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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6* x 9” black and white photographic prints are available for « ly photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Learning 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMÎ ROLE OF THE EOF RECEPTOR LIGAND VEIN IN PATTERNING THE DROSOPHILA WING DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Robert J. Wessells, B.S. The Ohio State University 2000 Dissertation Committee: Approved by Professor Amanda Simcox, Advisor Professor Arthur Burghes Professor Roy Tassava Professor Harald Vaessin Adviser Department of Molecular Genetics UMI Number 9971660 UMI UMI Microform9971660 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The Drosophila Epidermal Growth Factor Receptor (DER) Is the only known erbB-like receptor in Drosophila, Signaling through DER is transduced by the highly conserved Ras/MAPK cascade. DER signaling is required at multiple stages of development for the sun/ival, proliferation and differentiation of multiple cell types. Although DER is broadly expressed, its activity is tightly regulated by several extracellular proteins, including the activating protein, Vein (Vn). Vn is an EGF-like protein similar to the vertebrate neuregulins and plays a critical role in both the initial development of the wing imaginai disc and in the placement and differentiation of veins in the adult wing. Hypomorphic mutations in vn cause loss of vein material, while overexpression of vn leads to the formation of ectopic veins. I examined the role of vn in wing vein patterning by analyzing the regulation of vn expression in the third instar wing disc. In contrast to the embryo, where vn is a target of DER signaling and functions as part of a positive feedback loop, DER signaling in the wing pouch does not induce vn expression and, in fact, is capable of repressing it. Instead, vnisa target of the conserved signaling molecule Hedgehog in the wing pouch. The transcription factor Cubitus interruptus (01), a homolog of vertebrate Gli proteins, acts downstream of Hedgehog in this activation. Vn thus acts as a secondary signaling molecule which is instrumental in Hh-dependent patterning of the central wing. ÎÎ In order to examine the relationship between i/n and other genes in wing vein patterning and to potentially identify new factors involved in Vn/DER signaling, I conducted a screen to identify genes which were able to enhance or suppress an extra-vein phenotype caused by overexpression of vn. Sixty-three known genes and eight uncharacterized loci were shown to modify the extra-vein phenotype, including a putative regulator of disc growth and one which encodes a regulator of cell size. Characterization of these loci will add further to the understanding of wing vein development. tii Dedicated to my two ongoing developmental experiments, Taran and Teilo IV ACKNOWLEDGMENTS I would like to thank my advisor, Amanda Simcox, for her advice and career guidance over the years. I would also like to thank the members of the Simcox lab, past and present, for their friendship and many hours of useful discuusion, advice and shared reagents: Gary Grumbling, Bruce Schnepp, Tim Donaldson, Deyra Rodriguez, Ivan Tesic, Shu-Huei Wang and Tom Jacobsen. I owe a special debt of gratitude to my family, who although spread out across the country, always find the time to encourage and support each other's endeavours: My mother, Faye, my father, Mike, and my brother, Dave. Most of all, I would like to thank my wife, Meredith, for putting up with my antics and supporting my career path. I would like to thank my committee members for their expert guidance and willingness to take time out to give me advice at various time during my graduate career. Harald Vaessin, Roy Tassava and Arthur Burghes. In addition, I would like to thank the members of the Vaessin and Seeger labs for their many useful comments and shared reagents. VITA May 1,1971 .......................................... Bom - Williamsburg, VA 1993...................................................... B.S. Zoology, Miami University 1993 - present. ...................................... Graduate Research and Teaching Associate, The Ohio State University PUBLICATIONS 1. R.Wessells*, Grumbling, G.*.,Donaldson, T, Wang, S.-H.and Simcox, A. (1999). Tissue-specific regulation of Veln/EGF-receptor signaling In Drosophila. Developmental Biology 216:243-259. FIELD OF STUDY Major Reid: Molecular Genetics VI TABLE OF CONTENTS E âflê Abstract ................................................................................................................ii Dedication ............................................................................................................ iv Acknowledgments ................................................................................................ v Vita...................................................................................................................... vi List of Figures ....................................................................................................... x Chapters: 1. Introduction ................................................................................................ 1 1 .A The Drosophila Epidermal Growth Factor Receptor Pathway. .........1 1 .A.1 Components of the DER Pathway-Receptor and Signal Transduction................................................................................... 2 1 .A.2 Components of the DER Pathway-Extraceltular Regulators 5 1.8 Roles of Vn/DER Signaling In Development ................................... 8 1.0 Wing Development ....................................................................... 17 1.C.1 Wing Disc Proliferation .................................................................. 2 0 v ii 1 .C.2 Wing Vein Patterning ...................................................................... 21 1 -C.3 Vein/intervein Differentiation ............................................................29 2. Materials and Methods ................................................................................ 32 2.A. Stocks.............................................................................................32 2.B. Gene Expression Analysis .............................................................. 33 2.C. Genetic Screen ............................................................................... 35 2.C. 1 Phenotypic Scoring ......................................................................... 35 2.D. Gene Cloning by Plasmid Rescue ...................................................38 3. Tissue-specific Regulation of vn Transcription.............................................. 39 3 .A. Regulation of vn Transcription...........................................................39 3.A.1. vn is Regulated by Hh Signaling in the Wing Pouch ......................... 39 3.A.2 vn is an Effector of Hh Signaling ........................................................ 46 3.A.3 Relationship of Other Transcription Factors to vn Transcription in the Wing ........................................................................... 49 3.A.4 vn is Not a Target of DER Signalingin the Wing ................................ 53 3.B. Discussion....................................................................................... 57 4. A Genetic Screen for Modifiers of an Extra-vein Phenotype Caused By Overexpression of vn....................................................................... 63 4.A. Screen Results ................................................................................ 65 4.A.1 DER Pathway Members Modify the UAS-vnl. 1 Phenotype ........... 65 4.A.2 X Chromosome. ............................................................................... 73 4.A.3 Second Chromosome-Left Arm ....................................................... 74 VÎÜ 4.A.4 Second Chromosome-Right Arm ..................................................... 84 4.A.5 Third Chromosome-Left Arm ............................................................ 86 4.A.6. Third Chromosome-Right Arm ........................................................