of cell death, which leads us to hypothesize that miRNAs processed by Dcr-1 are likely involved in normal maintenance and proliferation of stem cells in Drosophila ovaries. To investigate the roles of miRNAs in GSCs directly, we have cloned GSC-enriched miRNAs by using GSC-like tumor ovaries. This has allowed us to recover many known miRNAs species, as well as some novel miRNAs. Currently, we are studying how some of these miRNAs might contribute to GSC self-renewal and proliferation.

879C Identification of New miRNA Pathway Components. Arthur Luhur, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. microRNAs (miRNAs) play key roles in gene silencing by regulating mRNA stability and translation. Following transcription of miRNA genes by RNA polymerase II, primary miRNA transcripts are folded into stem loop structures that are then cleaved by both nuclear and cytoplasmic factors including the RNaseIII enzyme Dicer to yield double stranded ~22 nucleotide intermediates. Further unwinding and processing of these intermediates results in the selective degradation of one strand leaving the mature single stranded miRNA to interact directly with members of the Argonaute (Ago) protein family. Mature miRNAs can hybridize to partially complementary sequences within the 3' untranslated region (UTR) of cellular mRNA targets, which leads to the initiation of mRNA degradation or translational interference. While the pathway that processes miRNA primary and intermediate transcripts has been identified and well studied in a variety of models organisms, much of the molecular machinery underlying miRNA mediated mRNA degradation and translational repression are less well known. In particular, the mechanisms by which the 3' UTR of target cellular mRNAs are processed and rapidly screened for complementarities with mature miRNAs have been poorly characterized. We set out to identify factors that play roles in these processes. We separately expressed two miRNAs (mir-318 and mir-277) in subsets of developing photoreceptors. These flies, which have moderate rough eyes, were then crossed to the Deficiency Kit and the progeny were screened for modifications of the rough eye. Single gene disruption mutations were used to identify genes that played a role in gene silencing. Of particular interest were loss-of-function mutations in genes that suppressed the effects of each microRNA. These are likely to be in genes that normally function within the miRNA pathway. We identified a number of genes that code for factors that function in miRNA-mRNA targeting, mRNA 3’UTR processing and translational repression. Our list of identified factors includes several RNA helicases, RNA binding proteins and members of the translational initiation machinery. The roles that these genes play in mRNA silencing by microRNAs will be discussed. POSTERS: RNA Biology 387

880A A sensitive reporter system for testing miRNA targeting applied to the regulation of Hox protein transcript and protein levels by Hox cluster-encoded microRNAs. Adam Paré, Derek Lemons, William McGinnis. Dept Cell & Developmental Biol, Univ California, San Diego, La Jolla, CA. In Drosophila there are few examples of specific microRNA regulation of endogenous target transcript/protein expression levels. Most evidence still consists of in vitro assays or imaginal disc assays using potential 3' UTR targets fused to reporter genes, coupled with overexpression of a putative miRNA regulator. The extent to which these assays faithfully predict endogenous miRNA targeting is unclear. We present a Gal4 driven reporter system for assaying miRNA binding site function in 3' UTRs. This system offers several advantages: 1) it allows screening for both mRNA and protein downregulation, 2) it relies on endogenous miRNAs at their normal concentration and in their normal expression patterns, 3) the expression levels of the reporters can be tuned to a wide range of levels, and 4) 3' UTR target-reporter constructs can be quickly generated, and introduced into flies at specific genomic locations. We test reporters containing 3' UTRs of the Hox genes Scr and Antp, and compare their expression patterns to endogenous patterns of Scr and Antp transcripts and protein. We also correlate these data with the expression patterns of miR-10 and miR-iab-4, microRNAs that are encoded in the Hox gene clusters and that have been predicted as regulators Scr and Antp, respectively. Finally, we examine embryos transheterozygous for wild type and mutant 3' UTRs fused to different reporters to detect subtle changes in target transcript and protein levels when specific miRNA target sites are mutated.

881B MicroRNA Mediated Translational Repression in the Ovary. John C Reich, Mark J Snee, Paul M Macdonald. Molecular Cell and Developmental Biology, University of Texas, Austin, US. MicroRNAs are small regulatory RNAs which target genes through sequence complementarity, leading to post-transcriptional gene regulation. Mutations in components of the microRNA pathway are often lethal, demonstrating that microRNAs have an essential role during development, but there is no direct evidence that microRNA repression occurs in the ovary. To determine if microRNAs can repress translation in the ovary, we constructed GFP reporter transgenes with or without copies of synthetic targets for miR-312 inserted into their 3' UTR. GFP levels were monitored by confocal microscopy and quantitative western blots. The presence of the miR-312 target sites leads to reduced GFP with no corresponding reduction in mRNA level. Two lines of evidence support the conclusion that the reporter mRNA is subject to microRNA-dependent translational regulation. The temporal pattern of repression - enhanced at later stages of oogenesis - mirrors the temporal pattern of miR-312 expression, and mutations in genes required for processing microRNAs relieve repression. In other cells, transcripts repressed by microRNAs are localized to P-bodies, which contain proteins involved in microRNA function. Sponge bodies are ovarian structures that contain many P-body proteins. Given the similar protein composition between the two structures, we asked if microRNA-regulated transcripts and components acting in microRNA function are concentrated in sponge bodies. To monitor the distribution of a repressed mRNA we used a second type of reporter transgene, with the oskar coding region with or without miR-312 target sites. These transgenes also contain multiple binding sites for MS2 coat protein (MCP), allowing detection of the mRNA by GFP tethering. The repressed transcripts and proteins involved in repression are not concentrated in sponge bodies, but are instead localized in small cytoplasmic puncta. We conclude that miRNA-dependent translational repression does occur in the ovary, but largely or completely outside of sponge bodies.

882C Analysis of the role of roX1 RNA 5' sequences in X chromosome targeting. Ying Kong, Victoria H. Meller. Department of Biological Sciences, Wayne State University, Detroit, MI. roX RNAs are involved in dosage compensation of the sex chromosomes in Drosophila. Dosage compensation equalizes expression of X-linked and autosomal genes. In Drosophila it involves a two-fold increase of transcription from the single male X chromosome. This is mediated by the MSL complex, composed of the male-specific lethal (MSL) proteins and two noncoding roX RNAs, roX1 and roX2. The MSL complex binds to hundreds of sites along the male X chromosome. roX1 and roX2 RNAs are redundant for the localization of MSL complex to the male X chromosome. A global decrease of X-linked gene expression is observed in the absence of both roX transcripts. roX1 has multiple regions necessary for function. Studies have identified a large 5' region and a small 3' stem loop that are necessary, but the central portion of roX1 can be deleted with no loss of function. Analysis of roX1 mutations suggests that the 5' roX1 sequence contributes to MSL X-localization. Multiple redundant elements are proposed to be present throughout this region. To investigate the function of these 5' roX1 elements, I created and analyzed roX1 transgenes carrying different sections of the 5' end. To control for differences due to the insertion site, transgenics were created using the site-specific ΦC31 integrase system. Males carrying a roX1 allele missing large portion of the 5' end display ectopic MSL binding and reduced binding on the X chromosome. roX1 transgenes with different fragments of the 5' end rescue male survival and MSL recruitment to the X chromosome to different extents. While different portions of roX1 5' end have some redundancy, they also display unique activities. Our results suggest that the 5' roX1 sequences function in X chromosome recognition and binding of the MSL complex. 388 POSTERS: RNA Biology

883A In vivo analysis of nonsense mediated mRNA decay. Kimberly A Frizzell, Shawn G Rynearson, Mark M Metzstein. Department of Human Genetics, University of Utah, Salt Lake City, UT. Premature translation termination codons (PTCs) can result from errors in transcription, genomic mutations, or splicing errors. Truncated protein products created from these transcripts could negatively affect normal biological processes. Nonsense mediated mRNA decay (NMD) is a cellular process that targets mRNAs containing PTCs for degradation. Core NMD genes, known from other organisms, are present in Drosophila, and have been shown in vitro and in vivo to be involved in the process of NMD. To better elucidate the mechanisms of NMD in Drosophila, we have designed a genetic screen of the X and 3rd chromosomes to identify mutations affecting NMD. We are using e22c-GAL4 driving FLP in the embryonic epidermis to generate homozygous mutant cells in heterozygous animals, and an NMD-sensitive DsRed expressing transgene to detect mutant cells. This construct is targeted by the NMD pathway, reducing its level of expression, and thus, in the absence of NMD, expression levels and hence fluorescence increase. This system allows us to screen L3 larvae for those containing increased red fluorescence in an otherwise light red background. The reporter construct is sensitive to both mutations in core and auxiliary components of the NMD pathway and the mosaic approach bypasses lethality caused by NMD gene mutations, and allows us to identify alleles of known NMD genes or new NMD genes that may have been missed due to lethality. The reason the DsRed mRNA is upregulated in NMD mutants is that the transgene contains an SV40 3’ UTR, which we have found is targeted by the NMD pathway for degradation. To better understand why the SV40 3’ UTR is targeted, we have designed a series SV40 3’UTR deletion transgenic constructs. We plan to use these to narrow down the sequences of the SV40 3’ UTR that are targeted by NMD in vivo.

884B Drosophila PTB/hnRNPI promotes formation of high-order oskar RNP complexes and represses oskar translation. Florence Besse1,2, Sonia Lopez de Quinto1,2, Anne Ephrussi1. 1) Developmental Biology, EMBL, Heidelberg, Germany; 2) contributed equally to the work. Embryonic axis specification in Drosophila depends on asymmetric localization and controlled translation of mRNAs encoding cell fate determinants. oskar mRNA, which encodes the posterior determinant responsible for germline and abdomen formation, is transported in a translationaly repressed state to the posterior pole of the oocyte, where it is specifically translated. Translational silencing of the unlocalized mRNA is essential, as ectopic expression of Oskar disrupts establishment of the anterior-posterior axis of the embryo. We identified the Drosophila homologue of Polypyrimide-Tract-Binding protein (PTB, also called hnRNPI) in a GFP- protein-trap screen, as a protein that colocalizes with oskar mRNA during all stages of oogenesis. Using both genetic and biochemical approaches, we have shown that PTB is a bona fide component of the oskar RNP complex in vivo. Moreover, we found that PTB function is required within the nucleus of germ cells for efficient polarization of the oocyte microtubule cytoskeleton and oskar mRNA transport. More specifically, we found that Oskar protein is prematurely expressed in ptb oocytes, and that this phenotype is rescued by a strictly cytoplasmic version of the protein. Furthermore, PTB directly binds to oskar mRNA, and associates with multiple regions of oskar 3’UTR, suggesting a direct role of PTB in oskar translation repression. Finally, PTB function is required for oligomerization of oskar mRNA molecules. We propose that PTB promotes formation of high-order RNP complexes in which oskar translation is silenced.

885C Identifying tissue specific regulatory proteins associated with the early spliceosome. Thomas Carr, Alexis Nagengast. Dept Biochemistry, Widener Univ, Chester, PA. The process of splicing regulates gene expression in a highly conserved manner from flies to humans and maximizes the output of a genome by generating different protein forms from the same gene sequence. The critical initial definition of intron-exon borders depends on splicing factors recognizing regulatory sequences within a pre-mRNA to promote or prevent splice site usage. Although over 200 spliceosome-associated proteins have been identified, exactly how these proteins function with one another in a living organism remains unclear. Because of the wide spectrum of genetic manipulations and biochemical analysis possible in Drosophila melanogaster, the testis of flies provides an ideal in vivo model system to identify tissue specific regulatory proteins. To identify these proteins, transgenic flies have been generated that express a Tandem Affinity Protein (TAP) tag cloned in frame to the early splicing factor U1-70K. U1-70K, an SR-like protein and component of the U1 snRNP, plays an important role in early splicing when intron-exon borders are first determined. After dissecting the testes of transgenic flies, the early pre-catalytic spliceosome will be purified using the TAP tag and the complex will be separated by SDS-PAGE. Individual bands will be isolated and their identity will be determined by LC-MS/MS. By identifying these proteins and examining their function in vivo, we will gain insight into the mechanisms underlying tissue specific splicing. POSTERS: RNA Biology 389

886A Bicaudal-C regulates nos expression during oogenesis. Chiara Gamberi, Paul Lasko. Biology, McGill University, Montreal, Quebec, Canada. Bicaudal-C (BIC-C) functions during oogenesis to establish anterior to posterior polarity in the Drosophila oocyte. Homozygous Bic-C mutant females arrest oogenesis at stage 10. Heterozygous Bic-C mutant females produce embryos with a range of patterning defects, the most severe being bicaudal embryos. In wild-type oocytes, NOS protein is restricted to the pole plasm through multiple levels of translational repression that limit the temporal period and spatial domain of its expression. We have obtained evidence that BIC-C, which binds to a specific region of the nos 3’ UTR, participates in regulating nos translation. Unlike in wild-type oocytes where NOS is undetectable before the end of oogenesis, in Bic-C homozygous mutants NOS protein accumulates in the oocyte, starting as early as stage 4. Embryos produced by Bic-C/+ heterozygous mothers also exhibit ectopic NOS, which is present in NOS-positive particles throughout the embryo, and ectopically at or near the anterior pole. This suggests that NOS derepression may underlie the observed bicaudal phenotype. BIC-C negatively regulates its own mRNA by recruiting the CCR4 deadenylase complex through a direct interaction with NOT3/5, which results in a reduction of its poly(A) tail length. We are investigating whether BIC-C affects nos expression through a similar mechanism.

887B Is symmetric arginine dimethylation of Sm proteins critical for Tudor anchoring? Graydon Gonsalvez1,2, A. Gregory Matera1,2. 1) Dept of Biology, University of North Carolina, Chapel Hill, NC; 2) Dept of Genetics, Case Western Reserve University, Cleveland OH. Germ cell development in Drosophila begins with the formation of a specialized cytoplasm in the posterior of the oocyte known as the pole plasm. Tudor protein is essential for proper assembly of the pole plasm. In stage 9 oocytes, Tudor localizes to the posterior pole and proper stablilization/anchoring therein is required for germ cell specification. Tudor is the founding member of a family of proteins that share a common structural motif (the Tudor domain) that was recently described as a methyl-binding motif. Dart5/ capsuleen is the Drosophila ortholog of human PRMT5, an arginine methyltransferase that post-translationally modifies spliceosomal Sm proteins. Interestingly, we found that Tudor was delocalized within oocytes of dart5/csul mutants. In C. elegans, Sm proteins have been shown to play a critical role in germ cell specification. We therefore hypothesized that Sm proteins, via their methyl modifications, are responsible for anchoring Tudor at the posterior pole. In the absence of Dart5, Sm proteins are un-methylated and are therefore unable to anchor Tudor, inhibiting proper assembly of pole plasm. To test this hypothesis we have created fly strains that express various GFP-tagged Sm proteins. We find that GFP-SmB and GFP-SmD3 co-localize with Tudor and Oskar at the posterior pole. To further demonstrate that SmB and SmD3 are components of the pole plasm, we show that both proteins can be targeted to sites of ectopically-induced pole plasm. Current studies are underway to biochemically and genetically characterize the Tudor-Sm interaction.

888C A novel role for klumpfuss as an RNA-binding protein. Erica J. Hutchins, Barbara J. Zaffo, Jamie C. Rusconi. Department of Biological Sciences, University at Albany, Albany, NY. klumpfuss (klu) is important for many developmental processes in Drosophila, e.g. it has been shown to be required for neuroblast determination in the developing nervous system and the proper regulation of apoptosis in the pupal retina. klu and its mammalian homolog Wilm’s Tumor Suppressor-1 (WT-1) are unique within the EGR family of transcription factors in that they contain an additional zinc-finger nucleic acid-binding domain. Previously, klu was thought to function primarily as a transcriptional regulator, binding DNA with its multiple zinc-finger domains and its expression restricted to the nucleus. However, we have recently found that klu is expressed in the cytoplasm as well, suggesting a novel role for klu in addition to transcriptional activation or repression. Interestingly, WT-1 is also expressed outside of the nucleus, where it appears to function as a shuttling protein. Using an RNA-binding protein immunoprecipitation method followed by microarray (RIP-Chip), we have shown that klu associates with RNA. Here, we will present data detailing the klu RNA-binding domain, the RNAs we’ve identified that klu binds, and the region of the klu protein that is responsible for its RNA binding capabilities. Together, these data will provide new implications for the biological significance of klu and its role in neural development and apoptosis. 390 POSTERS: RNA Biology

889A In vitro selection and characterization of the RNA binding sites for the RNA binding protein Bruno. Brad Reveal, Paul Macdonald. Molecular Cell and Developmental Biology, University of Texas, Austin, TX. Oskar (Osk) is the posterior body patterning determinant in Drosophila melanogaster oocytes. oskar (osk) mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein accumulates. Osk then acts to recruit other factors and nucleates assembly of polar granules, which act in posterior body patterning and germ cell formation. Expression of osk is regulated by mRNA localization and translational control. Repression prior to localization is mediated by Bruno (Bru), which binds to two discrete regions of the osk 3’UTR called AB and C. Bru contains three RNA Recognition Motifs (RRMs). The first two RRMs reside in the middle of Bru’s primary sequence and bind with specificity to the AB and C regions. The third RRM is in the C-terminal region of Bru, and along with flanking amino acid sequence, also binds with specificity to the AB and C regions. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was carried out using full length Bru and the fragments containing either the first two RRMs (RRM1+2) or the third RRM (RRM3+) to better define Bru binding sites. The RRM1+2 and RRM3+ selections each produced two or more distinct RNA motifs to which the peptides can bind. Aptamers selected for binding the full length Bru contain one of each class of binding site, with the RRM1+2 site positioned 5’ and immediately adjacent to the RRM3+ site. Notably, mRNAs regulated by Bru contain several of the different possible combinations of sites. Thus, Bru binding sites appear to be assembled combinatorially, with the full length protein folding to form an extended RNA binding pocket that can bind to multiple RNA substrates.

890B RNA localization through RNP transport particles in Drosophila ovaries. Yiyin Ho, Elizabeth Gavis. Dept of Molecular Biology, Princeton University, Princeton, NJ. The asymmetric distribution of proteins is essential in Drosophila embryonic development for the subsequent differentiation and function of many specialized cell types. The localization of maternal mRNAs during oogenesis is required in order to establish and pattern both the anterior-posterior (A-P) and the dorsal-ventral (D-V) body axis. These mRNAs contain localization signals that direct their transport in complexes to their target destination. Localized mRNAs are packaged into ribonucleoprotein (RNP) particles, which are presumed to contain many copies of the mRNA. However, it is unknown whether a single RNP particle contains more than one kind of mRNA transcript. We are taking two different approaches to elucidate the localization patterns established by the functional makeup, interactions, and the transport process of RNP complexes that mediate RNA localization during Drosophila oogenesis. First, we have designed a transgenic strategy to label two different mRNA species simultaneously in vivo, each with a different fluorescent protein. By using live confocal imaging, we will determine whether multiple mRNAs can cohabit the same particle for either transport or anchoring. Secondly, we will take a systematic approach to identifying the mRNAs of localization complexes by carrying out immunoprecipitation of localization factors and microarray hybridization to identify the cohort of mRNAs that bind specifically to different localization factors.

891C The changing dynamics of localized fluorescently labeled gurken mRNA in Drosophila. Angie Jaramillo1,2, Timothy Weil2, Elizabeth Gavis2, Trudi Schupbach1,2. 1) HHMI; 2) Department of Molecular Biology, Princeton University, Princeton, NJ. During Drosophila oogenesis, the targeted localization of gurken (grk) mRNA leads to the establishment of the axis polarity of the egg. In early stages of oogenesis, grk is found at the posterior of the oocyte, whereas in the later stages grk is positioned at the dorsal anterior corner of the oocyte. In order to visualize real time localization and anchorage of endogenous grk mRNA in living oocytes, we have utilized the MS2-MCP system. We show that MCP-GFP tagged endogenous grk properly localizes within wild type oocytes and behaves aberrantly in mutant backgrounds. Fluorescence Recovery After Photobleaching (FRAP) experiments of grk mRNA particles in egg chambers reveal a difference in the dynamic state of localized grk mRNA between young and older egg chambers. grk particles, as a population, are highly dynamic molecules, exhibiting high fluorescence recovery, in young stages. As oogenesis progress, grk mRNA steadily loses its dynamic nature. This difference in grk mobility is attenuated in K10 and sqd mutants such that mislocalized grk in older stages remains much more dynamic. Also by compromising dynein motor enzyme activity, we show that this can lead to mislocalization and altered dynamics of grk mRNA. Taken together, we have observed the nature of localized grk mRNA in live oocytes and propose that its maintenance changes from a dynamic to a static process. Currently, we are performing studies to investigate the roles of potential anchors important for the maintenance of grk mRNA. POSTERS: RNA Biology 391

892A Understanding the Role of Symplekin in Histone pre-mRNA Processing. Deirdre C Tatomer1, Mindy Steiniger3, Eric J Wagner3, Sarah Kennedy5, Matthew R Redinbo3,5, William F Marzluff1,2,3,4, Robert J Duronio1,2,4. 1) Department of Biology, UNC-Chapel Hill, Chapel Hill, NC; 2) Curriculum in Genetics and Molecular Biology, UNC-Chapel Hill, Chapel Hill, NC; 3) Department of Biochemistry and Biophysics, UNC-Chapel Hill, Chapel Hill, NC; 4) Program in Molecular Biology and Biotechnology, UNC-Chapel Hill, Chapel Hill, NC; 5) Department of Chemistry, UNC-Chapel Hill, Chapel Hill, NC. Cell division requires the production of histone proteins to package newly synthesized DNA into chromatin. This is achieved by increasing transcription, processing and stability of histone mRNA during S phase. Histone mRNAs are unique in that they have no introns and are not polyadenylated, instead ending in a conserved stem loop. Production of this unique mRNA 3’ end is directed by two cis elements, the stem loop and HDE in the 3’ UTR. A number of trans acting factors, such as SLBP and the U7 snRNP, bind to these cis elements and stimulate pre-mRNA processing. After a single cleavage event, the processed mRNA is ready for export and translation. We recently performed a genome wide RNAi screen in S2 cells that identified 24 proteins necessary for histone mRNA production, including Symplekin, a scaffold protein previously shown to be involved in canonical polyadenylation. Symplekin is a 150 kDa protein with a conserved NH2-terminal heat domain that is enriched in the histone locus body, a recently defined sub-nuclear organelle containing histone pre-mRNA processing factors that is associated with the histone locus. We are performing genetic and structure/function analyses in Drosophila to determine more precisely the roles played by Symplekin in histone mRNA biosynthesis and in development.

893B Purification and characterization of mRNA localization complexes. James Wilhelm, Risa Maruyama, Elena Monfort-Prieto. Dept Biological Sciences, Univ California San Diego, La Jolla, CA. While a great deal is known about how proteins are sorted to various membrane bound compartments, very little is known about how proteins are sorted to particular domains within the cytoplasm. One mechanism for localizing cytoplasmic proteins is to transport the mRNA encoding the protein to the desired location, so that the synthesis of the protein is spatially restricted to particular cytoplasmic regions. Establishment of these domains is further enhanced by a translational control mechanism that ensures that only the properly targeted messages are translated. This sorting mechanism has been implicated in processes as diverse as stem cell differentiation, regulating synaptic strength in neurons and embryonic pattern formation - underscoring the importance of understanding mRNA localization for medicine, neuroscience, and developmental biology. While the importance of this method of sorting cytoplasmic proteins is clear, it is unclear how localized messages are targeted to different domains or how many cellular or developmental processes utilize mRNA localization. Recent work on mRNA localization indicates that a large percentage of mRNAs are targeted to discrete subcellular sites (Lécuyer E et al Cell 2007). One implication of this work is that there should be specialized subunits that are responsible for recognizing a particular class of transcripts and sorting them to their appropriate destination. However, while a great deal of effort has gone into identifying components of the localization machinery, very few known subunits are found at novel locations. In order to address this problem, we have purified biochemically distinct localization complexes and identified all of their subunits by MUD-PIT mass spectrometry. This work has identified several subunits of the complex that are either tissue specific or are localized to novel intracellular sites. We are now generating mutations in these subunits to determine their function as well as conducting screens for the transcripts that are recognized by these components of the localization complex.

894C Antiviral RNAi Suppressors from plant and insect viruses interfere with siRNA and rasiRNA pathways in Drosophila. Bassam Berry1, Delphine Fagegaltier1, Safia Deddouche2, Ronald van Riji3, Raul Andino3, Jean-Luc Imler2, Olivier Voinnet4, Christophe Antoniewski1. 1) CNRS/Inst Pasteur, Paris, France; 2) IBMC, 15 rue René Descartes, 67084 Strasbourg Cedex, FR; 3) University of California, San Francisco, CA 94143-2280; 4) IBMPC, 12 rue du Général Zimmer - 67084 Strasbourg Cedex, France. Plants and Insects employ RNA silencing as a virus defense mechanism. To counteract host response, viruses encode Viral Suppressors of RNA silencing (VSR). In addition to blocking antiviral RNAi, VSRs perturb host siRNA and miRNA pathways, inducing developmental defects in plants. However, a similar function has not been established in animals. Likewise, while VSRs from insect viruses were shown to potently inhibit antiviral RNA in flies, whether or not they act on other small RNA related pathways remained unexplored. We established Drosophila transgenic lines expressing VSRs from plants, insects or mammalian viruses. Some of these lines, including B2, P19, P15 and P25, were hypersensitive to Flock House or Drosophila C viruses, underscoring a cross- kingdom effect of these proteins on antiviral RNAi. We further characterized the effects of each VSRs on endogenous siRNA-, miRNA- and rasiRNA-mediated silencing. B2, DCV, P19, P15, P21 and Tas were able to suppress RNAi induced by an inversed repeat transgene, as well as RNAi induced by dsRNA or siRNA injection in embryos. Unlike in plants, none of the tested VSRs induced obvious developmental defects or suppressed the silencing activity of the bantam miRNA, suggesting that they do not interfere with the miRNA pathway in flies. In contrast, B2 and P19 interfered with rasiRNAs derived from the Su(Ste) locus as well as from retrotransposons. In addition, they suppressed the variegation of an heterochromatic marker and dramatically affected the distribution of histone H3-K9 methylation on polytene chromosomes, pointing out a direct role of rasiRNAs in heterochromatin maintenance. Our data demonstrate that viral suppressors from distant kingdoms share the ability to interfere in fly with siRNA and rasiRNA, but not miRNA pathways. 392 POSTERS: RNA Biology

895A Antisense transcription of Drosophila telomeric retrotransposon HeT-A. Alla Kalmykova1, Sergey Shpiz1, Dmitry Kwon1,2, Yakov Rozovsky1. 1) Dept of Molecular Genetics of Cell, Institute of Molecular Genetics, Moscow, Russia; 2) Dept of Molecular Biology, Moscow State University, Moscow, Russia. Telomeres in Drosophila are maintained by transpositions of specialized telomeric retroelements HeT-A, TART and TAHRE rather than by telomerase activity adding short DNA repeats to chromosome ends in other eukaryotes. The abundance of HeT-A, TART and TAHRE transcripts in ovaries is strongly upregulated owing to mutations in the RNAi (RNA interference) genes spn-E, aub, piwi and vasa locus. Short RNAs of telomeric retrotransposons belong to long size class (26-29 nt) of rasiRNAs (repeat-associated short interfering RNA). Mutations of RNAi genes eliminate both sense and antisense HeT-A and TART rasiRNAs in the ovaries. Little is known about the HeT-A antisense expression. Using rapid amplification of cDNA ends (5’ RACE) we identified transcription initiation site for HeT-A antisense transcripts. It localizes upstream of sense transcription start site in the 3’ region of the element. Antisense promoter activity of HeT-A 3’ region was confirmed in cell culture transfection experiments. As a result of sequence analysis of 5’ RACE products we conclude that antisense transcripts are transcribed from different HeT-A copies rather than from a single master copy. Interestingly, that antisense HeT-A transcript abundance is not affected by RNAi gene mutations in spite of the presence of sense HeT-A rasiRNAs in the ovaries. We propose that only coding sequences are the targets of rasiRNA-mediated RNA silencing machinery.

896B The role of Drosophila fmr1 gene in rasiRNA silencing. Mikhail Klenov. Dept Animal Molecular Gen, Inst Molecular Genetics, Moscow, Select Country. Silencing of genomic repeats, including transposable elements, in Drosophila melanogaster is mediated by short RNAs (piRNAs) interacting with proteins of the Piwi subfamily. piRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. Here we show that Drosophila fmr1 gene, which encodes homolog of human fragile X syndrome protein, is indispensable for rasiRNA silencing in ovaries. In Drosophila, dFmr1 is also known to be involved in germline stem cells (GSCs) development and oocyte specification. We revealed that dFmr1 acts together with PIWI protein, but not AUB and Ago-3. We consider the possible role of rasiRNAs in GSCs fate.

897C Expression profiling of rasiRNA-affecting mutants. Alina Korbut, Sergey Lavrov, Vladimir Gvozdev. Dep. of Molecular Genetics of the Cell, Institute of Molecular Genetics, Moscow, Moscow, Russian Federation. Repetitive sequences, including transposable elements, form a considerable part of eukaryotic genome. Overexpression of transposable elements usually leads to mutations and chromosomal rearrangements, so they should be under keen cellular control. Silencing of genomic repeats, including mobile elements, in Drosophila melanogaster germline is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with Argonaute proteins of the Piwi subfamily; rasiRNA-based silencing differs from both the siRNA and microRNA pathways. rasiRNA-mediated regulation of expression has been shown for many target sequences but all of them represent different types of repeats. It is not known whether some unique cellular genes are regulated by this pathway. A possible approach for identification of new targets of rasiRNAs is expression profiling of germinal tissues of flies carrying mutations in genes involved in rasiRNA-mediated silencing. These mutations lead to drastic increase of amount of transcripts from repetitive elements and should also boost an amount of mRNA of presumable single-copy rasiRNA targets. We performed expression profiling of ovaries of flies carrying mutations in Homeless and Armitage genes using Drosophila transcriptome microarrays. Our analysis revealed significant alterations in abundance of some transcripts in homozygous mutant flies in comparison to heterozygous control flies. We identified genes that are upregulated and downregulated in homozygous mutant flies. Upregulated genes may be subjected to rasiRNA-mediated silencing. Downregulation may reflect unexplored activating function of the rasiRNA pathway components or some type of indirect interactions. A number of affected genes participate in RNA metabolism in drosophila ovary. Further research includes validation of putative targets and investigation of the role of short RNAs in regulation of their expression. POSTERS: RNA Biology 393

898A A Genome-wide RNAi Screen Identifies Novel Factors Required for Histone pre-mRNA Processing. Eric J Wagner, Brandon Burch, Ashly Godfrey, Harmony Salzler, William Marzluff, Robert J Duronio. Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC. The replication-dependent histone mRNA is the only non-polyadenylated messenger RNA. Replacing the polyA tail is an evolutionarily conserved stem loop (SL). This 3' end results from a single processing event between the SL and the histone downstream element (HDE). The SL is bound by the Stem Loop Binding Protein (SLBP) and the HDE is recognized by a small nuclear RNA called the U7snRNA. Despite our understanding of the biochemistry of this processing event, little is known about the additional proteins involved. Flies that are hypomorphic for SLBP null for U7snRNA exhibit histone pre-mRNA processing defects that arise due to inefficient cleavage of the message at the normal cleavage site. This aberrant processing creates histone mRNA with polyA tails due to cryptic polyadenylation signals downstream of the cleavage site. We posit that generation of these polyadenylated histone messages is a general phenotype of aberrant processing. In this study, we created a screening reporter comprised of a histone gene with the ORF for EGFP cloned downstream. Flies transgenic for the reporter and either hypomorphic for SLBP or null for U7snRNA show robust GFP signal in response to the transcriptional readthrough of the reporter processing site and subsequent translation of the histone-GFP fusion protein. We screened 22,000 dsRNAs at the DRSC and identified 24 genes that scored as GFP positive. Within these 24 genes are all 5 previously known factors as well as many that are novel. We are analyzing three factors not previously known to play a role in histone pre-mRNA processing using a combination of biochemical and genetic approaches. We have created nuclear extracts from S2 cells knocked down of these factors and are studying the effect on in vitro histone pre-mRNA processing. Moreover, we are analyzing flies that have P-element insertions within these genes for effects on processing in vivo as well as localization of processing factors.

899B Identifying tissue specific alternative splicing events. Tim Rudolph1, Neha Sirohi2, Alexis Nagengast1. 1) Dept Biochemistry, Widener Univ, Chester, PA; 2) Dept Biology, Widener Univ, Chester, PA. The process of splicing regulates gene expression in a highly conserved manner from flies to humans and maximizes the output of a genome by generating different protein forms from the same gene sequence. The regulated selection of alternative splice sites allows genes to be appropriately turned on and off in different tissues, sexes or stages of development. However, little is known about the earliest and most critical steps of tissue-specific alternative splicing, when intron and exon borders are first recognized and defined by the spliceosome. The testis of Drosophila melanogaster provides an ideal in vivo model system to examine splicing regulation in a tissue specific manner because a large number of specialized factors are required for the dramatic morphological change from a round spermatocyte to an elongated spermatid in the process of spermatogenesis. A high level of transcription and presumably post-transcriptional modification in the form of splicing occurs in these spermatocytes prior to spermatid development. We are developing RT-PCR assays to detect and characterize these testis-specific alternative splicing events. Additionally, to verify that splicing plays a critical role in spermatogenesis, we are conducting RNAi knock down experiments of known splicing factors specifically in testes. We will use the RT-PCR assays to address the nature of splicing misregulation under knock down conditions. 394 SPEAKER AND AUTHOR INDEX

This index includes names of speakers for the Opening General Session and for Plenary Sessions I and II and names of all authors of programmed abstracts. The number following an author’s name refers to the abstract program number. A, B, or C following a number indicates a poster presentation. The presenting author of an abstract is noted with an asterisk.

A Angel, Anna, 587B Bach, Erika, 165C, 352A, 394A Angela, Truesdale, 452B Bachtrog, Doris, 668B, 685A* Abed, Louka, 783C Anna, Grygoruk, 637A Bacon, Elizabeth M., 861C* Abmayr, Susan, 469A Anne, Hart, 96 Badouel, Caroline, 200B* Aboukhalil, Anton, 31 Anne, Joel, 484A* Badrinath, Krishan, 461B* Abou-Zied, Akram, 286A* Antipin, Maxim, 708C Bae, Esther, 300C, 309C Abramov, Yuri A., 277A, 329B Antoniewski, Christophe, 755B, 877A, Baehrecke, Eric, 6 Abt, Dawn, 50 894C Baig-Lewis, Shahana, 393C Abulimiti, Abuduaini, 590B Anxolabéhère, Dominique, 132, 271A Baker, Catherine, 493A* ACERT National ESR Center, 702C Aonuma, Hiroka, 759C, 764B Baker, Nicholas E., 570C Acharya, Usha, 364A Apel, Ido, 264C* Baker, Phill, 463A Ackerson, Larry, 637A Apfel, Katharina, 782B Bakhtojarov, George, 653B Acosta, Rafael, 259A Apidianakis, Yiorgos, 723C* Balamurugan, Kuppusamy, 368B Adachi, Yoshitsugu, 417C* Appleton, Kate, 126 Balbi, Mirjam, 33 Adams, Haley, 292A April, Duckworth, 96 Baldini, Regina, 723C Adams, Melanie, 463A Aquadro, Charles F., 654C, 676A Banerjee, Paromita, 91* Affolter, Markus, 77 Arai, Hideaki, 764B Banerjee, Soumya, 555C* Aggarwal, Kamna, 750C Araujo, Helena, 347B* Banerjee, Utpal, 545B, 775A Agnès, François, 7 Arbeitman, Michelle, 13, 147 Baoussis, Vassilis, 167B Agrellos, Rodrigo, 347B Arbelaez, Pablo, 125 Barbash, Daniel A., 51, 537C, 654C, Ahamed, Tarnima, 869B Archer, Trevor, 279C 706A, 718A Ahlquist, Paul, 761B Arguello, J. Roman, 114* Bardin, Allison, 17* Ahmad, Kami, 523A Armento, Alexander, 478A Bari, Khadeejah, 869B Ahmed, Atique, 797B Armstrong, Douglas, 574A Barolo, Scott, 310A Aigaki, Toshiro, 398B Armstrong, J. Douglas, 662B Barrio, Rosa, 851B Airoldi, Stephanie, 470B* Armstrong, Jennifer, 544A* Bashaw, Greg J., 145, 553A Aït-Ahmed, Ounissa, 267C* Arnosti, David N., 26* Basler, Konrad, 204C, 392B Akasaka, Takeshi, 800B* Arora, Kavita, 547A* Bass, B. Paige, 739A Akbar, Mohammed, 238A* Arsham, Andrew M., 140* Basu, Sanjay, 324C Akbari, Omar, 309C, 333C* Artabazon, Nicholas, 254B* Batavia, Mariska, 850A Akiyama, Takuya, 436A Ashburner, Michael, 2 Bateman, Jack R., 120*, 274A Aldridge, Amanda C., 505A Ashe, Hilary, 324C, 510C Batterham, P., 669C Alekseyenko, Art, 767B Ashim, Mukherjee, 96 Baumgardt, Magnus, 143*, 577A Alexandre, Kelly, 385A* Ashton, Jonathan, 539B Baxley, Ryan M., 126 Alfonso, Catalina, 524B* Ashton-Beaucage, Dariel, 794B* Bayes, Alex, 662B Al-Hasan, Yazan M., 619A* Asmar, Joelle, 760A Baylies, Mary, 127, 467B, 468C Allen, John M., 298A Astier, Martine, 63 Bayston, Laura, 324C Allikian, Michael, 803B Astigarraga, Sergio, 548B* Beachy, Philip A., 80 Alm, Christina, 105 Aten, Eric, 65 Beaton, Amy, 769A Al-Ramahi, Ismael, 841A Atikukke, Govindaraja, 197B* Beaucaire, Michael C., 505A Altenhein, Benjamin, 562A Atkinson, Nigel, 323B, 619A, 621C Beausoleil, Sean A., 793A Amy, Walker, 96 Atkinson, Nigel S., 623B, 624C, 643A Beaver, William, 768C* An, Ding Ding, 723C Auld, Douglas, 123* Becalska, Agata N., 395B* Anderson, David, Plenary Session II Austin, Christopher, 123 Beck, Lothar, 232A Anderson, Jason, 407B* Avila, Frank, 639C* Beckerle, Mary C., 256A, 462C Anderson, Michael, 492C* Awasaki, Takeshi, 731B Beckervordersandforth, Ruth, 562A Anderson, W. Ray, 84* Axelsson, Elin, 82 Beckett, Karen, 468C Andino, Raul, 894C Ay, Ahmet, 26 Beckingham, Kate, 612C Andrei, Pisarev, 780C Ayala, Aidee, 394A* Beckingham, Kathleen M., 494B*, 648C Andrejka, Laura, 539B Ayukawa, Tomonori, 356B* Becnel, Jaime, 635B* Andres, Andrew, 327C, 363C, 563B Azad, Priti, 536B*, 811A Behr, Matthias, 437B* Andrew, Bergen, 452B Beitel, Greg, 217A, 730A Andrew, Deborah J., 265A, 439A, Bejsovec, Amy, 174C 442A, 443B B Belenkaya, Tatyana Y., 390C Bell, William, 766A Andrews, Gracie L., 146* Babak, Tomas, 105 Bellen, Hugo, 622A, 819C Andrews, Hillary K., 602B Babaoglan, Burcu, 569B* Bellen, Hugo J., 11, 432C, 602B, 784A Andrioli, Luiz P., 334A* Babcock, Daniel T., 646A* SPEAKER AND AUTHOR INDEX 395

Beller, Mathias, 101*, 123 Boissonneau, Elisabeth, 7 Busuttil, Rita, 863B Bender, Michael, 373A, 474C, 634A Boitard, Simon, 693C Buszczak, Michael, 506B* Benes, Helen, 136 Boll, Werner, 16* Butcher, Shawn M., 611B* Benes, Judith, 543C, 747C Bolotov, Pavel, 770B Buthchar, Jon, 791B Benjamin, Nikette, 383B* Boltz, Kara, 613A Ben-Shahar, Yehuda, 647B*, 858C Bonaccorsi, Silvia, 151 Benzer, Seymour, 829A, 847A Bongiorni, Silvia, 187A* C Berezhkovskii, Alexander M., 242B Bonini, Nancy, Opening General Cabral, Larry, 616A Berg, Celeste A., 402C Session Cachero, Sebastián, 574A Berger, Christian, 556A* Bono, Jeremy M., 686B* Cadigan, Kenneth, 294C, 316A, 753C Berger, Edward, 293B Boozer, Daniel E., 774C* Cadigan, Kenneth M., 389B Berger, Michael, 31 Borevitz, Justin O., 687C Cai, Yu, 218B Berger, Susanne, 232A Bornemann, Douglas, 78*, 785B Caillouette, Katie L., 244A, 248B Bergland, Alan, 696C* Bortnick, Rachel, 549C* Calder, Brent, 863B Bergman, Casey, 672C Bosco, Giovanni, 275B, 498C Call, Gerald, 545B* Bergmann, Andreas, 8, 202A, 741C, Bose, Anasua, 571A*, 576C, 580A Call, Gerald B., 775A 742A Bosveld, Floris, 92 Callan, Matthew A., 604A* Bergner, Laura M., 505A Botas, Juan, 102, 819C, 841A Calleja, M., 845B Berhane, Hebist, 388A Botham, Crystal M., 799A, 842B Callender, J., 363C Berman, Allison, 736A Bou Aoun, Richard, 724A* Camara, Nicole, 23* Bernardo, Travis, 293B* Bouchard Kerr, Phoenix, 471C* Campbell, Gerard, 396C* Berry, Bassam, 755B, 894C* Boue, S., 110 Cantera, Rafael, 851B Besquillo, Lauren, 327C Bousum, Adam, 255C* Cantin, Greg, 808A Besse, Florence, 884B* Boutros, Michael, 82, 386B, 388A Cao, Xuesong, 350B Betrán, Esther, 665B, 671B, 677B Boyle, Michael J., 402C* Capkova Frydrychova, Radmila, Bhabha, Gira, 803B Boyle, Nicole, 670A 279C*, 281B Bhambhani, Chandan, 316A* Boyle, Patrick J., 108 Caplan, Jemila, 167B Bhattacharya, Abhishek, 570C* Boyles, Randy, 563B* Capri, Michèle, 267C Bhogal, Balpreet, 805A* Bradley, Pamela, 439A Capy, Pierre, 714C Bhutkar, Arjun, 674B Brawley, Crista, 69, 511A Cardoso-Moreira, Margarida, 687C* Bi, Xiaolin, 181A Brech, Andreas, 240C Cardozo, Timothy, 352A Bickel, Ryan D., 697A* Brenman, Jay, 12* Carlson, Darby, 864C, 873C Bickel, Sharon E., 186C*, 192C Brennan, Lesley, 729C Carlson, John R., 9, 651C Bidla, Gawa, 746B Bricker, Daniel, 363C Carlson, Joseph W., 121, 784A Bidwai, Ashok, 571A, 576C, 578B, 580A Brideau, Nicholas, 706A* Carlson, Kimberly, 864C, 873C Bier, Ehan, 93 Brill, Julie, 179B, 235A, 241A Carmon, Amber, 449B*, 667A, 856A Bier, Ethan, 347B, 432C, 768C, 804C Brisson, Jennifer, 47* Carneiro, Katia, 347B Bieri, Kevin, 643A Bristow, Chris A., 24 Carney, Ginger, 613A* Biggin, Mark, Plenary Session II, 125, Brodsky, Michael, 30*, 210C, 787A* Carney, Ginger E., 614B, 618C 296B, 306C Brody, Thomas, 304A Carr, Thomas, 885C* Biggin, Mark D., 319A, 326B Broihier, Heather, 581B Carreno, Sébastien, 152* Bilder, David, Plenary Session I, 53, Brooks, Elizabeth S., 15* Carrington, E., 542B 76, 240C Broomall, Kathleen, 557B Carroll, Pamela M., 527B Birchler, James, 332B, 526A, 530B* Brower-Toland, Brent, 529A Carvalho, Bernardo, 112* Birnbaum, Morris, 135 Brown, Julie, 816C Casad, Michelle E., 802A* Birse, Ryan, 139 Brown, Nicholas H., 517A Casanova, Jordi, 382A Biryukova, Inna, 589A* Brown, Nick, 648C Casanueva, M. Olivia, 70 Bishop, Clifton, 571A, 576C, 578B, Brown, Seth, 180C Casas-Tinto, Sergio, 18*, 103* 580A Brumbarova, Tzvetina, 522C Caséé, Thiago, 334A Bjorum, Sonia, 612C* Brummel, Ted, 847A Casper, Abbie, 485B* Blacket, Mark, 52 Brunet, Frédéric, 114 Cassidy, Justin, 375C Blackman, Kwesi, 503B Brusentsova, Irina, 272B Casso, David J., 431B* Blackshear, Perry J., 472A Bryantsev, Anton, 282C* Castañeda Sortibrán, America Nitxin, Blair, Seth, 85 Buechling, Tina, 386B* 874A Blanchette, M., 110 Buescher, Marita, 438C* Castellanos, Felix, 725B* Blanchette, Marco, 35 Bui, Chris, 545B Castelo, R., 110 Blitzer, Andrea, 237C Bulanin, Denis, 477C Castrezana, Sergio, 765C Blumenstiel, Justin, 275B Buldoc, Clare, 202A Caygill, Elizabeth E., 564C* Blumenthal, Edward M., 861C, 862A Bulyk, Martha, 31 Celestrin, Kevin, 29 Blute, Todd, 763A Burch, Brandon, 898A Celniker, Susan, Opening General Bodmer, Rolf, 94, 137, 139, 482B, Burg, Martin G., 774C Session, Plenary Session II, 240C, 800B, 801C*, 804C Burgess, Jason, 241A* 319A Boettiger, Alistair N., 242B Busick, Christopher, 772A Celniker, Susan E., 121, 300C, 769A Bogard, Nicholas, 212B Busser, Brian, 31* 396 SPEAKER AND AUTHOR INDEX

Celotto, Alicia, 831C Cheng, Shen, 797B Cox, Jordan, 245B Cenci, Giovanni, 151, 280A* Cheng, Ya-Jen, 707B* Cox, Rachel, 99* Cesario, Jeffry, 199A* Cherry, Christopher, 508A* Coyne, Jerry A., 712A, 713B Cespedes, Albert, 775A Cherry, Sara, Plenary Session I, 135, Crawley, Tim, 801C Cetera, Maureen P., 335B 727A, 733A, 735C, 736A, 757A, 761B Crest, Justin, 173B* Cha, Guang-Ho, 819C* Chi, Ai-Pei, 565A Cripps, Richard, 282C, 463A*, 479B Chan, Chih-Chiang, 439A Chia, William, 177C, 218B, 438C, Cripps, Richard M., 308B Chan, C. M., 837C 606C Crocker, Justin, 39* Chan, Gina, 527B Chien, Cheng-Ting, 560B Crook, Meaghan, 504C Chan, Ho Yin Edwin, 820A Chien, Jeff, 856A Croshaw, Dean A., 682A* Chan, H. Y. Edwin, 837C Chihara, Takahiro, 559A* Crozatier, Michèle, 19, 732C Chan, Jason W. H., 300C Chin, Mei-Ling, 156 Cruces, Martha P., 839B* Chan, Stacy, 545B Chiolo, Irene, 521B* Cruz-Moreno, Beatriz, 93 Chan, Tammy P., 853A* Chiu, Chichia, 26 Cryderman, D., 542B Chan, Wing Man, 820A* Cho, Lucy, 842B Cryderman, Diane E., 525C*, 840C* Chandrasekaran, Chitra, 665B* Choe, Kwang-Min, 216C Cui, Jun, 188B* Chang, Christine, 869B Choi, Hyowon, 29 Cukier, Holly, 102 Chang, Howard, 96* Choi, Kwang-Wook, 226A, 413B Curio-Penny, Beatrice, 525C, 809B, Chang, Hsiao-Han, 688A* Choi, Sekyu, 161B* 840C Chang, Hwei-yu, 688A Chopra, Vivek S., 287B* Currie, Benjamin, 598A Chang, Jinhee, 294C, 316A Christensen, Ryan, 30 Curtis, Brenda J., 546C* Chang, Mikyung, 294C*, 316A Christine, Rushlow, 34 Curtis, Daniel, 834C Chang, Pao-Ju, 599B Chu, Hou-Cheng, 326B Curtiss, Jennifer, 404B Chang, Qing, 642C Chu, Xiaowen, 860B Cuttell, Leigh, 728B Chang, Yung-Heng, 796A Chung, Jongkyeong, 161B, 162C Chang, Yu-Yun, 160A* Chung, Se-Yeon, 439A* Changkakoty, Binita, 391A Chung, Wei-Jen, 339C D Chanut-Delalande, Helene, 453C Ciapponi, Laura, 280A Dabbouseh, Noura, 79 Chao, Ju-Lan, 414C Ciufo, Stacy, 770B Daburon, Virginie, 19, 732C Charbonnier, Enrica, 348C* Clandinin, Thomas R., 216C Daga, Andrea, 261C Charlton-Perkins, Mark, 588C Clark, Andrew, 42, 112, 673A, 690C, Dahanukar, Anupama, 651C Charlton-Perkins, Mark A., 572B* 699C Dahmann, Christian, 213C* Charriere, Guillaume, 723C Clark, Andrew G., 676A Damen, Wim G. M., 421A Charroux, Bernard, 823A Clark, Denise V., 838A* Daniel, Lachaise, 691A, 709A Chatterjee, Shreyasi, 821B* Clark, Ivan, 403A* Daniels, Richard, 637A Chattopadhyay, Abanti, 582C, 590B* Clark, Karen, 770B Danusastro, Crissy, 481A Chau, Johnnie, 507C* Clark, Kathleen, 462C* Dascenco, Dan, 549C Chaudhuri, Anathbandhu, 827B Claude, Desplan, 369C DasGupta, Ramanuj, 391A Chauhan, Chhavi, 128* Clemens-Grisham, Rachel A., 362B* Dasgupta, Ujjaini, 364A* Chavoshi, Tina, 806B* Cohen, Leah, 292A Datta, Rhea, 408C* Cheema, Amrita, 545B Cohen, Robert S., 212B Dauback, Sarah, 136 Chekunova, Anna, 653B* Cohen, Stephen, Plenary Session I Dauer, Michael, 544A Chen, Chun-Hong, 46 Colazzo, Germana, 181A Dauwalder, Brigitte, 615C Chen, Elizabeth, 233B Cole, Michael, 180C David, Van Vactor, 96 Chen, Guang-Chao, 565A* Colley, Nansi Jo, 60, 244A, 248B Davidson, Jean M., 171C* Chen, Hungwen, 560B Colley, William C., 302B Davidson, Stuart, 326B Chen, Jianhui, 773B Collier, Glen, 273C Davies, Claire, 139 Chen, Jianming, 20 Collier, Simon, 65*, 433A Davis, Alisa, 368B Chen, Jiong, 545B Collins, Ann, 102 Davis, Andrew, 868A Chen, Jyh-Horng, 781A Colosimo, Pamela, 387C* Davis, Greg, 47 Chen, Ming, 779B Connacher, Robert, 466A Davis, Kailah, 772A Chen, Nan, 855C Cook, Orna, 432C* Dayringer, Evan, 26 Chen, Rui, 573C*, 582C, 590B Cook, Tiffany, 423C, 572B, 588C, 601A de Belle, J. Steven, 15, 626B, 628A, Chen, Ting, 791B Cooke, Evan, 411C 629B, 630C Chen, Tony, 226A Cooley, Lynn, 263B, 470B de Cuevas, Margaret, 511A Chen, Xiaohua, 368B Cooper, Thomas, 841A Deddouche, Safia, 744C, 755B*, 894C Chen, Xin, 32*, 500B, 501C Copf, Tijana, 625A* Dedrick, Mariah, 621C Chen, Xiofei, 361A* Coppey, Matthieu, 242B*, 378C Deery, William J., 494B Chen, Xue-Wen, 328A Coquin, Laurence, 124, 854B* de Haro, Maria, 841A* Chen, Ying, 114 Corby-Harris, Vanessa, 681C* Delaage, Michèle, 267C Chen, Zhihong, 202A, 741C Costa, Gonçalo, 195C Delidakis, Christos, 167B, 320B Cheng, Jun, 68* Costantino, Benjamin, 363C* Delmarre, Valérie, 132 Cheng, Longqiu, 390C Couble, Pierre, 522C Deng, Lisa, 185B Cheng, Louise, 144*, 593B Courey, Albert, 38, 345C SPEAKER AND AUTHOR INDEX 397

Deng, Wu-Min, 88, 358A, 397A*, 478A Durant, Melissa, 541A* F Dennis, Jared, 97 Durnbaugh, Sarah D., 496A Denton, Donna, 719B* Duronio, Robert, 385A Fabian, Lacramioara, 179B DePace, Angela, 296B Duronio, Robert J., 171C, 892A, 898A Fabrizio, James, 504C Dervisefendic, Azra, 138 Dushay, Mitch, 734B Fagegaltier, Delphine, 894C Desai, Ridhdhi, 214A* Dushay, Mitchell, 746B Fakhouri, Walid, 26 Desplan, Claud, 288C Dutta, Sudeshna, 6* Falkenburger, B., 824B Desplan, Claude, 575B, 586A, 600C Duttaroy, Atanu, 867C Fan, Yun, 8* de Vanssay, Augustin, 132 Dylla, Layne, 404B Fanto, Manolis, 823A* Devenport, Danelle, 517A Farajian, Reza, 548B Dey, Sudhansu, 155 Farkas, Robert, 5* Dialynas, George, 840C E Farr, C., 845B DiAngelo, Justin, 135* Eanes, Walter, 850A Fasulo, Barbara, 280A DiAntonio, Aaron, 637A Earley, Judy, 803B Favors, Sheena E., 496A, 497B*, 505A Diao, Fengqiu, 632B* Eastman, Deborah, 295A* Feala, Jacob, 854B Diaz, Manuel, 128 Ebrahimi, Saman, 174C Feala, Jacob D., 124* Díaz-Benjumea, Fernando J., 143 Egger-Adam, Diane, 338B Fedic, Robert, 472A* Dickson, Barry J., 10, 145 Egorova, Ksenia, 495C* Feitosa, Natália M., 421A Dilks, Stacie, 66* Eid, Jean-Pierre, 728B Feldman, Renny, 100 Dina, Kulikova, 290B Eimer, S., 824B Felix, T. M., 756C* DiNardo, Stephen, 66, 234C, 509B* Eisen, Michael, 27, 28, 296B*, 306C, Fenckova, Michaela, 726C Dinardo, Steve, 515B 319A, 672C Feng, Jing-jing, 220A Ding, Tian, 202A Eisen, Michael B., 326B Ferdousy, Faiza, 620B*, 827B Ding, Yun, 114 Eisenman, Robert, 131 Ferguson, Chip, 70 Dingwall, Andrew, 128 Eisman, Robert, 54* Fernandes, Isabelle, 453C Dingwall, Andrew K., 546C Ejsmont, Radoslaw K., 119* Fernandes, Joyce, 555C, 557B Dinkins, Michael, 451A Ekas, Laura, 352A* Fernandez, Harvey, 530B Dionne, Heather, 240C Eldon, Elizabeth, 372C Fernandez-Funez, Pedro, 18, 103 Do, Minh-Tu, 775A Eldon, Elizabeth D., 481A Ferrandon, Dominique, 724A, 760A Dobbs, Wesley, 818B Eleftherianos, Ioannis, 744C* Ferree, Patrick M., 51*, 718A Dobens, Leonard, 473B Eleiche, Aliaa, 471C Ferreira, Álvaro, 2 Dockendorff, Thomas C., 91 Elena, Modestova, 290B Ferrer, Pierre, 152, 453C Dodson, Mark W., 822C* Elgin, Sarah, 270C*, 529A, 767B Filardo, Paolo, 107 Dolezal, Tomas, 726C*, 859A Elhanany, Hadas, 224B* Filipski, Alan, 684C Domingo, V., 845B Eliana, Romero, 819C Filone, Claire Marie, 727A* Domingues, Celia, 720C* Elias, Maria C., 256A* Fink, Gerald, 74 Dominguez, Catherine, 766A Ellis, Jeremy, 547A Finley, Russell L., Jr., 197B Dominguez, Dalziel, 682A Ellis, Lisa L., 614B* Fischer, Janice, 500B Dominica, Carly, 810C Emerson, J. J., 687C Fischer, Karin, 149, 844A Doms, Robert, 727A, 757A Emes, Richard, 662B Fischer, Utz, 782B Dong, Chen-Yuan, 781A Enell, Lina, 14 Fisher, Bill, 240C, 319A Dong, Y., 828C Eng, Thomas, 278B Fisher, Karin A., 516C Dorighi, Kristel M., 130* Engel, Jeff, 870C* Fisher, William, 300C Dorr, Meredith, 555C, 557B* Engels, William, 116* Fitzpatrick, Carrie A., 182B Dospoy, Patrick, 803B Engels, William R., 776B Flatt, Thomas, 849C* Dottermusch, Christine, 223A* England, James H., 596B* Flores, Carlos, 116, 776B* Douglas, Dimlich, 96 English, Bevin C., 496A* Flores, Heather A., 654C* Dowd, Susan R., 175A Enriquez, Jonathan, 19* Florindo, Claudia, 195C Doyen, Cécile, 522C Ephrussi, Anne, 109, 884B FlyBase Consortium, 789C Doyle, Kristy, 65, 433A* Erdeniz, Naz, 393C FlyBase Consortium and The Gene Draper, Moon, 621C* Erfurth, Maria-Luise, 365B*, 549C Ontology Consortium, 792C Drewell, Robert A., 298A, 300C, 309C Erives, Albert, 39, 180C* Foley, Brad, 616A Driscoll, Michael, 207C Ershova, Eugenia V., 866B Fontana, Joseph R., 357C* Drummond, Emma, 122 Erturk-Hasdemir, Deniz, 750C Fontenele, Marcio, 347B Drummond-Barbosa, Daniela, 71, 155, Escaron, Claire, 728B Forde, Renee, 867C* 513C Estes, Patty, 106 Fortini, Mark, 76 Duan, Hong, 20, 468C Evans, Cory J., 775A Foussard, Hélène, 152 Dubois, Laurence, 19 Evans, Timothy A., 145* Fowlkes, Charless, 296B, 319A Dubrovskaya, Veronica, 293B Evans-Holm, Martha, 784A Fowlkes, Charless C., 125 Dubrovsky, Edward, 293B Eyun, Seong-il, 666C* Fox, Donald T., 231C Duchaine, Jean, 794B Fox, Rebecca M., 265A* Duchesneau, Christopher, 667A Franc, Nathalie, 728B*, 737B Dunham, Joseph, 694A Francis, Nicole, Plenary Session I Dunkelberger, Brian, 626B* Frank, Amanda, 704B 398 SPEAKER AND AUTHOR INDEX

Fransson, Fredrik, 587B Geis, Stephinie, 729C Greenberg, Anthony, 42* Fraser, Scott, 55 Gelbart, William, 674B, 786C Gregory, Stephen, 174C* Freer, Stephanie, 628A Gendron, Patrick, 794B Grevengoed, Elizabeth, 443B Frei, Christian, 855C* Genissel, Anne, 694A Griesinger, C., 824B Frei, Erich, 20 Georgiev, Pavel, 269B* Griffin, Ruth, 274A Freilich, Sarah, 509B Georgive, Oleg, 368B Grigorian, Melina, 465C* Freund, Yoav, 768C Gergen, Peter, 29* Grimm, Stefan, 414C Frise, Erwin, 769A* Gerhold, Abigail, 198C*, 201C Grimmler, Matthias, 782B Frizzell, Kimberly A., 883A* Gesualdi, Scott, 349A* Gross, Steven P., 243C* Fu, Dechen, 283A* Geyer, Pamela, 538A, 540C Grossfeld, Paul, 800B Fujiwara, Hiroo, 399C Geyer, Pamela K., 126, 840C Grossman, Tamar R., 804C* Fukumoto, Shinya, 759C Ghabrial, Amin, 440B* Grotewiel, Michael, 865A Fuller, Margaret, 32, 178A, 493A, Ghezzi, Alfredo, 619A Gruber, Jonathan, 41* 501C Ghosh, Sanjay, 109* Grushko, Olga G., 667A* Fuller, Margaret T., 344B Ghosh, Sohini, 273C* Grygoruk, Anna, 239B* Furlong, Eileen E., 315C Giagtzoglou, Nikolaos, 602B Gryzik, Tanja, 405C Furrer, Michael, 33* Giangrande, Angela, 560B Gstaiger, Matthias, 855C Furriols, Marc, 382A Gibbs, Richard, 573C Gubb, David, 751A Furukubo-Tokunaga, Katsuo, 627C Gibson, Greg, 801C, 816C Guenier, Anne-Sophie, 794B Futschik, Andreas, 693C Giebultowicz, Jadwiga, 868A Guerin, Colleen M., 466A* Fuwa, Takashi J., 360C Giebultowicz, Jaga, 611B Guerrero, Paola, 533B* Giedroc, David, 368B Guichard, Annabel, 93* Gilbert, Matthew, 532A* Guillemin, Karen, 799A, 842B G Gilsohn, Eliezer, 450C* Gumulak-Smith, Juliann, 67 Gim, Byung Soo, 344B Gundelach, Justin, 797B Gagneur, Julien, 315C Gindhart, Joseph, 245B Gunsalus, Kristin C., 474C Gajewski, Kathleen M., 464B* Ginn, Cody, 863B Günther, Viola, 368B Gajula Balija, Madhu, 824B* Girardot, Charles, 315C Guntur, Ananya, 558C* Galiana-Arnoux, Delphine, 755B Gisselbrecht, Stephen, 31 Guo, Ming, 46, 100*, 822C Galko, Michael, 64, 376A Gleason, Jennifer M., 698B* Gurbich, Tatiana, 668B* Galko, Michael J., 646A Glise, Bruno, 436A Gusella, James F., 594C Gall, Megan, 483C Godfrey, Ashly, 898A Guss, Kirsten, 148* Gallant, Peter, 33, 166A Godt, Dorothea, 517A Gutierrez, Luis, 336C* Gamberi, Chiara, 886A* Goetz, Sara E., 298A* Gvozdev, Vladimir, 495C, 897C Gamble, Caitlin, 31, 141, 608B Golden, Krista, 361A Gvozdev, Vladimir A., 277A Gamliel, Amir, 804C Goldman, Thomas, 13* Gygi, Steven P., 793A Gandille, Pierre, 7 Goldstein, Jeffery, 803B* Ganesh, Sunil, 740B Goldstein, Lawrence S. B., 252C Gangemi, Andrew J., 411C Golovnin, Anton, 269B H Gangopadhyay, Anu, 294C Golshani, Peyman, 832A Ganguly, Atish, 100 Haase, Erin, 573C Goltsev, Yury, 45* Gao, Guanjun, 181A* Haberman, Adam, 57* Gomez-Velazquez, Melisa, 18, 103 Garaulet, Daniel L., 418A* Habets, Ron L. P., 652A* Gonda, Rebecca, 366C* Garbe, David S., 553A Häcker, Udo, 59 Gong, Lei, 54 Garcia, Anamaria, 863B* Hackney, Jennifer, 473B* Gonsalves, Foster, 352A Garcia-Bellido, Antonio, Opening Haddad, Dominik M., 825C* Gonsalvez, Graydon, 887B* General Session Haddad, Gabriel, 346A, 536B, 592A Gonzalez, Eduardo, 404B* Gardano, Laura, 200B Haddad, Gabriel G., 846C Gonzalez, Katie, 127 Gardner, Kylee, 873C Haerry, Theodor, 349A Gonzalez-Reyes, Acaimo, 454A Gardner, Kylee M., 864C* Haerry, Theodor E., 812B Goodman, Anya, 270C Garesse, R., 845B Hafen, Ernst, 205A, 741C, 762C, 785B Goraksha, Pankuri, 154* Garfinkel, Mark, 286A Hab-Cordes, Eva, 633C Gornostaev, Nick, 659B Garza, Dan, 327C, 818B, 834C Hagen, Joshua, 339C Goryacheva, Irina, 708C Gates, Julie, 227B Haghighi, Pejmun, 610A Götze, Sandra, 392B Gatti, Maurizio, 151, 280A Hahn, Matthew, 115, 694A Gould, Alex, 144, 593B Gaul, Ulrike, 36, 83 Hales, Karen G., 496A, 497B, 505A Govind, Shubha, 167B, 725B, 795C Gauze, Larisa, 653B Halligan, Daniel, 672C Grady, Stephanie, 613A Gavis, Elizabeth, 890B, 891C Halme, Adrian, 198C, 201C* Graham, Suzanne, 139 Gavis, Elizabeth R., 107, 246C, 330C, Hamada, Masakazu, 4 Graham, Thomas G. W., 335B* 395B Hamann, Bernd, 125 Granadino-Goenechea, Begoña, 18 Gavrialov, Orit, 346A Hamazaki, Jun, 836B Grant, Jennifer, 593B* Gebelein, Brian, 284B, 297C*, 588C Hamilton, Andrew, 704B Grant, Seth G. N., 662B Gee, Gretchen V., 815B* Hammonds, Ann, 319A, 769A Graveley, Brenton, 35* Gehring, Walter, 417C Han, Kyung-An, 490A, 636C Gray, Carolyn, 220A SPEAKER AND AUTHOR INDEX 399

Han, Linzhu, 353B* Hester, Svenja, 122 Huber, Wolfgang, 82 Han, Mira, 115 Heydemann, Ahlke, 803B Hudson, Andrew M., 263B Hanna, Sheri, 757A* Hickman, Francis E., 505A Huelsmann, Sven, 648C Hansen, Ingrid, 745A* Hild, Marc, 818B Hughes, Michael, 549C Hardin, Paul E., 615C Hill, Erin, 699C* Hughes, Robert, 819C Hardy, Rich W., 735C Hiller, Mark, 292A Hughes, Tia, 434B* Hare, Emily, 27, 28* Hines, K., 542B Huisinga, Kathryn, 529A Hariharan, Iswar, 198C, 201C, 208A, Hines, Karrie A., 525C Hunt, Alan J., 68 817A Hipfner, David, 235A Hursh, Deborah, 87, 285C Harmon, Daniel, 860B Hirai, Shinobu, 764B Husain, Nicole, 216C* Haroon, Suraiya, 244A* Hirotaka, Kanuka, 759C Hutchins, Erica, 740B Harris, Adron, 621C Hlavina, Wratko, 770B Hutchins, Erica J., 888C* Harris, Harriet, 729C* Ho, Margaret, 309C, 560B* Harris, Kathryn, 215B*, 217A Ho, Margaret C. W., 300C* Harris, Laura, 122 Ho, Venus, 104 I Harris, Nathan, 225C* Ho, Yiyin, 890B* Ilya, Mertsalov, 290B Harris, Robin, 510C* Hoch, Michael, 437B Imler, Jean-Luc, 744C, 755B, 894C Harrison, Douglas, 355A, 400A Hoffman, Kristi, 582C Inamdar, Ajinkya C., 276C Harrison, Douglas A., 353B Hoffmann, Ary, 52 Inamdar, Arati, 827B* Harshman, Lawrence, 848B, 864C, Hoffmann, Jules, 724A, 744C, 755B Inglese, James, 123 873C Hofmeyer, Kerstin, 414C, 548B Iovino, Nicola, 36* Hart, Amy B., 826A* Hogan, Jesse, 255C Ishikawa, Hiroyuki O., 356B Hart, Craig, 532A, 534C, 535A Hogan, Justin, 65 Ishimoto, Takashi, 731B Hartenstein, Volker, 446B, 465C, 567C, Hogg, Grant, 800B Ismat, Afshan, 442A* 775A Hohl, Amber, 274A* Issigonis, Melanie, 511A* Hartl, Tom, 275B, 498C* Holbrook, Scott, 609C* Ito, Naoto, 594C Hartmann, Britta, 110* Holloway, David, 427A Ivanov, Andrey, 301A* Haselkorn, Tamara S., 49* Holloway, David M., 426C* Iwaki, D. David, 431B Hashimoto, Carl, 752B Holmberg, Sara H., 496A Iyer, Venky, 27, 28 Hashimoto, Yumi, 731B Holtzman, Stacy, 788B* Hatfield, Steve, 149 Holz, Anne, 232A Hauling, Thomas, 746B* Homem, Catarina, 58* J Haussler, David, 40 Hong, Joung-Woo, 129, 291C Hawley, Scott, 275B Hong, Yang, 777C* Ja, William W., 847A* Hay, Bruce, 46* Hongay, Cintia, 74* Jackson, George, 821B, 832A Hay, Ron, 324C Honjo, Ken, 627C* Jacobs, J. Roger, 380B Hayashi, Shigeo, 458B Hooven, Louisa, 611B Jacobs, Roger, 367A Hayes, Mike, 872B Horabin, Jamila, 317B* Jacobsson, Linn, 289A* Hays, Thomas S., 218B Hori, Kazuya, 360C Jacquier, Caroline, 877A* Hazelrigg, Tulle, 230B Horn, Thomas, 82*, 386B Jaeckle, H., 824B He, Jianxin, 723C Horner, Vanessa, 188B Jaeckle, Herbert, 101 He, Yuchun, 784A Hornick, Emma, 540C Jaeger, Savina, 31 Heath, Ben, 657C Hoseinovic, Neda, 834C Jaffray, Ellis, 324C Heffer, Alison M., 420C* Hoskins, Roger A., 784A Jagla, Krzysztof, 63 Heidmann, Stefan, 193A Hossain, Noor, 367A* Jahren, N., 542B Heifetz, Yael, 264C, 474C*, 476B Hou, Shuling, 303C James, R. Andrew, 698B Heitzler, Pascal, 589A Houle, David, 700A* Jang, Eric, 27 Helenius, Iiro T., 730A Howard, Julie, 122 Jannat, Habiba, 293B Hemati, Nahid, 68 Hoxha, Valbona, 615C* Jaramillo, Angie, 891C* Hempel, Leonie, 486C* Hoy, Ronald, 474C, 476B Jarman, Andrew, 574A*, 603C Henderson, Abigail, 409A* Hozumi, Shunya, 398B Jasper, Heinrich, 137 Hendrix, Dave, 129 Hozumi, Syunya, 399C Javier, Anna, 483C Henrich, Vincent, 363C Hsia, Cheryl, 655A* Jean-Luc, Da Lage, 691A Henriquez, Clara, 296B Hsiao, Hui-Yi, 288C* Jenkins, John, 670A Henriquez, Clara N., 125, 319A Hsiao, Yun-Ling, 599B Jenny, Andreas, 388A* Herman, Tory, 583A, 609C Hsouna, Anita, 441C* Jepsen, Kristen, 804C Hernandez, Greco, 37* Hsu, Hwei-Jan, 71*, 513C Jevede, Harris, 96 Hernández-Sierra, R., 845B Hsu, Tien, 219C, 222C, 441C, 464B Jia, Jianhang, 350B Herrera, Salvador C., 157A Hu, Xiaobang, 596B Jiang, Changan, 822C Herrmann, Bethany R., 838A Hua, Haiqing, 368B* Jiang, Jin, 350B, 351C Hersh, Bradley, 299B* Huang, Haixia, 46 Jiang, Nan, 534C* Herzig, A., 824B Huang, Juan, 777C Jiang, Zifeng, 683B*, 710B Herzog, Sabine, 193A* Huang, Min-Yu, 296B Jiménez, Gerardo, 378C Hesse, Shayla, 4 Huang, Zhiyu, 435C Jin, Zhigang, 878B* 400 SPEAKER AND AUTHOR INDEX

Jindra, Marek, 475A* Karandikar, Umesh, 578B, 582C Klichko, Vladimir, 747C John, Reinitz, 780C Karess, Roger, 539B Klingseisen, Anna, 405C* Johnsen, Holly, 300C Karlsson, Daniel, 143, 577A* Knowles, David, 296B Johnson, Aaron, 477C Karpen, Gary, 521B, 531C, 767B Knowles, David W., 125, 319A Johnson, Brooke, 640A Karpen, Gary H., 118 Knudsen, Bruce, 797B Johnson, Cassidy B., 494B, 648C* Karpinar, D. P., 824B Koch, Carmen M., 338B* Johnson, Eric, 730A Karr, Timothy, 657C* Koehrn, Kara M., 496A Johnson, Erik, 12 Karr, Timothy L., 679A Koelsch, Verena, 422B* Johnson, Glynnis, 122 Kashevsky, Helena, 278B Koles, Kate, 807C Johnson, Oralee, 635B Kassis, Judy, 541A Kolodkin, Alex, 442A Johnson, Robert W., 302B* Kaszuba, David W., 125 Kondrashov, Alexey, 113 Johnson-Schlitz, Dena, 116 Kataoka, Hiroshi, 852C Kondrashov, Alexey S., 667A Johnston, Laura A., 158B, 159C, Katzen, Alisa L., 182B Kondrashov, Fyodor A., 113* 183C, 209B, 564C Kaufman, Thomas, 54 Konduri, Vanaja, 648C Johnston, Robert, 288C, 575B* Kaufman, Thomas C., 788B Kong, Ying, 882C* Jones, Bradley W., 302B Kaur, Manpreet, 247A König, Tina, 213C Jones, Christopher, 270C Kavi, Harsh, 526A* Konikoff, Charlotte, 664A Jones, Christopher J., 631A, 644B, Kavita, Arora, 34 Konopova, Barbora, 475A 645C Kawazu, Shin-ichiro, 759C Konsolaki, Mary, 834C Jones, Leanne, 519C Kawecki, Tadeusz, 849C Koonin, Eugene, 113 Jones, Melanie, 865A* Kazgan, Nevzat, 12 Kopp, Artyom, Plenary Session I, Jones, Richard, 543C Kazutaka, Akagi, 337A* 697A, 704B Jones, R. S., 542B Keesling, David, 499A* Korbut, Alina, 897C* Jones, Whitney, 174C Keightley, Peter, 672C Korenberg, Julie R., 804C Jongens, Thomas, 805A Kelleher, Erin, 689B* Kornberg, Thomas B., 431B Joseph, Penel, 503B Kelliher, Tim, 515B Kosman, David, 768C Joshi, P., 542B* Kellis, Manolis, Plenary Session II Kostuchenko, Margarita, 269B Josowitz, Rebecca, 245B* Kelly-Tanaka, Kathleen, 282C Kovalick, Gae E., 871A* Josse, Thibaut, 132 Kemp, Sadie, 574A Koyfman, Hannah Cohen, 185B Jowdy, Casey, 550A* Kennedy, Cameron, 767B Kozlov, Konstantin N., 780C* Joyce, Eric F., 189C*, 191B Kennedy, Sarah, 892A Kracklauer, Martin, 500B* Juarez, Michelle, 258C* Kennell, Jennifer A., 389B* Kraft, Robert, 563B Juhasz, Gabor, 5 Kent, Daisey Y., 318C* Krahn, Michael P., 142* Jukam, David, 288C, 369C* Keränen, Soile, 296B Kramer, Helmut, 57, 238A, 241A Justiniano, Steven, 791B Keränen, Soile V. E., 125, 319A* Kramer, Sunita G., 456C, 457A, 466A Kern, Andrew, 40* Krantz, David E., 15, 239B, 637A Kertesz, Michael, 36 Krasnow, Mark, 21, 440B K Ketel, C., 542B Krause, Henry, 105 Keung, Benison, 447C Kreitman, Martin, 661A, 663C Kabil, Hadise, 848B* Khammari, Asma, 7 Kremer, Susan, 393C Kadam, Snehalata, 75 Kharchenko, Peter, 767B Kreuz, Andrew, 259A* Kadandale, Pavan, 61* Khuong, Thang M., 652A Krishan, Badrinath, 555C Kadrmas, Julie L., 256A Kibanov, Mikhail V., 277A* Krishnan, Harish, 619A Kagesawa, Tatsuo, 656B* Kidd, Thomas, 146, 255C Krishnan, Natraj, 868A* Kaguni, Laurie S., 843C Kiger, Amy, 61, 97, 251B Krivega, Ivan, 269B Kaguni, L. S., 845B Kim, Ah-Ram, 303C* Kroiss, Matthias, 782B* Kahali, Bhaskar, 571A, 576C*, 578B Kim, Changsoo, 512B Kronhamn, Jesper, 289A Kalamarz, Marta, 795C* Kim, Eunha, 775A Krstic, Dimitrije, 16 Kalamegham, Rasika, 487A* Kim, Hyeeun, 615C Krupinski, Tomasz, 730A* Kalderon, Daniel, 518B Kim, Il-Man, 802A Krzemien, Joanna, 732C Kalmykova, Alla, 895A* Kim, Jee-Eun, 578B* Kucherenko, Mariya, 844A Kalmykova, Alla I., 329B Kim, Jiyoung, 512B Kugler, Jan-M., 321C* Kambris, Zakaria, 752B Kim, Sung Yun, 512B* Kuleck, Gary, 270C Kaminski, Mary F., 701B* Kim, Sunhong, 162C Kulikov, Alex, 653B, 658A*, 659B*, Kampinga, Harm, 92 Kim, Wonho, 162C* 708C* Kanda, Hiroshi, 208A Kim, Woongki, 579C* Kumar, Justin, 22, 203B, 407B, 408C, Kang, Min-Ji, 95* Kim, Yoosik, 242B, 378C* 409A, 415A, 416B, 879C Kang, Ruth, 247A Kim, Young-Cho, 636C Kumar, Kritika, 188B Kannan, Ramakrishnan, 556A Kim, Young-Joon, 10 Kumar, Sharad, 719B Kanwar, Ritu, 76 Kiparaki, Marianthi, 320B* Kumar, Sudhir, 664A, 680B, 684C*, Kao, Ling-Rong, 170B Kirov, Nikolai, 25 772A, 773B Kapadia, Bhaveen, 121 Kiryutin, Boris, 770B Kundu, Mukta, 304A* Kapelnikov, Anat, 474C, 476B* Kitts, Paul, 770B Kunttas, Ezgi, 580A* Kapustin, Yuri, 770B Klenov, Mikhail, 896B* Kunttas-Tatli, Ezgi, 571A SPEAKER AND AUTHOR INDEX 401

Kuoy, Edward, 775A Lembong, Jessica, 81, 322A* Lopes, Francisco J. P., 426C Kuraishi, Takayuki, 731B* Lemke, Steffen, 660C* Lopes, Hedibert F., 679A Kuranaga, Erina, 721A, 836B Lemmon, Zachary, 596B, 738C* Lopez de Quinto, Sonia, 884B Kurashima, Rick, 27 Lemons, Derek, 419B*, 880A Loppin, Benjamin, 522C* Kuroda, Mitzi, 767B LeMosy, Ellen, 401B, 451A* Lorigan, James, 703A* Kutzer, Megan, 99 Leonard, Dobens, 452B Lott, Susan E., 661A* Kuzin, Alexander, 304A Leopold, Pierre, 133* Lou, Xi, 137 Kwon, Dmitry, 895A Leptin, Maria, 107, 228C, 422B Lovato, TyAnna, 463A Kwon, Jae Young, 651C Lerit, Dorothy A., 246C* Low, Peter, 5 Kylsten, Per, 734B Lesly, Shera, 203B* Low, W. Y., 669C* Kyriakakis, Phillip, 783C Letsou, Anthea, 833B Lowe, Nick, 122 Leung, Wilson, 529A Loza Coll, Mariano A., 597C* Levi, Boaz, 440B Lu, Chenggang, 32, 501C* L Levine, Benjamin, 452B* Lu, Han, 76 Levine, Michael, 287B, 291C Lu, Wen, 70* Lacin, Haluk, 581B* Levine, Michael S., 129* Luan, Haojiang, 778A* Lackey, Melinda, 202A Levine, Mike, 45, 283A Lubensky, David K., 412A LaFave, Matthew C., 268A* Levis, Robert W., 784A* Ludwig, Michael, 663C LaFever, Leesa M., 513C* Leyssen, Maarten, 825C Ludwig, Michael Z., 661A LaFlamme, Brooke A., 702C* Li, A., 828C* Luengo, Cris, 296B Lai, Eric, 339C* Li, Hsun, 876C Luengo Hendriks, Cris L., 125*, 319A LaJeunesse, Dennis, 260B*, 640A* Li, Hui, 753C* Luhur, Arthur, 879C* Lambeth, Stacey, 404B Li, Ling, 131 Lujan, Ernesto, 785B* Lan, Lan, 212B Li, Wei-Ru, 796A* Lukinova, Nina, 736A Landry, Christian, 646A Li, Xiaolei, 323B*, 624C Lundell, Martha, 558C, 863B Langley, Sasha, 531C* Li, Xiao-Yong, 326B Luo, Jianyuan, 499A Lani, Aaron, 596B Li, Xiaoyong, 306C Luo, Liqun, 559A Lanzaro, Gregory, 45 Li, Xin, 114 Luo, N., 845B Laprise, Patrick, 217A* Li, Xingguo, 126 Luong, Nancy, 139 Larracuente, Amanda, 690C* Li, Yi-Ju, 599B Ly, Cindy, 11, 622A*, 819C Lasko, Paul, 37, 321C, 340A, 886A Li, Ying, 741C Lyalin, Dmitry, 807C* Lavine, Mark, 728B Li, Yumei, 573C, 582C* Lavrov, Sergey, 897C Li, Yun, 73 Lavrov, Sergey A., 277A Li, Zhouhua, 218B* M Lawal, Hakeem, 620B, 827B Liang, Hsiao-Lan, 25* Lawless, George, 832A Ma, Lina, 574A Libert, Sergiy, 853A Layalle, Sophie, 133 Mabuchi, Megumu, 607A Li-Chin, Yao, 34 Lazaga, Nelson, 642C MacAlpine, David, 278B Ligoxygakis, Petros, 3 Lazebny, Oleg, 658A, 659B, 708C MacArthur, Stewart, 306C* Li-Kroeger, David, 284B*, 297C Lazzaro, Brian P., 749B Macdonald, Paul, 889A Lilley, Kathryn, 122 Lebois, Evan, 810C Macdonald, Paul M., 881B Lim, Hui-Ying, 94* Lecuyer, Eric, 105* Machado, Carlos, 683B, 710B* Lin, Chiao-Ying, 781A* Lee, Anne M., 120 Machado, Carlos A., 682A Lin, Chii-Wann, 781A Lee, Cheng-Yu, 141, 361A, 608B MacIntyre, Ross, 449B, 856A* Lin, Nianwei, 527B* Lee, Ethan, 155 MacIntyre, Ross J., 667A Lin, Sung-Jan, 781A Lee, Gina, 161B Mack, Paul, 474C Lin, Xinhua, 86, 390C* Lee, Heuijung, 285C Maddox, Cynthia, 292A Lin, Yong Qi, 11 Lee, Hyun-Gwan, 490A* Maeda, Reo, 398B*, 399C Linder, Jodell E., 758B* Lee, Jeffrey, 104 Maeder, Morgan, 295A Lipshitz, Howard, 325A Lee, Kyu-Sun, 153 Maggert, Keith, 524B, 528C, 533B Lipsick, Joseph S., 539B Lee, Laura, 155, 196A, 492C Maglott, Donna, 770B Litvinova, Oksana, 136* Lee, Noemi E., 775A Maheshwari, Shamoni, 537C* Liu, Dongmei, 184A* Lee, Sung Bae, 162C Mahowald, Anthony, 286A Liu, Feng, 305B* Lee, Tammy, 520A Maia, Andre F., 169A Liu, Hsiao-Yun, 25 Lee, Thai, 282C Maidment, Nigel, 637A Liu, Jiandong, 482B Lee, Tom, 202A* Maines, Jean, 73* Liu, Lei, 858C Lee, Yuan-Ming, 561C* Makki, Rami, 732C* Liu, Songmei, 431B Legent, Kevin, 410B* Malartre, Marianne B. Y., 551B* Liu, Ya-Hsin, 312C Legrand, Delphine, 691A*, 709A* Malik, Bilal R., 662B* Liu, Yajuan, 350B* Lehmann, Ruth, 382A Malik, Jitendra, 125, 296B, 319A Long, Anthony, 41 Lei, Elissa P., 108* Mallozzi, Carolyn A., 56 Long, Apple G., 190A* Leips, J., 756C Manage, Kevin, 483C Long, Manyuan, 114, 679A, 687C Leips, Jeff, 701B, 705C Mandal, Lolitika, 465C Lopatto, David, 270C Lemaitre, Bruno, 752B Marchand, Virginie, 109 Lopes, Francisco, 427A* 402 SPEAKER AND AUTHOR INDEX

Marcus, Jeffrey, 434B McGregor, Alistair P., 421A* Minden, Jonathan S., 175A Mardon, Graeme, 573C, 582C McKay, James, 554B Mindrinos, Michael, 723C Marenda, Daniel R., 411C*, 546C McKay, K. M., 828C Ming, Liang, 325A* Maria, Samsonova, 780C McKearin, Dennis, 73 Minoda, Akiko, 767B* Marie-Louise, Cariou, 691A, 709A McKim, Kim, 199A Mintseris, Julian, 793A Marion, David J., 126* McKim, Kim S., 189C, 190A, 191B Miron, Mathiu, 37 Mark, Kankel, 96 McLean, Janna, 502A* Mirouse, Vincent, 12 Mark, Wu, 637A McLean, Reid, 502A Mistry, Hemi, 569B Markov, Alexander, 708C McMahon, Amy, 55* Mistry, Hemlata, 148 Markow, Therese, 43, 689B, 765C McNally, Elizabeth, 803B Mitrofanov, Vladimir, 653B, 658A, 659B Markow, Therese A., 49, 681C, 686B McNamara, Lucy, 670A* Mittelman, David, 102* Marques, Ana, 194B* McNamee, Laura M., 210C* Miura, Masayuki, 721A, 836B Marshall, Monique, 265A McNeil, Gerald, 270C Mizrachi, Ilene, 770B Martín, David, 851B McNeil, Gerard, 247A* Mkrtchyan, Marianna, 446B Martin, Francisco A., 157A* McNeill, Helen, 200B Mlodzik, Marek, 388A Martin, Paul, 257B McQuibban, Angus, 104 Mockler, Todd, 611B Martin Bermudo, Maria D., 551B McQuilton, Peter, 789C* Moki, Takeshi, 731B Martinho, Rui, 194B Mechler, Bernard M., 484A Molina, Camilo, 557B Martinho, Rui Gonçalo, 382A Mecklenburg, Kirk L., 598A* Mondal, Ashis, 136 Martins, Torcato J., 169A* Medina, Paul, 12 Monfort-Prieto, Elena, 893B Marty, Thomas, 194B Medioni, Caroline, 63* Montell, Craig, 649A Maruyama, Rika, 443B* Medranda, Giorgio, 29 Montooth, Kristi, 50 Maruyama, Risa, 893B Medrano, Vilma R., 56 Monzo, Kate, 175A*, 808A Marzluff, William, 898A Megraw, Timothy L., 170B* Moon, Seok Jun, 649A* Marzluff, William F., 892A Meier, Kerstin, 423C Moore, David J., 636C* Masly, John P., 711C* Meier, Rudolf, 28 Moore, Jocelyn, 340A* Mason, James, 279C, 281B* Meiklejohn, Colin, 50* Morales, Jorge, 725B Mason, James M., 472A Meisel, Richard, 115* Moran, Nancy, 765C Masuyama, Kaoru, 14 Meister, Marie, 732C Moran, Nancy A., 49 Matakatsu, Hitoshi, 85* Melicharek, David, 411C Morata, Gines, 157A, 722B Mateos San Martín, Daniel, 739A Meller, Victoria H., 882C Morgan, Rachel, 383B, 384C* Matera, A. Gregory, 887B Mellinger, Laurel, 511A Morillo, Jose, 32 Mathews, Sam J., 228C Melnikova, Larisa, 269B Morillo, Santiago A., 370A* Matsuno, Kenji, 356B, 360C, 398B, Mendoza-Ortiz, Miguel, 566B* Moriyama, Etsuko N., 666C 399C, 656B Meng, Xiangdong, 787A Moriyama, Hideaki, 594C Matsushima, Y., 845B Mentelová, Lucia, 5, 195C* Morley, Samantha, 872B* Matt, Nicolas, 724A Merkle, Julie, 196A* Morris, Deanna, 552C* Matunis, Erika, 67, 69, 508A, 511A Merriam, John, 363C, 446B Morris, Lucy, 514A* Matute, Daniel, 712A* Merriam, Lillian, 120 Morrison, Holly A., 240C* Matute, Daniel R., 713B* Metta, Muralidhar, 790A Mortin, Mark, 87 Matzkin, Luciano, 43* Metzstein, Mark, 25 Morton, C. J., 669C Matzkin, Luciano M., 686B Metzstein, Mark M., 445A, 883A Morton, David B., 362B, 650B Maughan, Bill, 293B Meyer, Heiko, 633C Moser, Melissa, 396C Maurange, Cedric, 144, 593B Meyer, Régis, 267C Moser, Theresa, 733A* Maynard, Jason C., 163A* Mezey, Jason, 703A Moshelion, Menachem, 264C Mazzoni, Esteban O., 600C Mezey, Jason G., 702C Moshkovich, Nellie, 108 McBride, Carolyn, 714C* Miailhe, Jérome, 152 Motiwale, Mansi, 671B* McBride, Sean M. J., 91 Michalak, Katarzyna, 747C Mottier, Violaine, 151* McCall, Kim, 739A*, 763A Michelson, Alan, 31 Mueller, Stefanie, 755B McCartney, Brooke, 237C Michelson, Andrew, 250A Muffat, Julien, 829A* McCartney, Brooke M., 56 Middleton, Ginnene, 411C Mugue, Nikolai, 715A*, 716B* McClure, Kim, 90 Mijares, Michelle, 483C Mukherjee, Subhas, 867C McConnell, Gretchen H., 488B* Milan, Neil, 48* Mulinari, Shai, 59* McCulloch, Andrew, 854B Miles, Cecelia, 663C* Muller, Anne C., 248B* McCulloch, Andrew D., 124 Miles, Wayne, 324C* Müller, H.-Arno, 403A, 405C McDaniel, Ivy, 544A Millard, Tom, 257B Müller, Jürg, 336C McDermott, Jeffrey, 469A Miller, Adam, 583A* Munjaal, Ravi P., 494B McDonald, Elizabeth, 423C*, 588C, Miller, David F. B., 788B Murakami, Ryutaro, 398B 601A Miller, E., 542B Murakami, Satoshi, 568A Mc Gillivray, Shauna, 93 Miller, Steven W., 307A* Murata, Shigeo, 836B McGinnis, William, Opening General Mills, Kathryn, 719B Murillo-Maldonado, Juan Manuel, 379A* Session, 258C, 311B, 419B, 655A, Milverton, Joanne, 174C Murphy, Terence, 770B* 768C, 880A Min, Jeong-Hye, 636C Musaró, Mariarosaria, 280A McGregor, Alistair, 444C Min, Kyung-Jin, 153 Muyskens, Jonathan B., 842B* SPEAKER AND AUTHOR INDEX 403

Myat, Monn Monn, 447C, 455B Nowak, Scott J., 127* P Myrick, Kyl, 786C* Nowotarski, Stephanie, 227B* Noyes, Marcus, 30, 787A Paaby, Annalise, 52* Nuzhdin, Sergey, 47, 694A, 697A, 714C Padash Barmchi, Mojgan, 59 N Nuzhdin, Sergey V., 616A* Paddibhatla, Indira, 795C Nystul, Todd, 72* Nadar, Christina, 795C Page-McCaw, Andrea, 4, 185B Nagalakshmi, Vidya, 474C Pai, Chi-Yun, 333C Nagarkar, Sonal, 193A O Paladino, Elana A., 327C* Nagengast, Alexis, 885C, 899B Palladino, Michael, 831C* Nair, Jayan N., 107* O'Connor, Kate, 569B Pallanck, Leo, 104 Nakajima, Yuichiro, 721A* Ocorr, Karen, 800B, 801C Palmer, Colin, 509B Nakamura, Naosuke, 807C Odenwald, Ward, 304A Pan, Lei, 134* Nakamura, Yukio, 656B O'Donnell, Janis, 620B, 638B, 827B Panchanathan, Sethuraman, 772A Nakanishi, Yoshinobu, 731B O'Donnell, Mike P., 553A* Pancratov, Raluca, 391A* Nakato, Hiroshi, 436A Oenel, Susanne-Filiz, 223A Panin, Vlad, 807C Nakayama, Hiroshi, 731B Ogawa, Nobuo, 326B* Pantoja, Mario, 844A* Nallamothu, Gouthami, 219C* Ogden, Stacey K., 431B Panz, Mareike, 633C* Nam, Sang-Chul, 226A* Oh, Byung-Ha, 321C Papoulas, Ophelia, 808A* Nambu, John, 743B Ohlstein, Benjamin, 448A* Pappano, William N., 80 Napoletano, Francesco, 823A Ohsako, Takashi, 491B Paquette, Nicholas, 750C Naps, Keith, 164B* Ohyama, Tomoko, 11, 622A Paré, Adam, 311B, 655A, 768C, 880A* Nässel, Dick R., 14 Okado, Kiyoshi, 759C* Paredes, Silvana, 528C* Natalia., Burdina, 290B Okamura, Katsutomo, 339C Parisi, Michael, 678C, 680B Natarajan, Rajalaxmi, 584B* Okegbe, Tishina C., 515B* Park, Cheol, 797B Neal, J. T., 842B Okumura, Takashi, 399C* Park, Jung, 35 Nelson, Dylan, 641B Olcese, Ursula, 317B Park, Peter, 767B Neto da Silva, Ricardo M., 158B* Oldham, Sean, 139* Park, Sangbin, 78 Neufeld, Thomas, 154, 160A, 164B, Olenina, Ludmila, 495C Park, Soo, 121 834C Olenkina, Oxana, 495C Parker, Louise, 547A Neufeld, Thomas P., 140 Olga, Simonova B., 290B* Parker, M. W., 669C Newfeld, Stuart, 477C*, 664A* Oliveira, Ligiane, 334A Parsa, Bayan, 121 Newfeld, Stuart J., 772A Oliveira, Marcos T., 843C* Parthasarathy, Neela, 105 Newton, Fay, 574A Oliveira-Silva, Adriana, 347B Parthasarthy, Akhila, 549C Ng, Chen-Siang, 704B* Oliver, Brian, Plenary Session II, 101, Pashaj, Anjeza, 864C, 873C* Ng, H. L., 669C 123, 486C, 487A, 489C, 678C, 680B Pastor-Pareja, José C., 1* Ngo, Kathy, 775A Oliver, Daniel, 333C Pasyukova, Elena, 692B Nguyen, Bac T., 15 Oliver, Daniel K., 342C* Pasyukova, Elena G., 866B Nguyen, David, 567C Olson, Aaron, 236B Patananan, Alexander N., 775A Nguyen, Elizabeth, 642C Olson, Emily R., 391A Patch, Kistie, 229A* Nguyen, Hanh, 432C, 468C Olson, John M., 775A* Patel, Leena, 367A, 380B* Nguyen, Minh, 547A Olson, Sara, 35 Patel, Niapm H., 707B Nicchitta, Christopher V., 163A Önel, Susanne-Filiz, 232A Patel, Rajesh, 456C Nichols, Charles D., 635B Ong, SengKai, 176B* Patel, Reena, 607A Nien, Chung-Yi, 25 Onorato, Thomas, 503B Paterno, Shelley, 99, 506B Nightingale, Barbara, 554B Opazo, F., 824B Paternostro, Giovanni, 124, 854B Niimi, Teruyuki, 788B Opiyo, Stephen O., 666C Paterson, Bruce, 111 Nikitina, Ekatherina A., 830B* Ordaz Tellez, Maria Guadalupe, 874A Paul, Jennifer T., 815B Nilsen, Steven, 641B* Orenic, Teresa, 477C Paul, Sarah, 217A Nilson, Laura, 471C Orfanos, Zacharias, 62 Paululat, Achim, 633C Nilsson, Patrik, 587B Orr, Allen, 117 Pavlovic, Maja, 734B* Nirenberg, Marshall, 301A Orr, William, 747C* Payne-Tobin, Anna, 230B Nishikawa, Minori, 656B Orr-Weaver, Terry, 74, 185B, 278B Payre, Francois, 152, 257B, 444C*, Nisoli, Ilaria, 823A Orsi, Guillermo, 522C 453C* Niwa, Ryusuke, 852C Orso, Genny, 261C* Peabody, Nathan C., 632B Nizet, Victor, 93 Ortiz, Zachary, 4 Pearce, Elspeth, 266B Nogueira, Cristina, 185B* O'Shea, Michelle, 106 Pearse, Elspeth, 328A* Nolan, Nicole, 626B Ospina, Jason, 250A Pearson, John, 454A* Nolan, Nicole W. C., 628A O'Tousa, Joseph, 738C Pechmann, Matthias, 421A Noll, Markus, 16, 20 O'Tousa, Joseph E., 596B, 598A Peden, E., 110 Nomie, Krystle J., 341B* Ott, Alice, 228C* Peifer, Mark, 58, 225C, 227B, 231C, Norvell, Amanda, 220A* Ottenwaelder, Birgit, 790A 385A Nouaud, Danielle, 271A* Overton, Lewis J., 268A Peller, Cassandra R., 862A* Novakova, Milena, 726C Ovetsky, Michael, 770B Pellikka, Milena, 214A, 216C, 221B Pendin, Diana, 261C 404 SPEAKER AND AUTHOR INDEX

Pendred, Julia, 593B Prazak, Lisa, 29 Reich, John C., 881B* Peng, Emmeline, 853A Preat, Thomas, 625A Reichhart, Jean-Marc, 748A* Peng, Jamy, 521B Presgraves, Daven C., 711C Reid, David, 842B Peng, Jianlan, 573C Preston, Christine, 116 Reinitz, John, 303C, 428B Peng, Lixin, 114 Pret, Anne-Marie, 7* Reis, Tânia, 817A* Pennetier, Delphine, 732C Price, Hilary, 255C Reischl, Joachim, 423C Pennington, Matthew W., 412A* Priest, Henry, 611B Reiter, Lawrence, 811A Peralta, S., 845B Prince, Frederic, 417C Rembold, Martina, 228C Perdigoto, Carolina, 17 Prohaska, Sonja, 684C Renkawitz-Pohl, Renate, 223A, 232A Pereanu, Wayne, 567C Prokopenko, Sergei, 177C* Resenchuk, Sergey, 770B Pereira, Fátima, 195C Promislow, Daniel E. L., 758B Restifo, Linda L., 563B Perez, Alma, 102 Pruitt, Kim, 770B Restrepo, Simon, 204C Pérez, Coralia, 851B Purrier, Sheryl, 247A Reuter, Rolf, 228C Perrimon, Norbert, 424A Pyrowolakis, George, 77, 348C Reveal, Brad, 889A* Petersen, Andrew J., 525C Reynolds, Steven H., 172A* Petersen, Melissa, 809B Rezende, Gustavo, 45 Peterson, Brant, 27*, 28 Q Rhea, Jeanne, 634A* Peterson-Nedry, Wynne, 393C Rhrissorrakrai, Kahn, 474C Qian, Li, 137, 482B Petrella, Lisa N., 263B Ribeiro, Carlos, 10 Qin, Wei, 111 Peyre, Elise, 823A Ribeiro, Inês, 97*, 251B Querings, Silvia, 150 Pfeiffer, Barret, 240C, 607A Richard, Magali, 410B Quesneville, Hadi, 271A Pfeiffer, Barret D., 319A Richardson, Brian, 467B* Quintin, Jessica, 760A* Pfleger, Cathie, 156 Rickert, Christof, 562A Quirin, Magali, 728B Pflugfelder, Gert O., 414C Riddle, Nicole, 529A*, 767B Phadnis, Naina, 262A* Riechmann, Veit, 107 Phadnis, Nitin, 117* R Riedel, D., 824B Phelps, Melissa, 54 Riesgo-Escovar, Juan, 379A, 566B Phin, Sopheap, 78 Raafat, Kimia, 544A Rinaldo, Francesca, 4 Pi, Hai-Wei, 599B* Rachamaduggu, Rakesh, 557B Rincon-Limas, Diego E., 103 Piazza, Nicole, 872B Radford, Sarah J., 190A Rinner, Olivier, 855C Pick, Leslie, 84, 420C Radtke, Tina, 437B Rio, D., 110 Pickup, Amanda, 325A Radyuk, Svetlana, 747C Rio, Donald, 35 Pidoux, Josette, 877A Rafiqi, Matteen, 660C Río, Xavier, 671B Piel, Jess, 129 Raftery, Laurel, 324C RioSingh, R., 110 Piel, Jessica, 291C* Rahmani, Sahar, 361A Ritchie, Michael G., 698B Pierre, Susan, 607A Rahme, Laurence, 723C Rivlin, Patricia, 474C, 476B Pignoni, Francesca, 168C, 314B Rajendran, Vani, 202A Roberts, David, 385A Pimentel, Emilio, 839B Ramachandran, Kapil V., 623B* Roberts, Stephen P., 630C Pinto, Belinda, 538A*, 540C Ramazani, Rosie B., 623B Robin, C., 669C Pirraglia, Carolyn, 455B* Ramos, Hector, 772A Robinow, Steven, 331A, 642C* Pirrotta, Vincenzo, 767B Ramsay, Gary, 182B Roche, Andrea, 370A Pitsouli, Chrysoula, 424A* Rana, Anil, 92 Rochlin, Kate, 468C* Platt, Jeffery, 4 Ranade, Swati, 314B Rockman, Howard, 803B Plaza, Serge, 257B, 417C, 444C, 453C Rand, David, 50 Rockman, Howard A., 802A Pletcher, Scott, 745A, 848B, 860B Rasmuson-Lestander, Åsa, 289A Rodrigues, Aloma, 165C* Pletcher, Scott D., 138, 853A Rassis, Raquel, 113 Rodriguez, Deyra, 259A Plummer, Timothy, 4 Rataiczak, Holly, 555C Rodriguez-Arnaiz, Rosario, 874A* Pocklington, Andrew J., 662B Ratnaparkhi, Anuradha, 832A* Rogers, Edward M., 231C* Poh, Yu-Ping, 717C* Ratnaparkhi, Girish, 38* Rogers, Greg, 385A Pohl, Jascha B., 643A Rauser, Cassie, 181A Rogulja-Ortmann, Ana, 595A* Pokrywka, Nancy, 230B* Ravulapalli, Anandarao, 111* Rollins, Janet, 503B* Polesello, Cédric, 152 Rawls, John, 499A, 552C Roman, Cherezov, 290B Pollard, Daniel, 672C* Ray, Anandasankar, 9* Roman, Gregg, 615C Pollock, John, 554B* Ray, Sanchali, 57, 238A Romero-Calderon, Rafael, 637A Ponce, Alberto, 345C Rayburn, Lowell, 634A Rong, Yikang, 181A Poole, Angela, 104 Read, Renee D., 98* Ronshaugen, Matthew, 44*, 655A Poon, Peter, 860B* Real, Joseph, 596B Ronsseray, Stéphane, 132* Posakony, James, 605B Rebay, Ilaria, 79, 335B, 370A, 375C Root, Cory M., 14* Posakony, James W., 305B, 307A, Redinbo, Matthew R., 892A Roote, John, 122 357C, 597C Reed, Jamian D., 605B* Rose, Patrick P., 735C* Posenau, Trevor, 67 Reed, Laura, 816C* Rosenbaum, Erica E., 60*, 244A, 248B Poulton, John, 358A*, 397A Reed, Nanette, 797B Roshina, Natalia V., 866B* Powell, Lynn, 574A Rees, Johanna, 122 Rosset, Roland, 267C Prantera, Giorgio, 187A Reeves, Nick, 605B Rottig, Carmen, 785B SPEAKER AND AUTHOR INDEX 405

Rovani, Margritte K., 182B* Sayal, Rupinder, 26 Sharakhova, Maria, 272B, 675C Roy, Soumit, 371B* Schaefer, Gritt, 223A Sharma, Yagya V., 390C Roy, Swarnava, 535A* Schaeffer, Stephen, 674B* Sharp, David, 303C Rozas, Natalia S., 60 Schäfer, Gritt, 232A* Shashidhara, L. S., 556A Rozovsky, Yakov, 895A Schaffner, Walter, 368B Shaw, Pang Chui, 820A Ruaud, Anne-Françoise, 857B* Schejter, Eyal, 89 Shcherbata, Halyna, 149, 844A Rübel, Oliver, 296B Scherr, Heather, 202A Shcherbata, Halyna R., 516C* Rudolph, Tim, 899B* Schickedanz, Adam, 569B Shearer, Andrew, 294C Ruiz, Oscar E., 445A* Schiller, Ben, 300C, 309C* Shelly, Spencer, 736A* Ruohola-Baker, Hannele, 149, 516C, Schlenke, Todd, 48 Shen, Jie, 414C 844A Schloetterer, Christian, 693C*, 790A* Shen, Kate, 363C, 563B Rusconi, Jamie, 740B* Schmidt, Paul, 52, 850A* Shen, Ying, 766A Rusconi, Jamie C., 888C Schmidt, Rebecca, 4, 797B Sheng, Rebecca, 67 Ruse, Cristian, 808A Schmidt-Ott, Urs, 660C Sheng, Xuting Rebecca, 69* Rushlow, Christine, 25 Schmucker, Dietmar, Plenary Session Sher, Noa, 278B* Russell, Steve, 122 I, 365B, 549C Sherazee, Nyssa A., 643A* Russo, Susan, 674B Schneeman, Anette, 755B Shibutani, Shu, 171C Rusten, Tor Erik, 240C Schneider, Daniel, 385A Shields, Alicia, 344B* Ryan, Kathryn M., 308B* Schneiderman, Jonathan I., 523A* Shilo, Benny, 89 Rybina, Olga Y., 692B* Schoenfeld, Brian P., 91 Shin, JuHyun, 211A* Ryder, Ed, 122* Schoenfeld, Joshua, 220A Shingleton, Alexander, 206B*, 207C* Ryder, Pearl, 803B Scholl, Maureen R., 624C* Shinzawa, Naoaki, 759C Rynearson, Shawn G., 883A Schongalla, Malcolm, 740B Shiratsuchi, Akiko, 731B Ryoo, Hyung Don, 95, 720C Schroeder, Mark, 83* Shlevkov, Evgeny, 722B* Ryoo, Hyung-Don, 754A Schubiger, Gerold, 90 Short, Sarah M., 749B* Ryu, Hyoje, 549C Schulz, J. B., 824B Shouche, Yogesh, 675C Schulz, Robert A., 464B Shpiz, Sergey, 895A Schulze, Karen L., 11, 602B, 784A Shpiz, Sergey G., 329B* S Schulze, Sandra R., 809B*, 840C Shubeita, George, 243C Schüpbach, Trudi, 81, 891C Shvartsman, Stanislav, 378C Sabin, Leah R., 761B* Schwank, Gerald, 204C* Shvartsman, Stanislav Y., 24, 81, Sackton, Katharine, 188B Schwartz, Yuri, 767B 242B, 322A Sackton, Timothy, 673A* Schwedes, Christoph C., 618C* Sibon, Ody, 92* Sage, Brian, 137* Schweisguth, François, 17 Sibon, Ody C. M., 771C Sage, Brian T., 875B Schweizer, Felix, 832A Sickmann, Albert, 782B Saint, Robert, 174C Schwinkendorf, Daniela, 166A* Siddiqui, Saira, 869B Saltz, Julia, 617B* Scott, Matthew, 253A Sidor, Clara, 132 Salz, Helen, Opening General Session Screen Team, 2R, 194B Silva, Elizabeth, 737B* Salz, Helen K., 507C Secombe, Julie, 131* Silverman, Neal, 730A, 750C* Salzer, Claire, 22* Seeds, Andrew, 354C Simcox, Amanda, 259A, 791B* Salzler, Harmony, 898A Seeger, Mark, 550A Simirenko, Lisa, 296B Samsonova, Maria, 428B Segal, Eran, 36 Simmons, Grant, 585C* Sánchez, Jonatan, 851B Sehgal, Amita, 637A Simon, Anne, 637A* Sánchez-Herrero, Ernesto, 418A Seibert, Janina, 591C* Simon, J. A., 542B Sanchez-Martinez, A., 845B* Seigle, Jacquelyn, 831C Simone, Robert, 234C* Sanders, Laura, 147* Seinen, Erwin, 771C* Simonette, Rebecca A., 494B Sandoval, Efren, 258C Seisenbacher, Gerhard, 205A*, 762C* Simpson, Ian, 574A Sandoval, Natalie A., 372C* Sekelsky, Jeff, 268A Simpson, Pat, Plenary Session II Sang, Tzu Kang, 821B Selva, Erica M., 390C, 810C* Singh, Amit, 413B* Santiago-Martínez, Edgardo, 456C*, Semeriva, Michel, 63 Singh, Nadia D., 676A* 457A Sen, Aditya, 285C* Singhania, Aditi, 31 Sapira, Sagi, 723C Senoo-Matsuda, Nanami, 159C* Sinha, Saurabh, 787A Sarhan, Moustafa, 343A* Sens, Kristin, 233B* Sink, Helen, 20 Sarov, Mihail, 119 Serbus, Laura, 249C* Sirohi, Neha, 899B Sarpal, Ritu, 214A, 221B* Sergienko, Vyacheslav, 653B Sisneros, David, 502A Sasamura, Takeshi, 398B Serway, Christine, 626B Sisson, John C., 175A, 808A Sass, Miklos, 5 Serway, Christine N., 628A* Situ, Shufen, 141, 608B Satish, Aruna, 718A* Serysheva, Ekatherina, 388A Sitz, Daniel, 775A Sato, Makoto, 568A Sexton, Travis, 400A* Sivatchenko, Anna, 833B* Saville, Ken, 270C Shah, Arpit, 411C Skeath, James, 148, 569B, 581B, Savla, Urmi, 543C* Shao, Huanjie, 469A 584B Savva, Yiannis, 35 Shapiro, Peter J., 754A* Skorupa, Danielle A., 138*, 853A Savvateeva-Popova, Elena V., 830B Shapiro Kulnane, Laura, 507C Small, Chiyedza, 167B* Sawyer, Jessica, 225C Sharakhov, Igor, 272B*, 675C* Small, Kate R., 373A* 406 SPEAKER AND AUTHOR INDEX

Small, Stephen, 429C, 430A Stinchfield, Michael, 664A Tang, Huaping, 752B* Smith, Helen, 498C St. Johnston, Daniel, 12, 122 Tang, Wei, 607A* Smith, Helen F., 275B* Stocker, Hugo, 205A, 762C Tang, Xiaofang, 390C Smith, John, 489C* Stockwell, Sarah, 42 Tång Hallbäck, Erika, 813C* Smith, Temple, 674B Stormo, Gary, 30 Taniguchi, Kiichiro, 398B, 399C Smith-Bolton, Rachel K., 208A* Stramer, Brian, 257B Tanner, Elizabeth, 739A, 763A* Smolinski, Sara, 774C Strobel, Lisa L., 630C Tanneti, Shree N., 191B* Snee, Mark J., 881B Stronach, Beth, 366C Tanzi, R. E., 828C Snowflack, Danielle R., 330C* Strope, Pooja K., 666C Tare, Meghana, 413B Somers, W. Gregory, 606C Stultz, Brian, 87*, 285C Tarnay-Cogbill, Jolene, 331A* Son, Wonseok, 413B Stümpges, Birgit, 437B Tarone, Aaron, 694A* Sonenberg, Nahum, 37 Sturgill, David, 678C*, 680B Tatar, Marc, 137, 153, 696C, 815B, Song, Haiyun, 392B* Su, Jessica, 46 875B Song, Ho-Juhn, 834C* Su, Ying, 250A* Tatomer, Deirdre C., 892A* Song, Yan, 520A Subramanian, Vijayalakshmi, 186C Tatusova, Tatiana, 770B Sopheap, Phin, 34 Sullivan, David, 856A Tavantzis, Naoum, 835A* Soplop, Nadine, 457A* Sullivan, William, 173B, 249C Tavares, Álvaro, 195C Soplop, Nadine H., 456C Summers, Kyle, 811A* Tcybulko, Eugenia A., 866B Sorourian, Mehran, 677B* Sun, Jianjun, 478A* Technau, Gerd M., 556A, 595A Sorrentino, Richard P., 464B Sun, Lin, 530B Technau, Gerhard, 562A, 591C Soshnev, Alexey A., 126 Sun, Xiaoping, 530B Teixeira, Luís, 2* Soto, Claudio, 103 Sun, Y. Henry, 312C, 313A, 414C, Ten Hagen, Kelly G., 460A Soukup, Sandra F., 751A* 425B, 561C, 796A Tepass, Ulrich, 214A, 215B, 216C, Soula, Cathy, 436A Sun, Yishan, 858C* 217A, 221B Sousa-Nunes, Rita, 606C* Sung, Carl, 642C Terman, Jonathan, 435C* Southall, Noel, 123 Sunkel, Claudio, 169A Terriente, Javier, 143 Souvorov, Sasha, 770B Supatto, Willy, 55 Terry, Natalie, 509B, 515B Spana, Eric, 163A Surkova, Svetlana, 428B* Tesic, Ivan, 259A Sparrow, John, 62* Sustar, Anne, 90* Tetweiler, Gritta, 37 Spencer, Susan, 579C, 585C Swanson, Christina I., 310A* Texada, Michael J., 494B Spichiger, Chloé, 392B Sykiotis, Gerasimos, 818B Teysset, Laure, 132 Spindler, Shana R., 567C* Symonenko, Alexandr V., 866B Thackeray, Justin, 379A Spirov, Alexander, 427A Szafer-Glusman, Edith, 178A* Theopold, Ulrich, 746B Spirov, Alexander V., 426C Szeri, Flora, 331A Therrien, Marc, 377B, 794B Spitzer, Ayelet, 869B Sznajder, Jacob I., 730A Thomas, Chenel, 709A Sporn, Peter, 730A Sztalryd, Carole, 101 Thomas, Chrisna V., 479B* Spradling, Allan, Plenary Session II Thomas, John B., 98 Spradling, Allan C., 72, 99, 480C, Thomas, Ruth, 104 506B, 514A, 784A T Thomassin, Hélène, 877A Sprecher, Simon, 586A* Thor, Stefan, 143, 577A, 587B Tabata, Tetsuya, 568A Sprenger, Frank, 150 Thorpe, Lauren M., 56 Tabchi, Simon, 644B*, 645C Spriggs, Helen, 122 Thummel, Carl, 857B Tabone, Christopher, 629B* Springston, Mastafa, 816C Tian, Ai-Guo, 88* Tabone, Christopher J., 15 Spyros, Artavanis-Tsakonas, 96 Tian, Sufang, 401B* Taghli-Lamallem, Ouarda, 804C Srinivasan, Shrividhya, 130 Tien, An-Chi, 599B Tajiri, Reiko, 458B* Stadelmann, Laura, 120 Timmermans, Wendy, 650B Takaesu, Norma, 477C Staiger, Dominik, 368B Ting, Chau-Ti, 688A, 707B, 717C Takakazu, Yokokura, 96 Stamm, Joyce, 270C Tipping, Marla, 374B*, 783C Takashima, Shigeo, 446B* Stanley, E. Richard, 371B Tittermary, Sara, 254B Takeda, Shugaku, 324C Stapleton, Mark, 121* Todeschini, Anne-Laure, 132 Takemori, Nobuaki, 491B* Starks, Adrienne, 705C* Tolstoy, Igor, 770B Talamillo, Ana, 851B* Stathopoulos, Angela, 55 Tolwinski, Nicholas, 387C Tamkun, John, Plenary Session I Stathopoulos, Angelike, 75* Tomancak, Pavel, 105, 119 Tamkun, John W., 130 Steffensen, Sören, 169A Tomioka, Takeyasu, 836B Tan, Change, 176B, 779B Steinhauer, Josefa, 381C*, 410B Tonning, Anna, 814A Tan, Guihong, 779B* Steiniger, Mindy, 892A Tonoki, Ayako, 836B* Tan, Jingwen, 545B Stenmark, Harald, 240C Tootle, Tina L., 480C* Tan, Julie, 235A* Stephenson, Edwin C., 276C* Torres, Tatiana, 790A Tanaka, Akemi J., 263B* Stern, David, 47, 444C Tosetto, Jessica, 261C Tanaka, Asami, 627C Stevens, Traci, 254B Tostões, Rui, 194B Tanaka, Keiji, 836B Stevens, Traci L., 798C Tour, Elvira, 311B* Tanda, Soichi, 766A* Stewart, Judith, 650B Tracy, Charles, 671B Tanentzapf, Guy, 517A* Stewart, Shannon, 229A Tran, PhuongThao, 775A Tang, Amy, 4*, 4, 797B*, 797B Stieper, Bradley, 207C Tran, Susan, 243C SPEAKER AND AUTHOR INDEX 407

Tranguch, Susanne, 155 Vibranovski, Maria D., 679A* Wang, Yan, 64*, 376A, 619A Treisman, Jessica, 381C, 410B, 548B Vichas, Athea, 406A* Wang, Ying, 483C* Trejo, Theodore, 4 Vied, Cynthia, 518B* Wang, Yi-Yun, 876C* Trivigno, Catherine, 812B* Vielmas, Erika, 481A* Wang, Yuan, 741C* Tsai, Yu-Chen, 414C* Vigil, Hollie, 502A Ward, Catherine, 46 Tsaur, Shun-Chern, 717C Vigneswaran, Krishanthan, 413B Ward, Ellen J., 172A, 516C Tsoi, Frankie Ho, 820A Vijg, Jan, 863B Ward, Robert, 229A, 236B, 266B, Tsui, Man-kin Marco, 354C* Viktorinova, Ivana, 213C 328A Tsujimura, Hidenobu, 764B* Villa-Cuesta, Eugenia, 875B* Waring, Gail, 459C Tsurudome, Kazuya, 610A Villella, Adriana, 818B Warren-Paquin, Maude, 610A* Tu, Meng-Ping, 183C* Villén, Judit, 793A Warrick, John, 835A Tu, Zhijian, 675C Vincent, Alain, 19, 732C Warrick, John M., 826A Tulina, Natalia, 511A Vining, Melissa, 439A Warrior, Rahul, 34*, 78, 483C, 785B Tummala, Hemachand, 811A Vining, Melissa S., 442A Watson, Annie, 777C Tung, Jennifer, 596B Visk, DeeAnn W., 592A* Watts, Thomas, 765C* Turan, Tolga, 483C Vitte, Clémentine, 271A Wawersik, Mattew J., 488B Turk, Rebekah, 292A Vivekanand, Pavithra, 335B Wawersik, Matthew, 67* Turki-Judeh, Wiam, 345C Vogler, Georg, 482B* Weasner, Bonnie, 415A* Turnbull, Doug, 730A Voinnet, Olivier, 894C Weasner, Brandon, 416B* Turner, Thomas, 695B* Volk, Talila, 224B, 450C Weaver, Carole, 252C* Turner, Tom, 714C Volkov, Ilya, 269B Weaver, Molly, 21* Tussey, Nathaniel, 644B, 645C* Volland, Dagmar, 591C Weber, Gunther, 296B Tweedie, Susan, 792C* Volpi, Silvia, 187A Weber, Gunther H., 125 Tyler, David, 339C von Hilchen, Christian M., 562A* Webster, Jane, 122 Tzou, Phoebe, 75 von Kalm, Laurence, 383B, 384C Wegener, Christian, 634A Von Stetina, Jessica, 155* Wehling, Misty D., 126 Voog, Justin, 519C* Wehrli, Marcel, 393C* U Vrailas-Mortimer, Alysia D., 411C Wei, Ho-Chun, 241A Vreede, Andrew, 778A Weil, Timothy, 891C Ueda, Hitoshi, 337A, 343A Vuilleumier, Robin, 77* Weil, Timothy T., 246C Ueda, Koichi, 731B Weiss, Linnea A., 651C* Ulvklo, Carina, 587B* Weiszmann, Richard, 769A Umetsu, Daiki, 568A W Welch, Aaron, 229A Unnerstall, Ulrich, 36 Welsh, Michael, 647B Urbach, Rolf, 591C Wagner, Eric J., 892A, 898A* Welsh, Michael J., 858C URCFG, UCLA, 775A Wagner, Jorj, 681C Welte, Michael, 243C, 262A Uv, Anne, 806B, 813C, 814A Wakabayashi, Atsuya, 30, 787A Wen, Hong, 539B* Wakabayashi-Ito, Noriko, 594C* Weng, Mo, 141*, 608B* Wakimoto, Barbara, 187A Werz, Christian, 741C V Wallenfang, Matthew, 869B* Wessells, R. J., 139 Wallrath, L. L., 542B Vaccari, Thomas, 76* Wessells, Robert J., 94, 804C, 872B Wallrath, Lori L., 525C, 538A, 540C, Valcarcel, J., 110 Whaley, Michelle A., 596B 809B, 840C Valenti, Philippe, 152, 444C Wharton, Keith, 439A Wan, Ken, 121 Valle, Denise, 45 Wharton, Robin, 341B, 520A Wandler, Anica, 842B van der Goes van Naters, Wynand, 9 Whitaker, S. Leigh, 572B Wandler, Anica M., 799A* van der Linde, Kim, 700A White, Benjamin, 778A Wang, Cheng-wei, 425B* Van Doren, Mark, 23, 67, 485B White, Benjamin H., 632B Wang, Chuan-Ju, 312C* Van Emden, Bernard, 772A* White, Kristin, 607A Wang, Dennis, 730A Van Goethen, Emeline, 728B Whitman, Stacey, 807C Wang, Hao, 602B van Riji, Ronald, 894C Whitworth, Alexander J., 104* Wang, Horng-Dar, 876C Varadi, Andras, 331A Wicker-Thomas, Claude, 698B Wang, J., 542B Vasiliauskas, Daniel, 600C* Wiesner, Julia, 782B Wang, Jing W., 14 Vassor, Valerie, 670A Wilhelm, James, 893B* Wang, Jing-Zi, 876C Vecomnskie, Kathryn, 504C* Wilk, Ronit, 325A Wang, Keqing, 582C, 590B Vedenina, Varvara, 715A, 716B Williams, Carrie, 620B Wang, Lan-hsin, 313A* Velichkova, Michaella, 251B* Williams, Elizabeth H., 80* Wang, Lihui, 3* Venken, Koen J. T., 432C, 784A Williams, Stephanie, 816C Wang, Liqun, 355A* Ventura, Gemma, 382A* Williamson, Linsay, 12 Wang, Liwei, 218B Veraksa, Alexey, 374B, 783C* Wilmington, Shameika, 538A, 540C* Wang, Qiong, 818B* Vermehren, Anke, 362B, 650B* Wilson, Beth, 569B, 581B Wang, Wen, 114 Vernì, Fiammetta, 151 Wilson, Rachel, Plenary Session II Wang, Xia, 630C* Vernier, Kaitlyn M., 798C* Wimmer, Ernst, 423C Wang, Xiaochen, 266B*, 328A Verstreken, Patrik, 11, 622A, 652A, Winbery, Brenda, 534C Wang, Xiaoling, 29 819C, 825C Windler, Sarah L., 53* 408 SPEAKER AND AUTHOR INDEX

Wingen, Christian, 437B Y Z Winkler, Clint, 345C* Wiora, Heather, 500B Yackle, Kevin, 775A Zaffo, Barbara J., 888C Wisotzkey, Robert, 664A Yacoub, Nasrine, 367A Zallen, Jennifer, 406A Witt, Lorraine, 284B, 297C Yakoby, Nir, 24, 81*, 322A Zanet, Jennifer, 257B*, 444C Wodarz, Andreas, 142, 338B Yamada, Kenta, 360C* Zapata, Monica, 245B Wojcinski, Alexandre, 436A* Yamada, Masaki, 491B Zare, Fariba, 814A* Wolf, Matthew, 803B Yamaguchi, Masamitsu, 731B Zarnescu, Daniela C., 106*, 604A Wolf, Matthew J., 802A Yamamoto, Masa-Toshi, 491B Zartman, Jeremiah J., 24* Wolfe, Scot, 30, 787A Yamamoto, Naoko, 731B Zeitlinger, Julia, 129 Wolfner, Mariana, 188B Yamamoto, Shinya, 602B* Zelentsova, Helen, 653B Wong, Alan S. L., 837C Yamanishi, Minoru, 594C Zelinger, Einat, 474C Wong, Azaria K. Y., 837C* Yamashita, Yukiko M., 68 Zeng, Lucy, 326B Wong, Laura, 642C Yan, Dong, 86*, 390C Zhai, Bo, 793A* Wong, Raymond, 179B* Yan, Hua, 156* Zhang, Cheng, 20* Woo, Jae-Sung, 321C Yang, Shuang, 114 Zhang, Jiuli, 170B Wood, Carla M., 488B Yao, Bing, 527B Zhang, Jun, 253A* Wood, Jamie, 299B Yao, Chi-Kuang, 11*, 622A Zhang, Liang, 236B* Wood, Jamie L., 302B Yao, Jih-Guang, 414C Zhang, Liping, 460A* Wood, Kathleen H., 505A* Yapici, Nilay, 10* Zhang, Tianyi, 168C*, 314B* Wood, Margaret, 292A* Yasugi, Tetsuo, 568A* Zhang, Weiguo, 118* Woolworth, Julie, 222C* Yates III, John, 808A Zhang, Yanping, 527B Workman, Michael, 423C, 601A* Yatsenko, Andriy, 844A Zhang, Ya-Qin, 123 Worley, Melanie, 208A Yazdani, Umar, 435C Zhang, Ying, 460A Wright, O'Neil, 638B* Ye, Fangfei, 703A Zhang, Yu, 678C, 680B* Wrobel, Carolyn, 375C* Ye, Jieping, 773B* Zhang, Yue, 114 Wu, Christine, 209B* Yeakley, Joanne, 35 Zhang, Zhongchun, 377B* Wu, C.-ting, 120, 274A Yemelyanova, Anastasia, 631A* Zhao, Huiwen W., 846C* Wu, G., 756C Yeo, Gene, 35 Zhao, Ruoping, 114 Wu, Joy, 545B Yeung, Y. G., 371B Zhao, Yun, 351C* Wu, Joy S., 559A Yin, Jerry C.-P., 776B Zhou, Bo, 390C Wu, June-Tai, 781A Yogev, Shaul, 89* Zhou, Dan, 346A*, 592A, 846C Wu, Ming, 1 Yoneda, Tomohiro, 764B Zhou, Meng-Ning, 237C* Wu, Samuel, 527B Yoo, Na-Eun, 430A Zhou, Pei, 192C* Wu, Tianyi, 459C* Yoo, OokJoon, 211A Zhou, Qi, 114 Wu, Yihui, 390C Yoo, Siuk, 301A Zhou, Qingxiang, 314B Wu, Yujane, 376A* York, John, 354C Zhou, Wenke, 777C Yoshida, Hideki, 105 Zhou, Ying, 743B* Yoshiyama, Takuji, 852C* Zhou, Zhaolan, 102 X Young, Lynn, 185B Zhu, Alan, 250A Xia, Ai, 675C Young, Richard A., 129 Zhu, C. T., 850A Xie, Baotong, 588C* Younossi-Hartenstein, Amelia, 446B Zhu, Yi, 581B Xie, Ting, 134, 878B Yu, Charles, 121 Zhuang, Shufei, 469A* Xie, Weiwu, 332B* Yu, Danyang, 429C* Zielke, Norman, 150* Xie, Xuanhua, 359B* Yu, Jenn-Yah, 149*, 172A, 516C Zimmerman, Sandra G., 56* Xie, Z., 828C Yu, Jessica, 393C Zimniak, Piotr, 136 Xiong, Wenjun, 79*, 370A Yu, Jianzhong, 397A Zinzen, Robert P., 315C* Xu, Dongbin, 741C, 742A* Yu, Kweon, 153* Zirbel, Luka N., 525C Xu, Jiang, 212B* Yu, Lin, 520A* Zmojdzian, Monika, 63 Xu, Jing, 243C Yuan, Rong, 527B Zoghbi, Huda, 102 Xu, Mu, 674B Yucel, Gozde, 430A* Zraly, Claudia, 128 Xu, Na, 447C* Yulia, Vorontsova, 290B Zraly, Claudia B., 546C Xu, Tian, 1 Yusuff, Tanzeen, 643A Zuberova, Monika, 726C, 859A* Xue, Jin, 346A zur Lage, Petra, 574A, 603C* Zweckstetter, M., 824B Zwiener, Jessica E., 853A KEYWORD INDEX 409

The following index is composed of keywords selected by presenting authors from a list in the Conference Call for Abstracts. Abstracts program number follow each keyword.

■ Cell Division and Growth Control epigenetic regulation 539B cell competition 157A 158B 159C histone variants and modifications 530B 531C cell growth 149 154 160A 161B 162C 163A 164B 165C 166A HP1interactor 167B 168C 537C centrosome hypoxia 169A 170B 536B checkpoint insulators/boundary elements 171C 172A 126 532A 533B 534C 535A compensatory growth nuclear lamina 198C 538A 540C cytokinesis polycomb/trithorax complexes 152 173B 174C 175A 176B 177C 178A 179B 130 131 541A 542B 543C developmental modulation remodeling complexes 180C 181A 182B 183C 127 128 544A 545B 546C endoreplication 150 ■ Cytoskeleton and Cellular Biology kinase/phosphatase/cyclin cell adhesion, cell-cell fusion 184A 64 kinetochores and cohesion cell morphology 185B 61 meiosis cell polarity 155 186C 187A 188B 189C 190A 191B 192C 53 65 213C 214A 215B 216C 217A 218B 219C 220A 221B 222C mitosis 151 193A 194B 195C 196A cell shape remodeling 63 oogenesis 197B characterization spindles and motors 263B 199A cytoskeleton tissue growth 56 58 59 66 223A 224B 225C 226A 227B 153 200B 201C 202A 203B 204C 205A 206B 207C 228C 229A 230B 231C 232A 233B 234C 235A 236B 208A 209B 237C tumor suppressors and oncogenes endocytosis 156 210C 211A 212B 238A 239B 240C

endoplasmic reticulum ■ Chromatin and Gene Expression 261C chromatin assembly epidermis 521B 522C 523A 258C chromatin structure intracellular transport 132 524B 525C 526A 527B 528C 529A 57 60 241A 242B 243C 244A 245B 246C 247A 248B 249C 250A 251B 252C 253A 410 KEYWORD INDEX lipid droplet neural degeneration 262A 92 95 96 99 100 103 104 819C 820A 821B 822C 823A 824B 825C 826A 827B 828C 829A migration 830B 831C 832A 833B 834C 835A 836B 837C 55 254B 255C 256A 257B neuromuscular disorders mitochondria 841A 260B muscle ■ Evolution and Quantitative Genetics 259A bacterial symbiont phosphorylation 49 54 chimeric genes sarcomere 114 62 evolution and development secretion 39 44 45 47 48 653B 654C 655A 656B 264C 265A 266B 657C 658A 659B 660C 661A 662B 663C 664A

evolutionary ecology ■ Drosophila Models of Human Diseases 681C anthrax genome evolution 93 42 46 115 665B 666C 667A 668B 669C 670A 671B 672C 673A 674B 675C 676A 677B 678C 679A bacterial-host interaction 680B 842B ncRNA evolution cancer 683B 98 795C 796A 797B 798C 799A phylogenetics cardiovascular 684C 94 800B 801C 802A 803B 804C population variation developmental disorders 40 41 43 50 52 685A 686B 687C 688A 91 97 102 805A 806B 807C 808A 809B 810C 689B 690C 691A 692B 693C 694A 695B 811A812B813C814A quantitative traits diabetes and obesity 696C 697A 698B 699C 700A 701B 702C 703A 704B 101815B816C 817A 705C drug discovery sexually antagonistic coevolution 818B 682A dystrophin speciation 844A 51 117 706A 707B 708C 709A 710B 711C 712A 713B 714C 715A 716B 717C 718A hyperoxia, tolerance, gene, regulation 846C ■ Genome and Chromosome Structure metabolism 838A centromere 118 267C mitochondrial diseases 845B DNA repair 116 268A mitochondrial DNA replication 843C duplication 276C muscular dystrophy 840C genome sequence 269B mutagenesis 839B KEYWORD INDEX 411 genome strucutre evolution stem cells 113 766A heterochromatin trade-offs 112 270C 271A 272B 758B isoforms transcriptional network 273C 756C pairing virus 275B 2 position effect variegation 277A ■ Neurogenetics and Neural Development replication axon guidance 278B 145 146 547A 548B 549C 550A 551B 552C 553A 554B segregation 274A CNS 144 555C 556A 557B 558C telomere 279C 280A 281B dendrites 559A

■ Immune System and Cell Death embryonic brain development 591C antiviral immunity 755B 757A eye development 590B caspases 8 719B 720C 721A 722B glia 560B 561C 562A cellular immunity 1 723C 724A 725B 726C 727A 728B 729C 730A hypoxia 731B 732C 733A 734B 735C 736A 737B 592A death mutants/genes neuronal morphogenesis 5 6 7 738C 739A 740B 741C 742A 743B 148 563B 564C 565A 566B 567C 568A endosymbiont neuronal specification 765C 143 147 569B 570C 571A 572B 573C 574A 575B 576C 577A 578B 579C 580A 581B 582C 583A 584B humoral immunity 585C 586A 587B 588C 3 4 744C 745A 746B 747C 748A 749B 750C 751A 752B postembryonic 593B 594C infection 759C programmed cell death 595A inflammation-like phenotype 762C sensory 596B 597C 598A 599B 600C 601A 602B 603C inhibitors of apoptosis (iaps) 753C 754A sensory organ development, proneural prepattern 589A innate immunity 760A 761B stem cells 141 142 604A 605B 606C 607A 608B mitochondria 763A synaptogenesis 609C 610A ras/MAPK signaling 764B 412 KEYWORD INDEX

■ Neurophysiology and Behavior female reproductive tract 474C 476B acps, sperm competition 639C follicle development 480C aggression 641B heart morphogenesis 482B autonomic nervous system 640A imaginal discs, embryonic and larval development 472A circadian rhythms 611B meiotic cell fate determination 74 courtship and mating 10 13 16 612C 613A 614B 615C 616A 617B mesodermal derivatives 618C 20 461B 462C 463A 464B 465C 466A 467B 468C 469A drug 643A metamorphosis 475A eclosion 642C myogenesis 19 479B homeostasis 12 619A 620B oogenesis 470B 471C 478A 481A 483C ion channels 621C 622A 623B 624C posterior spiracles 477C learning/memory 625A 626B 627C 628A 629B 630C 631A pre-gametogenic germ cell development 484A neuropeptides 632B 633C 634A sex determination 23 485B 486C 487A 488B 489C neurotransmitters 15 635B 636C 637A 638B sex-specific traits and molecules 490A 491B paralysis 644B 645C spermatogenesis 492C 493A 494B 495C 496A 497B 498C 499A 500B sensory 501C 502A 503B 504C 505A 9 14 646A 647B 648C 649A 650B 651C stem cells synapse 17 21 67 68 69 70 71 72 73 11 652A 506B 507C 508A 509B 510C 511A 512B 513C 514A 515B 516C 517A 518B 519C 520A ■ Organogenesis and Gametogenesis ■ Pattern Formation chorion gene amplification 473B axis specification 88 394A 395B 396C 397A 398B 399C 400A 401B ectodermal derivatives 437B 438C 439A 440B 441C 442A 443B 444C 445A cell migration and motility 446B 447C 86 402C 403A 404B 405C 406A endodermal derivatives eye disc 18 448A 22 89 407B 408C 409A 410B 411C 412A 413B 414C 415A 416B extracellular matrix/cell adhesion 24 449B 450C 451A 452B 453C 454A 455B 456C eye-antenna disc, eyeless, cut 457A 458B 459C 460A 425B KEYWORD INDEX 413 homeotics transcriptome analysis 87 417C 418A 419B 864C larval tracheoblasts 424A ■ RNA Biology nervous system miRNA 423C 111877A878B 879C 880A 881B non-Drosophila patterning non-coding transcripts 420C 421A 882C oogenesis nonsense mediated decay 81 883A planar cell polarity RNA binding proteins 85 884B 885C 886A 887B 888C 889A rhoGEF2 RNA localization 422B 105 106 107 109 890B 891C 892A 893B segmentation RNAi (RNA interference) 83 84 426C 427A 428B 429C 430A 108 894C 895A 896B 897C 898A transdetermination splicing and its regulation 90 110 899B wing disc 431B 432C 433A 434B 435C 436A ■ Regulation of Gene Expression

ABC transporters ■ Physiology and Aging 331A dietary restriction activators/coactivators 137 847A 848B 25 30 33 38 282C 283A 284B 285C endocrine function alternative splicing 849C 850A 851B 852C 35 lifespan regulation binding sequence 866B 326B locomotor impairment cis-regulatory modules 865A 319A metabolism coactivator/corepressor 135 138 139 853A 854B 855C 856A 857B 858C 316A 859A computational model of gene regulation nutrient sensing 322A 133 860B core promoters and general transcription factors nutrition 286A 287B 288C 289A 290B 291C 292A 861C 862A ecdysone oxidative damage 327C 328A 136 867C 868A 869B enhancers physiology of adult organs 26 27 28 29 31 129 293B 294C 295A 134 870C 871A 872B 296B 297C 298A 299B 300C 301A 302B 303C 304A 305B 306C 307A 308B 309C 310A 311B somatic mutation 312C 313A 314B 315C 863B gene targeting stress response 323B 140 873C 874A 875B 876C 414 KEYWORD INDEX genomic imprinting hedgehog 318C 80 350B 351C hindsight JAK/STAT 325A 352A 353B 354C 355A microRNAs JNK 36 366C 371B modifications of transcription factors MTF-1 320B 368B post-translational gene regulation notch 321C 76 356B 357C 358A 359B 360C repressors/corepressors phosphodiesterase 37 333C 334A 335B 336C 337A 338B 339C 340A 362B 341B 342C 343A 344B 345C 346A phototranduction, sphingolipid RNAi 364A 329B rap1 RNAi and cell death 377B 332B receptor tyrosine kinase sex determination 367A 75 378C 379A 380B 381C 382A 317B rho gtpases SUMO 383B 384C 324C synthetic interactions tissue-specific regulation of gene expression 82 32 toll-like receptor translational control 372C 330C warts/hippo pathway 369C ■ Signal Transduction wingless abl, eya 385A 386B 387C 388A 389B 390C 391A 392B 393C 79 wound healing arrestin 376A 374B atypical protein kinase C ■ Techniques and Genomics 361A 454 sequencing cytosolic tyrosine phosphatase 790A 375C cell lines dpp 791B 34 77 78 347B 348C 349A chipchip dscam 767B 365B computational analyses ecdysone 124 768C 769A 770B 771C 772A 773B 363C database energy 789C 792C 373A gene and transcript mapping eya 774C 775A 370A KEYWORD INDEX 415 gene disruption and targeting proteomics 119 776B 777C 778A 779B 121 microscopy RNAi 125 780C 781A 794B molecular interactions small compounds 782B 783C 123 mutational screens transcription factor specificities 784A 785B 786C 787A phosphoproteome transgenesis 793A 120 protein expression and interaction transgenics 122 788B 416 FLYBASE GENETIC INDEX TO ABSTRACTS

This is an index of the genes and natural transposable elements mentioned in the abstracts, prepared by FlyBase. Where there is a significant difference between the gene symbol used by the authors and the valid FlyBase gene symbol, we have listed both versions. This is a change in policy compared to previous years (when only the valid FlyBase symbol was given) and we welcome comments on the usefulness of this change.

Indexed terms are in bold. Numbers following each term refer to abstract program numbers: 1–156 are platform presentations and 157A–899B are poster presentations.

If you notice any omissions or errors in the index, please either visit the FlyBase demonstrations room during the meeting or email us at [email protected].

1360 ...... 529A Ance ...... 694A bab1 ...... 697A 18w ...... 372C, 481A Ani ...... 470B bab2 ...... 697A 22C10 ...... 558C Antp ...... 417C, 435C, 577A, babo ...... 349A, 547A 28S ...... 528C 587B, 595A, 880A baf ...... 540C 28SrRNA ...... 528C aop ...... 335B bai ...... 613A 4E-BP ...... 153, 37 ap ...... 143, 31, 577A, 587B bam ...... 507C, 512B, 513C, 4EBP ...... 876C AP-2 ...... 53 654C, 69, 73 5-HT7 ...... 635B apaf1...... 720C, 754A ban ...... 172A, 516C 5-HT[[7]]Dro ...... 635B Apc ...... 237C, 393C, 56 barr ...... 193A Aats-gly ...... 825C APC1 ...... 56 bas ...... 644B ab ...... 432C, 564C, 99 Apc2 ...... 237C, 56, 70 baz ...... 142, 215B abd-A ...... 297C, 309C, 339C, 694A aPKC ...... 142, 215B, 361A, 387C Bc ...... 746B Abd-B ...... 298A, 309C, 417C, 419B app ...... 85 bcd ...... 110, 188B, 242B, 326B, Abl ...... 177C, 231C, 79 AQP ...... 264C 378C,426C, 427A, ac ...... 126, 307A, 576C, 578B, aqz ...... 566B 430A, 660C 589A, 599B, 603C aret ...... 340A, 889A BEAF ...... 534C, 535A Ack...... 365B Argk ...... 273C BEAF-32 ...... 532A, 534C, 535A Acp36DE ...... 639C Ark ...... 7, 720C, 753C, 754A BEAF-32A ...... 532A Acp70A...... 10 arm ...... 214A, 221B, 222C, 316A, bel ...... 358A Act57B...... 282C 385A, 387C, 392B, ben ...... 196A, 750C Act88F ...... 62, 764B 393C, 476B, 56 beta-Spec ...... 226A Actn ...... 476B armi ...... 108, 132, 197B, 897C betaTub56D...... 788B Adgf-A ...... 726C, 859A Arp14D ...... 232A, 467B betaTub60D...... 788B ago ...... 560B Arp2 ...... 232A, 467B betaTub85D...... 788B Ago-3 ...... 896B Arp3 ...... 232A, 467B betaTub97EF...... 788B AGO1 ...... 877A Arp66B ...... 232A, 467B bgcn ...... 73 AGO2 ...... 108, 526A, 877A Arr2 ...... 360C bgm ...... 833B AGO3 ...... 896B As ...... 811A bhr ...... 127 agu ...... 647B ase ...... 574A, 603C bi ...... 345C, 396C, 414C, 432C AIF ...... 738C ash1 ...... 130 BicC ...... 886A AK ...... 273C atado ...... 194B Bj1 ...... 502A Akh ...... 634A Atg1 ...... 160A, 162C, 5, 61, 834C Blimp-1 ...... 337A, 343A, 545B Akt1 ...... 135, 153, 161B, 876C Atg13 ...... 160A bnl ...... 86 Alg10 ...... 810C atg14...... 164B bns ...... 483C alpha-Adaptin ...... 367A atg3 ...... 61 bocksbeutel ...... 540C alpha-Cat ...... 214A, 221B, 56 Atg6 ...... 164B bol ...... 187A, 493A alpha-Spec ...... 222C, 226A, 476B atl ...... 261C bond ...... 178A alphaTub67C...... 68, 788B ato ...... 284B, 314B, 411C, 412A, boss ...... 57 alphaTub84B...... 68, 788B 574A, 576C, 578B, 603C botv ...... 785B alphaTub84D...... 68, 788B Atpalpha ...... 217A br ...... 236B, 266B, 322A, 328A, alphaTub85E ...... 68, 788B aub ...... 132, 329B, 526A, 895A, 896B 402C, 475A, 479B, 481A alr ...... 90 Aut1 ...... 61 Br ...... 24, 81 amn ...... 627C Avl ...... 240C BR-C ...... 475A, 479B amon ...... 373A, 634A awd ...... 222C brat ...... 510C, 606C amos ...... 574A, 603C Axn ...... 385A, 393C Brd ...... 307A, 357C AMPK ...... 12 B ...... 332B Brd-C ...... 307A FLYBASE GENETIC INDEX TO ABSTRACTS 417 brk ...... 210C, 283A, 322A, 34, CG1652...... 639C closca ...... 382A 345C, 348C, 396C, CG1656...... 639C clu ...... 99 432C, 477C, 768C CG16708...... 364A cmet...... 280A brm ...... 128, 546C CG16752...... 10 cmi ...... 128 Bru ...... 889A CG16778...... 612C cn ...... 870C bsg ...... 504C CG16801...... 642C cnk ...... 377B bsk ...... 137, 201C, 210C 325A, CG17033...... 155 cnn ...... 151, 170B, 54, 788B 376A, 720C, 747C CG17575...... 639C cno ...... 225C bss ...... 645C CG1776...... 259A Cnx99A...... 5 btl ...... 21, 441C, 86 CG1837...... 731B col ...... 143, 19 Bub3 ...... 169A CG18811...... 808A comm ...... 146, 550A BubR1 ...... 280A CG2097...... 892A Coop ...... 392B bw ...... 277A, 523A, 870C CG30122...... 524B cos ...... 350B, 351C c ...... 259A CG30410...... 876C Cos2 ...... 350B, 351C c(3)G ...... 192C, 275B CG3074...... 451A Cp1 ...... 8 c-fos ...... 720C CG31190...... 146 Cp190 ... 108, 151, 269B, 333C, 342C Ca-P60 ...... 5 CG31670...... 141, 608B cp309 ...... 151 Ca-P60A ...... 5 CG31711...... 74 CP60 ...... 269B cac ...... 622A CG32150...... 597C cpa ...... 227B cact ...... 347B, 795C CG32595...... 255C cpb ...... 227B cad ...... 336C, 660C CG33936...... 398B cpo ...... 850A Cad74A...... 24 CG34364...... 382A Cpr47Ee ...... 299B CadN ...... 609C CG3590...... 838A crb ...... 215B, 217A, 218B, 226A, 53 Caf1 ...... 522C, 542B CG3748...... 540C Crc ...... 5 CalpA...... 347B CG4567...... 812B CrebA ...... 265A CaMKII ...... 490A CG4643...... 321C CRMP ...... 552C Cap-G ...... 193A CG4666...... 846C crp ...... 198C, 201C Cap-H ...... 193A CG4699...... 520A Cry ...... 545B Cap-H2 ...... 275B, 498C CG4701...... 496A cry ...... 495C car ...... 369C CG5149...... 23 csul ...... 484A cas ...... 143, 144, 587B CG5284...... 264C csw ...... 410B, 799A cato ...... 574A, 603C CG5316...... 281B ct ...... 425B, 452B Catsup ...... 620B, 638B CG5625...... 390C cta ...... 225C, 422B cav ...... 280A CG5933...... 74 CtBP ...... 316A, 34, 589A Cbl ...... 367A, 741C, 742A CG6014...... 90 Ctr1B ...... 368B Cbx ...... 498C CG6091...... 499A cul-5...... 385A Cct5 ...... 5 CG6680...... 752B cv-c ...... 434B cdc2 ...... 155 CG6708...... 260B CycB ... 155, 172A, 341B, 344B, 507C cdc2c ...... 150 CG6995...... 524B CycE ...... 150, 556A Cdc42 ...... 215B, 482B, 610A CG7217...... 747C CycJ ...... 197B cdk1 ...... 155 CG7263...... 738C CYP28A1 ...... 686B Cdk2 ...... 150 CG7447...... 450C CYP4D10 ...... 686B CecA1 ...... 38 CG7589...... 264C Cyp6a2 ...... 611B ced-6...... 728B CG8552...... 248B Cyp6g1 ...... 611B Cenp-C ...... 118 CG8580...... 127 D ...... 438C, 591C Cenp-meta ...... 280A CG8679...... 540C D-TACC ...... 151, 170B Cf2 ...... 282C, 464B CG8707...... 154 da ...... 558C CG10233...... 598A CG9000...... 809B dac ...... 143, 203B, 22, 407B, 587B CG10251...... 15 CG9001...... 809B Dad ...... 507C, 96 CG10413...... 264C CG9002...... 809B dAkt ...... 135 CG10743...... 548B CG9138...... 236B dally ...... 400A, 80 CG11206...... 548B CG9809...... 815B Dame\Gr68a ...... 716B CG11877...... 164B CG9997...... 639C Dana\Lhr ...... 706A CG11887...... 353B Chc ...... 53 dap ...... 149, 172A CG11940...... 648C Chd1 ...... 544A dAP-4...... 201C CG11968...... 154 chic ...... 177C, 376A Dark ...... 7, 753C, 754A CG12340...... 338B chico ...... 513C, 52 dASK ...... 720C CG12534...... 90 ci ...... 385A daw ...... 547A CG12928...... 382A cic ...... 378C dbb ...... 833B CG13222...... 299B cid ...... 118 dBlimp-1 ...... 343A CG14224...... 100, 828C CK2 ...... 571A Dbor\Gr68a ...... 716B CG14939...... 184A CkIIalpha ...... 571A Dbx ...... 581B CG14971...... 197B CkIIbeta ...... 571A Dcas ...... 565A CG15025...... 281B CkIIbeta2 ...... 571A dCBP ...... 835A CG15609...... 602B Clk ...... 826A dced-6 ...... 728B 418 FLYBASE GENETIC INDEX TO ABSTRACTS

DCERK ...... 364A DMyb ...... 182B dTak1 ...... 747C Dcp-1 ...... 211A, 719B, 8 dMyc ...... 131, 158B, 159C, 165C, dTCF ...... 389B Dcr-1 ...... 149, 172A, 317B, 516C, 166A, 180C, 209B, 33, 510C Dtex\Gr68a ...... 716B 877A, 878B DMYPT ...... 176B DTS5 ...... 781A Dcr-2 ...... 317B, 516C, 526A, 755B DNApol-gamma35 ...... 843C dUbc9 ...... 795C dCtBP ...... 589A DNaseII ...... 739A Dube3a ...... 811A Ddc ...... 638B, 694A, 697A dnc ...... 362B dUbqln...... 828C Ddok ...... 371B Dnov\Gr68a ...... 716B duf ...... 232A, 461B, 469A dec ...... 471C Dntf-2...... 671B Duf ...... 223A dec-1...... 471C dock ...... 365B Dvir\Gr68a ...... 716B DEcad ...... 221B Dok ...... 371B Dvir\Lhr ...... 706A decay ...... 646A dome ...... 625A Dvir\ras1 ...... 653B Deco ...... 483C dor ...... 241A dVMAT ...... 239B, 637A Def ...... 38 dos ...... 799A dVps45 ...... 369C dendos ...... 155 Dox-A2 ...... 746B dWIP ...... 233B Dere\Lhr ...... 706A Dox-A3 ...... 746B dx ...... 360C Dezo\Gr68a ...... 716B dp ...... 449B, 667A Dyak\Lhr...... 706A dFAP ...... 808A Dp110 ...... 865A Dys ...... 844A Dfd ...... 311B, 87 dPANK ...... 92 dzy ...... 228C Dfla\Gr68a ...... 716B dPGC ...... 815B e ...... 697A dfmr1 ...... 604A, 805A, 91 DPLP ...... 151 E(br)155 ...... 236B dFMRP...... 175A, 808A dpp ...... 134, 18, 204C, 25, 266B, E(bx) ...... 508A dfos ...... 18, 376A 285C, 34, 347B, 396C, E(spl) ...... 307A, 571A, dFOXO ...... 137, 153, 745A, 875B 410B, 432C, 477C, 481A, 576C, 578B dFz3 ...... 389B 507C, 510C, 573C, 664A, E(spl) mgamma ...... 295A Dg ...... 844A 703A, 722B, 8, 87 E(spl)-C ...... 307A dgo ...... 213C dPPCS ...... 92 E(spl)C ...... 571A DGrip91 ...... 151 dpr ...... 13 E(spl)M8 ...... 578B dHCF ...... 33 dprx5 ...... 747C E(tc) ...... 281B Dhod ...... 499A Dpse\GA14945...... 706A E(z) ...... 32, 542B DHR38 ...... 857B Dpt ...... 38, 750C E-cad ...... 455B Dhyd\Minos...... 784A dPTP61F...... 365B E1 ...... 156 dia ...... 177C, 58, 59 Dr ...... 31, 591C E23 ...... 327C Diap1 ...... 157A, 7, 753C, 754A, Dras1 ...... 653B E2f ...... 150, 171C, 478A 763A, 764B dream ...... 332B E2F1 ...... 150, 171C, 478A dIAP2 ...... 750C DREDD ...... 750C E2f2 ...... 539B Dif ...... 135, 38, 795C Dredd ...... 750C E75 ...... 293B Dilp2 ...... 71 DRhoGEF2 ...... 59 E75A ...... 337A dimm ...... 143, 587B Drice ...... 7, 719B, 742A, 753C, 8 ea ...... 401B Dip3 ...... 38 Drip ...... 264C eas ...... 94 djun ...... 376A drn ...... 398B east ...... 269B Dkan\Gr68a ...... 716B Dronc ...... 7, 719B, 720C, 722B, ec ...... 676A dl ...... 129, 283A, 347B, 38, 795C 742A, 753C, 764B, 8 eca ...... 613A Dl ...... 307A, 356B, 357C, 359B, drosha ...... 877A eco ...... 483C 369C 448A, 465C, 515B, 57, drpr ...... 728B, 731B EcR ...... 293B, 363C, 473B 478A, 583A, 584B, 585C, 602B Drs ...... 38, 751A, 752B 586A, 618C Dlac\Gr68a ...... 716B ds ...... 65, 85 ed ...... 569B, 579C, 585C dlg1 ...... 482B Dscam ...... 146, 35, 365B, 549C eff ...... 367A Dlit\Gr68a ...... 716B Dscam3 ...... 146 Egfr ...... 310A, 380B, 381C, 404B, Dll ...... 316A, 389B dsh ...... 213C, 388A, 439A 410B, 481A, 57, 585C, dlp ...... 400A, 80, 86 DSH3PX...... 365B 591C, 741C, 89, 98 dLsd1 ...... 546C Dsim\bam ...... 654C egg ...... 33, 338B Dlum\Gr68a ...... 716B Dsim\GstD1 ...... 669C eIF-4E ...... 37 dm ...... 131, 158B, 159C, 165C, 166A, Dsim\HP6 ...... 706A eIF4E-3 ...... 37 180C, 208A, 209B, 33, 510C Dsim\Lhr ...... 537C, 706A eIF4E-4 ...... 37 dMAN1...... 538A Dsim\OdsH ...... 707B eIF4E-5 ...... 37 Dmau\OdsH ...... 707B, 717C Dsim\unc-4 ...... 707B eIF4E-6 ...... 37 Dmau\unc-4 ...... 707B dSin3A...... 589A eIF4E-7 ...... 37 Dmef2 ...... 468C DSix4 ...... 409A, 416B eIF4G ...... 37 Dmet\Cyp28a1 ...... 686B Dsor ...... 410B Eip63E ...... 184A Dmet\Cyp4d10 ...... 686B Dsor1 ...... 410B Eip75B ...... 293B, 337A dMitf ...... 168C dspag ...... 500B EloA ...... 287B Dmon\Gr68a ...... 716B dSUR ...... 744C emc ...... 570C dMRP ...... 331A dsx ...... 147, 23, 486C, 487A en ...... 418A, 431B, 541A, 59, dmw ...... 451A dTAK ...... 720C 694A, 84 FLYBASE GENETIC INDEX TO ABSTRACTS 419 ena ...... 177C, 227B, 231C, gft ...... 385A His2A ...... 898A 456C, 798C Ggamma1...... 376A His2Av ...... 118, 531C endos ...... 155 giac ...... 500B His2B ...... 506B, 898A Epsin ...... 367A GiM ...... 152 His3 ...... 293B, 338B, 506B, 521B, ERK ...... 153 gl ...... 694A 522C, 525C, 526A, 531C, esc ...... 542B Gmd ...... 356B 542B, 898A esg ...... 488B Gmer ...... 356B His3.3A ...... 523A eve ...... 303C, 334A, 336C, 406A, GNBP1...... 3 His3.3B ...... 523A 428B, 581B, 661A, 83 Gos28 ...... 60 His4 ...... 530B, 898A Evi ...... 386B Gp93 ...... 163A hk ...... 57 ex ...... 200B, 369C Gpdh ...... 856A hkb ...... 340A, 378C, 769A exd ...... 297C, 413B, 420C, 577A, 87 Gpo ...... 856A hkl ...... 57 ey ...... 168C, 289A, 407B, 410B, GPO-1 ...... 856A HLHm3 ...... 571A 415A, 417C, 425B, 573C, 582C Gr32a ...... 715A HLHmgamma ...... 295A eya ...... 143, 168C, 22, 370A, 375C, Gr5a ...... 649A hlm ...... 554B 407B, 587B, 79 Gr66a ...... 649A, 651C hls ...... 526A eyg ...... 313A, 407B, 414C, 590B Gr68a ...... 715A, 716B HmgD ...... 318C eys ...... 216C grh ...... 258C Hmr ...... 718A f ...... 645C grim ...... 157A, 332B, 607A, hnt ...... 325A FAS 11 ...... 558C 753C, 764B hog ...... 255C Fas2 ...... 558C grk ...... 220A, 891C homer ...... 740B Fas3 ...... 263B gro ...... 345C, 576C Hop ...... 5 fat2 ...... 213C grp ...... 210C, 280A hop ...... 352A, 353B, 375C, 394A, fbl ...... 92 GSK-3B ...... 387C 400A, 488B, 511A, 568A, fit ...... 614B gskt ...... 487A 625A, 67, 70, 766A fj ...... 65 GstD1 ...... 669C how ...... 224B fkh ...... 443B GstD2 ...... 846C HP1 ...... 272B, 338B, 525C, flfl ...... 137, 606C GstS1...... 136 529A, 706A flower ...... 11 GstS1-1 ...... 136 HP6 ...... 706A Fmi ...... 439A gt ...... 26, 27, 326B, 334A, Hph ...... 855C Fmr1 ...... 106, 175A, 604A, 805A, 660C, 661A, 663C hpo ...... 369C, 397A, 6 808A, 896B, 91 GTPCH ...... 638B Hr38 ...... 857B Fmrf ...... 587B Gtr1 ...... 154 Hr51 ...... 642C FMRFa ...... 587B Gtr2 ...... 154 Hrb87F...... 35 FMRP ...... 106 gus ...... 321C hrp36 ...... 35 fog ...... 225C, 422B gw ...... 877A Hrs ...... 360C, 57 FoxK ...... 18 Gyc-89Da ...... 650B hsc3 ...... 5 foxo ...... 137, 138, 153, 745A, 875B Gyc-89Db ...... 650B Hsc70-3 ...... 5 fra ...... 146, 553A gypsy...... 269B Hsp70 ...... 103, 291C frag ...... 260B gypsy\su(Hw)BR ...... 108, 333C Hsp70Aa...... 103, 291C fred ...... 579C h ...... 334A, 346A Hsp70Ab ...... 103, 291C fru ...... 13, 147, 615C, 641B H2Av ...... 118, 531C Hsp70Ba...... 291C fs(1)K10 ...... 220A H2B ...... 506B Hsp70Bb ...... 103, 291C fs(1)N ...... 382A H3 ...... 506B, 521B, 526A, 531C Hsp70Bbb ...... 103, 291C ft ...... 200B, 369C, 65, 85 H3.3 ...... 523A Hsp70Bc...... 103, 291C ftz ...... 334A, 420C, 694A, 83, 84 H4 ...... 530B Hsp83 ...... 169A ftz-f1 ...... 337A, 343A, 420C, 84 hac1 ...... 754A Hsp90 ...... 169A fu ...... 350B, 351C hb ...... 426C, 427A, 429C, hth ...... 297C, 407B, 413B, futsch ...... 558C 520A, 660C 577A, 87 fz ...... 388A Hcf ...... 33 htl ...... 405C, 86 fz3 ...... 389B Hdac1 ...... 527B, 589A HtrA2 ...... 104 Fzr ...... 150 Hdc ...... 774C htt ...... 819C G-ialpha65A ...... 142 Hem ...... 467B, 565A ial ...... 199A GABA-B-R1 ...... 14 hep ...... 376A, 720C, 93 Iap2 ...... 750C GABA[[B]] ...... 14 heph ...... 884B Ice ...... 7, 719B, 742A, 753C, 8 Galphai ...... 142 HeT-A ...... 279C, 329B, 895A ico ...... 812B gammaTub23C ...... 151, 170B HGTX ...... 591C ics ...... 256A gammaTub37C ...... 151, 170B hh ...... 250A, 325A, 350B, 351C, ik2 ...... 750C gars ...... 825C 410B, 431B, 436A, 518B, IKK ...... 750C gbb ...... 134, 587B 573C, 59, 703A, 78, 8, 80 Ilk ...... 256A, 64 gcl ...... 340A hid ...... 157A, 210C, 332B, 527B, Ilp2 ...... 71 gcm ...... 560B 607A, 7, 753C, 764B imac ...... 825C gd ...... 401B Hira ...... 522C IME2...... 74 Gef26 ...... 228C, 377B HIS-C ...... 892A IME4...... 74 geminin ...... 150 His1 ...... 318C, 898A ImpE3 ...... 328A 420 FLYBASE GENETIC INDEX TO ABSTRACTS

Incenp ...... 190A, 199A Lsd-1 ...... 546C Mod(mdg4)-67.2 ...... 269B ind ...... 591C lwf ...... 861C, 862A Moe ...... 152 InR ...... 149, 52 lwr ...... 795C, 851B mof ...... 530B insc ...... 142 lz ...... 310A, 410B morgue ...... 743B insv ...... 605B M(1)15D ...... 183C mre11 ...... 181A inv ...... 541A M(2)60E ...... 183C MRLC ...... 234C IP[[3]]R ...... 379A M(3)66D ...... 183C MRP ...... 331A Ipk2 ...... 354C M(3)95A ...... 183C ms(3)K81 ...... 657C ire-1 ...... 95 Mad ...... 18, 180C, 34, 396C, msh ...... 591C Itp-r83A ...... 379A 477C, 516C, 587B MSL ...... 882C Jafrac2 ...... 753C mam ...... 360C, 518B msl-1...... 882C JAK ...... 375C, 400A, 488B, 511A, MAN1 ...... 538A, 540C msl-2...... 530B, 882C 568A, 625A, 67 Map60 ...... 269B msl-3...... 882C JNK ...... 137, 201C, 210C, 376A, MAPK ...... 242B, 378C, 461B msn ...... 376A 394A, 70, 720C MAPk-Ak2 ...... 762C Msp-300...... 224B jog ...... 648C Mat89Bb ...... 492C msps ...... 170B, 230B jp ...... 598A mats ...... 742A mst ...... 151 Jra ...... 376A Max ...... 180C MTF-1 ...... 368B kay ...... 18, 376A, 720C mbl ...... 219C mth ...... 869B ken ...... 398B mbmB ...... 626B, 628A Mtl ...... 610A kette ...... 467B, 565A mbr ...... 626B mtm ...... 251B, 61, 97 Khc ...... 243C, 245B, 249C, Mbs ...... 176B mtrm ...... 186C 252C, 497B Mcm5 ...... 191B mts ...... 185B, 250A, 388A, kirre ...... 223A, 232A, 461B, 469A Mcr ...... 724A 431B, 571A, 580A kis ...... 130, 411C Med ...... 324C, 34, 396C mtSSB ...... 843C kl-2 ...... 690C Mef-2...... 479B mus304 ...... 280A kl-3 ...... 690C Mef2 ..282C, 463A, 468C, 479B, 538A mus309 ...... 268A kl-5 ...... 690C mei-218...... 191B Mvl ...... 443B Klc ...... 245B, 497B mei-41...... 280A Myb ...... 182B, 539B Klp54D...... 190A mei-P22 ...... 191B Myc ...... 208A klu ...... 740B, 888C mei-S332 ...... 185B N ...... 17, 203B, 215B, 310A, 356B, kn ...... 143, 19, 587B Mer ...... 369C 357C, 358A, 360C, 369C, kni ...... 26, 432C mess ...... 260B 397A, 402C, 448A, 452B, koi ...... 224B Met ...... 475A 458B, 478A 515B, 563B, Kr ...... 26, 428B, 661A Mhc ...... 136, 234C, 282C, 62 569B, 57, 570C, 571A 576C, krz ...... 374B milt ...... 497B 580A, 583A, 584B 585C, 592A kst ...... 226A Minos...... 784A 605B, 76 kug ...... 213C mip130...... 539B nab ...... 143, 587B L ...... 413B mir-10 ...... 419B, 880A Nak ...... 361A l(1)dd4 ...... 151 mir-125 ...... 564C Nat1 ...... 483C l(1)sc...... 20, 568A mir-277 ...... 879C nau ...... 111, 19 l(2)gl ...... 605B mir-3 ...... 111 nbs ...... 181A l(2)NC136 ...... 886A mir-306 ...... 111 Nc ...... 7, 719B, 720C, 722B, lab ...... 18, 87 mir-309 ...... 111, 149 742A, 753C, 754A, 764B, 8 Lam ...... 272B, 342C, 734B, 809B mir-312 ...... 881B ncd ...... 199A LamC ...... 734B, 809B, 840C mir-318 ...... 111, 879C nclb ...... 485B Lar ...... 548B, 609C mir-7 ...... 149, 172A ndl ...... 401B lark ...... 247A mir-8 ...... 149, 389B nec ...... 751A lawc ...... 290B mir-iab-4 ...... 339C, 880A Nedd4 ...... 360C lea ...... 145, 457A mir-iab-4-3p ...... 339C, 880A nej ...... 826A, 835A lectin-46Ca ...... 639C mir-iab-4-5p ...... 880A NELF ...... 29 lectin-46Cb ...... 639C mira ...... 142, 606C NELF-A ...... 29 let-7...... 564C mit(1)15 ...... 280A NELF-B ...... 29 Lhr ...... 537C, 706A Mitf ...... 168C Nelf-E...... 29 lid ...... 131 mitoshell ...... 505A Nep4 ...... 633C Lim3 ...... 692B mjl ...... 487A nerfin-1 ...... 304A lin ...... 509B MK2 ...... 762C NetB ...... 255C lin19 ...... 385A Mlc2 ...... 259A neur ...... 167B, 307A, 357C Liprin-alpha ...... 548B, 609C mlck ...... 179B, 259A ninaE ...... 244A, 248B, 288C, 369C, Liprin-beta ...... 548B Mlp84B ...... 462C 596B, 598A, 60, 694A, 95 Liprin-gamma ...... 548B Mmp1 ...... 90 NK-2 ...... 301A loj ...... 613A Mnf ...... 18 Nk6 ...... 591C lok ...... 196A mnk ...... 196A nkd ...... 294C, 316A, 389B lqf ...... 359B, 367A Mnt ...... 180C nmd ...... 496A lrrk ...... 822C mod(mdg4) ...... 269B, 342C NMDA R1 ...... 621C FLYBASE GENETIC INDEX TO ABSTRACTS 421

Nmdar1...... 621C Pde6 ...... 362B Pros29 ...... 677B Nmdar2...... 662B Pde8 ...... 362B Pros35 ...... 677B nopo ...... 196A Pde9 ...... 362B Prosalpha3T ...... 677B norpA ...... 179B, 364A, 652A, 738C Pdi ...... 230B Prosalpha6T ...... 677B nos ...... 246C, 330C, 341B, 520A, PDK1 ...... 865A Psn ...... 100, 360C 660C, 73, 886A PDZ-GEF ...... 377B PTB ...... 884B NOT3/5 ...... 886A peb ...... 325A ptc ...... 250A, 431B Notch ...... 17 ped ...... 499A Pten ...... 205A Notum...... 294C pelo ...... 187A Ptp61F ...... 365B NPFR1 ...... 153 Pen ...... 262A, 628A pu ...... 871A Nplp1 ...... 577A pent ...... 77 Pu ...... 638B Ntf-2r ...... 671B per ...... 643A puc ...... 210C, 747C nub ...... 394A PGRP-LC ...... 4, 750C pum ...... 341B, 507C, 512B nuf ...... 67 PGRP-LCx ...... 750C put ...... 547A numb ...... 361A, 569B, 584B, 605B PGRP-LE ...... 750C Pxd ...... 838A NURF301 ...... 508A PGRP-SA...... 3 pxt ...... 480C NURF55 ...... 542B PGRP-SD...... 3 pyr ...... 75 nvd ...... 852C PhKgamma ...... 726C, 859A R ...... 225C, 228C, 377B, 404B nvy ...... 325A phl ...... 380B, 382A, 410B r2d2 ...... 132 Oamb ...... 490A pho ...... 543C Rab11 ...... 212B, 367A obst-A...... 437B phol ...... 543C Rab5 ...... 240C, 367A oc ...... 423C, 430A, 588C, phyl ...... 599B Rab7 ...... 251B 601A, 660C PI-3 kinase ...... 98 Rac ...... 455B, 468C, 551B, 610A, 70 odd ...... 83 Pi3K59F ...... 164B Rac1 ..... 455B, 468C, 551B, 610A, 70 OdsH ...... 707B, 717C Pi3K68D ...... 97 Rac2 ...... 610A Ogt ...... 460A Pi3K92E...... 865A, 98 RacGAP50C ...... 174C, 466A omb ...... 345C, 396C, 414C PI4KII ...... 241A rad50 ...... 181A, 280A Omi ...... 104 Pi4KIIalpha ...... 241A Raf ...... 380B, 410B opa ...... 285C Pi4KIIIalpha ...... 235A ran ...... 199A, 502A, 671B opo ...... 386B PINCH ...... 256A Ranbp21 ...... 877A Optix ...... 407B, 409A, 416B, 582C Pink1 ...... 104, 822C RanGap ...... 502A Or83b...... 860B Pins ...... 142 RanGEF ...... 502A Orc2 ...... 478A pip ...... 401B, 471C rap ...... 150 ord ...... 192C Pipe ...... 401B Rap1 ...... 225C, 228C, 377B, 404B Ory ...... 690C piwi ...... 108, 132, 329B, 507C, raps ...... 142 os ...... 312C, 355A, 400A, 625A 526A, 895A, 896B raptor...... 161B Osbp ...... 260B pk ...... 213C, 433A, 439A, 65 Ras ...... 1, 377B, 380B, 98 osk ...... 109, 881B, 884B, Pk61C ...... 865A Ras64B ...... 377B 887B, 889A Pk92B ...... 720C Ras85D ...... 1, 156, 377B, 380B, otd ...... 423C, 430A, 588C, 601A PKA ...... 632B 791B, 98 Ote ...... 540C Pka-R1 ...... 632B RasV12 ...... 791B otu ...... 487A, 489C PLC ...... 179B Rbf ...... 171C, 539B ovo ...... 286A, 438C, 444C, ple ...... 620B, 638B Rbf1 ...... 171C 453C, 785B plexB ...... 442A Rbsn ...... 240C ovoD ...... 785B pnr ...... 589A rDNA ...... 528C p130CAS ...... 565A pnt ...... 335B, 441C rec ...... 191B p35 ...... 157A pnut ...... 177C Rel ...... 38, 730A, 747C, 750C p53 ...... 210C polgamma ...... 843C Rep1 ...... 739A P58IPK...... 5 PolII...... 291C, 32 repo ...... 302B Pak ...... 365B, 455B polo ...... 169A, 539B Ret ...... 255C pall ...... 737B ponchik ...... 853A retinophilin ...... 598A, 728B pan ...... 386B, 389B, 392B, 477C Poxm ...... 20 rgn ...... 90 par-1 ...... 142, 821B, 88 Poxn ...... 647B Rh ...... 288C par-6 ...... 142, 215B PP2a ...... 388A Rh1 ...... 244A, 248B, 596B, 598A, 60 park ...... 104, 822C, 99 PP2A ...... 185B, 250A, 571A Rh3 ...... 288C, 369C pasha...... 877A Pp2A-29B ...... 250A, 571A Rh4 ...... 288C, 369C pav ...... 177C, 466A, 470B PP2A-B’ ...... 250A, 571A Rh5 ...... 288C, 369C, 586A, 600C Pax ...... 376A Ppcs ...... 92 Rh6 ...... 288C, 369C, 586A, 600C Pax-6 ...... 289A ppk11...... 858C Rheb ...... 154 Pax2 ...... 310A PPO ...... 746B Rho ...... 284B, 610A pbl ...... 174C, 177C, 811A pros ...... 142, 144, 572B, 588C, 606C rho ...... 284B, 297C, 768C, 89 Pc ...... 287B, 32, 501C, 527B Pros26 ...... 781A Rho-1 ...... 89 Pde11 ...... 362B Pros28.1 ...... 677B Rho-2 ...... 89 Pde1c ...... 362B Pros28.1A ...... 677B Rho-3 ...... 89 PDE4 ...... 362B Pros28.1B ...... 677B 422 FLYBASE GENETIC INDEX TO ABSTRACTS rho-7 ...... 104 scrib ...... 1 snf ...... 507C Rho1 ...... 174C, 177C, 229A, 254B, sd ...... 645C SNF1A ...... 12 383B, 384C, 422B, Sd ...... 502A snk ...... 401B 447C, 610A Sdc ...... 400A sNPF ...... 153 RhoA ...... 174C, 384C Sec23-IP ...... 248B sNPFR1 ...... 153 RhoGAP100F ...... 609C Sec61...... 140, 230B Snr1 ...... 546C RhoGEF2...... 422B, 59 Sec61alpha ...... 140, 230B, 266B snRNA:U7 ...... 892A, 898A rictor ...... 161B Sec61beta ...... 140, 230B snRNP70K ...... 885C rk ...... 871A Sec61gamma ...... 140 sns ...... 223A, 232A, 469A rl ...... 153, 242B, 378C, 461B Sema-2a ...... 442A so ...... 22, 370A, 375C, 407B, Rm62 ...... 108 sens ...... 284B, 297C, 389B, 588C 409A, 416B, 582C, 694A rn ...... 411C Ser ...... 356B, 359B, 413B, Socs36E ...... 511A robo ...... 145, 146, 456C, 515B, 83A Sod ...... 134 457A, 550A Set1 ...... 33 Sod2 ...... 867C Robo2 ...... 145, 457A sev ...... 572B, 741C sog ...... 283A, 768C robo3 ...... 145 Sgcd ...... 803B SoxN ...... 438C, 591C Roc ...... 385A sgg ...... 252C, 387C, 393C, SP ...... 10 Roc1a ...... 385A 410B, 821B SP1070 ...... 236B Roc1b ...... 385A sgl ...... 785B spdo ...... 569B, 584B, 602B, 605B Roc2 ...... 385A Sgt1 ...... 169A, 195C spi ...... 284B, 297C, 381C, 89 rok ...... 234C, 399C, 422B SH3PX1 ...... 365B Sple ...... 433A Rop ...... 819C shark ...... 371B spn-E ...... 108, 132, 329B, 526A, roX1 ...... 882C shg ...... 214A, 221B, 225C, 402C, 895A, 897C Rpd3 ...... 527B, 559A, 589A 404B, 455B, 456C, 518B, Spn27 ...... 751A RpII140 ..... 291C, 32, 38, 526A, 750C 56, 569B Spn27A...... 746B, 751A, 752B RpII15 ...... 291C, 32, 38, 750C shi ...... 390C, 53, 561C, 627C Spn77Ba...... 752B RpII18 ...... 291C, 32, 38, 750C shn ...... 34, 396C SPR ...... 10 RpII215 ...... 291C, 32, 38, 750C shot ...... 66 springer ...... 558C RpII33 ...... 291C, 32, 38, 750C sima ...... 855C sqd ...... 220A, 891C RpL14 ...... 183C Sin3A ...... 589A sqh ...... 179B, 234C, 371B, 399C, RpL19 ...... 183C sina ...... 385A, 599B, 797B 422B, 59, 842B rpr ...... 157A, 332B, 527B, 607A, sip3 ...... 95 sqz ...... 143, 587B 753C, 764B, 777C Six4 ...... 409A, 416B sr ...... 66 RpS3 ...... 183C skl ...... 607A, 753C srp ...... 485B RpS5a ...... 183C Skp ...... 385A ss ...... 575B rst ...... 469A skpA ...... 195C, 385A stan ...... 439A RSU-1 ...... 256A skpB ...... 385A STAT ...... 353B, 375C, 394A, 488B, rt ...... 807C skpC ...... 385A 511A, 568A, 625A, 67 Rtnl1 ...... 230B skpD ...... 385A Stat92E ...... 165C, 352A, 353B, ru ...... 89 skpE ...... 385A 375C, 394A, 488B, run ...... 334A, 406A, 83 skpF ...... 385A 511A, 568A, 625A, 67 Rya-r44F...... 728B sl ...... 379A stau ...... 606C S ...... 89 slbo ...... 452B Stbm ...... 213C, 439A S6k ...... 161B, 162C Slbp ...... 892A, 898A stc ...... 866B Sac1 ...... 241A sli ...... 145, 456C, 457A, 550A stck ...... 256A, 64 SAF-A ...... 524B slimb...... 560B Ste ...... 495C SAF-B ...... 524B Slit ...... 145, 550A stet ...... 89 sal ...... 396C slmb ...... 385A, 560B StIP ...... 353B salm ...... 288C, 396C, 432C, 694A slo ...... 323B, 619A, 624C stj ...... 622A san ...... 194B slomo ...... 450C Strn-Mlck...... 179B San ...... 483C slou ...... 31 Su(H) ...... 295A, 305B, 360C sano ...... 439A slp1 ...... 29, 294C su(Hw) ...... 269B satDNA ...... 51 slpr ...... 366C, 376A Su(Ste) ...... 495C, 894C sav ...... 369C, 397A, 742A sls ...... 462C, 62 Su(var)205...... 132, 272B, 318C, Sb ...... 383B, 384C Smad2 ...... 547A 338B, 525C, sc ...... 307A, 320B, 574A, 576C, SmB ...... 484A, 887B 529A, 706A 578B, 589A, 599B, 603C SMC1 ...... 186C, 192C Su(var)3-3 ...... 546C scab ...... 451A SmD3 ...... 887B Su(var)3-7 ...... 132 SCAR ...... 232A, 233B, 467B smg ...... 188B, 330C Su(var)3-9 ...... 318C, 521B, scb ...... 451A smo ...... 250A, 350B, 351C, 431B 525C, 527B Sce ...... 192C Smox ...... 547A Su(z)12 ...... 32, 527B, 542B Scgdelta ...... 803B smt3 ...... 851B sub ...... 199A scny ...... 506B smu ...... 626B Sumo ...... 851B Scr ...... 419B, 439A, 880A sn ...... 257B Sur ...... 744C scra ...... 174C, 177C, 470B sna ...... 422B, 768C, 769A sv ...... 310A FLYBASE GENETIC INDEX TO ABSTRACTS 423 sV23...... 459C Tl ...... 135, 347B, 374B, Usp36 ...... 506B svb ...... 438C, 444C, 453C 482B, 748A, 766A Vago ...... 755B svp ...... 144, 308B tll ...... 378C Vang ...... 213C, 439A swm ...... 431B TnI ...... 282C Vap-33-1 ...... 832A Sxl ...... 110, 317B, 487A, to ...... 615C VAPB ...... 832A 489C, 507C toe ...... 407B, 590B vas ...... 321C, 329B, 895A synj ...... 652A Toll ...... 482B, 748A vav ...... 551B Syx13...... 367A Toll-6 ...... 84 veg ...... 244A Syx16...... 367A Toll-7 ...... 372C vg ...... 148, 345C Syx17...... 367A tor ...... 378C, 382A, 660C vig ...... 329B Syx18...... 367A Tor ...... 133, 139, 834C Vinc ...... 214A Syx1A ...... 367A torp4a ...... 594C vls ...... 484A Syx4...... 367A Torso ...... 378C Vm26Ab...... 459C Syx5...... 367A toy ...... 289A, 407B, 415A Vmat ...... 239B, 637A Syx6...... 367A Tpi ...... 831C vnc ...... 483C Syx7...... 240C, 367A tra ...... 110, 485B, 489C vnd ...... 283A, 301A, 591C, 768C Syx8...... 367A tra2 ...... 487A Vps25 ...... 57 T48 ...... 422B tral ...... 175A Vps28 ...... 360C tacc ...... 151, 170B Tre ...... 649A Vps34 ...... 164B TAHRE ...... 895A Trf2 ...... 290B Vps35 ...... 390C Tak1 ...... 720C, 747C Trh ...... 439A, 441C Vps45 ...... 240C, 369C tam ...... 843C trio ...... 551B Vrp1 ...... 232A, 233B tan ...... 697A trk ...... 382A vsg ...... 470B TART-element ...... 895A trn ...... 84 w ...... 241A, 279C, 332B, 528C, TAS ...... 279C tRNA[[Ala]] ...... 533B 676A, 694A tau ...... 88 tRNA[[Asp]] ...... 533B W ...... 157A, 210C, 332B, 527B, Tbp ...... 292A tRNA[[Ser]] ...... 533B 607A, 7, 753C, 764B TCF ...... 477C tRNA[[Thr]] ...... 533B Wals ...... 321C tefu ...... 280A trol ...... 400A WASp ...... 232A, 365B Tel ...... 281B trx ...... 130, 287B WAVE ...... 232A Tep1 ...... 724A TSG101...... 57 wbl ...... 471C Tep2 ...... 724A tsh ...... 394A, 407B, 408C wdb ...... 250A, 571A Tep3 ...... 724A tsl ...... 382A Wde ...... 338B Tep4 ...... 724A Tsp ...... 450C wek ...... 283A Tep5 ...... 724A ttk ...... 332B, 402C, 478A wg ...... 208A, 294C, 301A, 312C, Tep6 ...... 724A tud ...... 887B 386B, 390C, 391A, 392B, TepI ...... 724A tum ...... 174C, 177C, 466A 410B, 518B, 56, 664A, TepII ...... 724A tw ...... 807C 722B, 78, 8, 80, 90 TepIII ...... 724A twe ...... 155, 187A wisp ...... 188B TepIV ...... 724A tweek ...... 652A wit ...... 96 TepV ...... 724A twi ...... 127, 129, 20, 255C, wls ...... 386B, 390C TepVI ...... 724A 422B, 479B Wnt ...... 664A TfIIA-L...... 292A tws ...... 250A, 571A wts ...... 369C, 397A, 6, tfiia-l ...... 292A U1-70K...... 885C 742A, 791B tfiia-s ...... 292A U7 snRNP ...... 892A wupA ...... 282C TfIIA-S ...... 292A U7snRNA ...... 898A Xbp1 ...... 265A, 5, 95 TfIIA-S-2 ...... 292A Uba1 ...... 156, 202A y ...... 126, 329B, 697A tfiia-s-2 ...... 292A ubc13...... 750C Yan ...... 335B TGF-beta ...... 18, 664A Ubc9 ...... 851B Ybb ...... 528C TH ...... 638B Ubqn ...... 100 yemalpha ...... 267C th ...... 157A, 7, 753C, 754A, Ubx ...... 299B, 311B, 339C, 418A, yrt ...... 217A 763A, 764B 498C, 543C, 577A, 595A yuri ...... 494B TH1 ...... 29 Uev1A ...... 196A, 750C z ...... 694A Thor ...... 153, 37, 876C Umbrea...... 706A zfh1 ...... 769A ths ...... 75 unc-104 ...... 825C zfh2 ...... 558C Tig ...... 462C unc-4 ...... 707B zip ...... 234C, 371B, 399C, 422B Timp ...... 454A Unc-76 ...... 245B zld ...... 25 tin ...... 308B, 482B, 769A upd ...... 312C, 400A, 625A zpg ...... 507C tio ...... 408C upd2 ...... 355A zw10 ...... 280A Tis11 ...... 472A upd3 ...... 355A, 400A zwilch ...... 280A Tkr ...... 612C usp ...... 293B, 363C, 473B Zyx102EF ...... 798C tkv ...... 322A, 81 Speed Up Your Research and Get Results Quicker!

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