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Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 5-1998 Nudel, an unusual mosaic protease involved in defining the embryonic dorsal-ventral axis of Drosophila Melanogaster Charles Chansik Hong Yale University. Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Hong, Charles Chansik, "Nudel, an unusual mosaic protease involved in defining the embryonic dorsal-ventral axis of Drosophila Melanogaster" (1998). Yale Medicine Thesis Digital Library. 2217. http://elischolar.library.yale.edu/ymtdl/2217 This Open Access Dissertation is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. Nudel, an Unusual Mosaic Protease Involved in Defining the Embryonic Dorsal-Ventral Axis ofDrosophila Melanogaster A Dissertation Presented to the Faculty of the Graduate School o f Yale University in Candidacy for the Degree of Doctor of Philosophy by Charles Chansik Hong Dissertation Director: Carl Hashimoto, Ph.D. May, 1998 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. © 1998 by Charles Chansik Hong All rights reserved. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Nudel, an Unusual Mosaic Protease Involved in Defining the Embryonic Dorsal-Ventral Axis ofDrosophila Melanogaster Charles Chansik Hong Yale University 1998 Dorsal-ventral polarity of Drosophilathe embryo is determined by positional information that originates outside of the embryo. This information, which is created during oogenesis by the somatic follicle cells surrounding the oocyte, is utilized following fertilization to trigger a spatially regulated serine protease cascade in the extraembryonic perivitelline space. This protease cascade, which appears to be activated only on the presumptive ventral side of the embryo, generates the ligand for the Toll transmembrane protein that polarizes the embryo by inducing ventral development This thesis presents evidence that nudel, a gene maternally required in somatic tissue for the formation of Toll’s ligand, plays a key role in the transmission of the positional information that determines embryonic dorsal-ventral polarity.nudel The gene was cloned and found to be expressed in follicle cells when they are secreting egg shell components during oogenesis. However,nudel function can be disrupted after fertilization, hours to days after follicle cells have degenerated, suggesting that the nudel gene product persists in the fertilized egg to play a direct role in producing Toll's ligand during embryogenesis. Thenudel gene encodes a large modular protein that structurally resembles extracellular matrix proteins. Consistent with its being an i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. extracellular matrix component, nudel is required for structural integrity of the egg. Moreover, the nudel protein contains several motifs that may bind serine proteases or their zymogens, as well as two regions that resemble a serine protease catalytic domain. Strikingly, catalytic activity of the serine protease domain which lies at the center of nudel’s primary structure appears to be required specifically for generating embryonic polarity. Based on these findings, it is proposed that nudel functions both to anchor and to trigger the protease cascade leading to Toll’s ligand, and is therefore an essential component of the extracellular cue that defines embryonic dorsal-ventral polarity. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS First of all, I would like to thank my advisor, Carl Hashimoto, whom I have grown to admire as a true gentleman and a scholar. In addition to his encouragement and guidance, I am grateful to have been the beneficiary of and witness to his gentleness, sincerity, thoughtfulness and creativity. I would also like to thank the past and present members of the Hashimoto and Wolin labs, especially Candra Smith, Kathy Winans and Ellen LeMosy, for invigorating discussions and for making my graduate study memorable and enjoyable. I am grateful to them especially for putting up with me. I am also grateful to the following individuals for providing invaluable reagents and helpful experimental advice: Kathryn Anderson, Yvonne DeLotto, Walter Gehring, Todd Laverty, Donald Morisato, Wendy Neckameyer, Trudi Schupbach, John Tamkun, Peter Tolias, and Gail Waring. In addition, I gratefully acknowledge the members of my qualifying committee (Doug Brash, Michael Stem, Alan Weiner and Steve Reeders) and my thesis committee (Michael, Lynn Cooley and Joe Madri) for their encouragement, guidance and patience. I also acknowledge the members of the “Invertebrate Meeting” group (the labs of Carl, Lynn, Michael, Tian Xu and Michael Tiemeyer) for their intellectual and technical input. In particular, I would like to thank Lynn for referring me to Carl back in 1991, and Doug Robinson for stimulating discussions and encouragement. I would also like to thank the members of Kathryn Anderson’s lab at Berkeley for their warm camaraderie. In addition, I am grateful to the members of both the Genetics department for their constant support and the Cell Biology department for adopting me as one of their own. One of the things I have grown to appreciate about science is the quality, both intellectual and otherwise, of my colleagues. 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I would like to express my deepest gratitude to the most important person in my life, my wife Stephanie, for her love, patience, constant support and prayers. I hope that I have been a blessing to her as she has been to me. I would also like to thank my twins, Sarah and Katie, and my oldest daughter, Emily, for making me feel so special. I almost feel as if Emily went through the Ph.D. program with me. In a sense Yale Medical School is her "alma mater" too, since it is the home of the Phyllis Bodel Child Care Center, without which completion of my graduate study would have been impossible! I would also like to thank my mother-in-law for her support and love, and for single handedly raising such a wonderful woman like Stephanie. Finally, I would like to thank my mother and my father, the true heroes of my life. Their perseverance, courage and love over the years have been and continue to be my inspiration. Of course, I thank God, without whom there would not be anything so beautiful as the fly! iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The heavens declare the glory of God; the flies* proclaim the work of his hands. Psalm 19:1 *altered from original "skies" without expressed written consent of its author ;) v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. "Natura enim non nisi parendo vincitur." At first men try with magic charm To fertilize the earth, To keep their flocks and herds from harm And bring new young to birth. Then to capricious gods they turn To save from fire or flood; Their smoking sacrifices bum On altars red with blood. Next bold philosophers and sage A settled plan decree, And prove by thought or sacred page What Nature ought to be. But Nature smiles—a Sphinx-like smile— Watching their little day She waits in patience for a while— Their plans dissolve away. Then come those humbler men of heart With no completed scheme, Content to play a modest part, To test, observe, and dream, Till out of chaos come in sight Clear fragments of a Whole; Man, learning Nature's ways aright, Obeying, can control. The changing Pattern glows afar; But yet its shifting scenes Reveal not what the Pieces are Nor what the Puzzle means. And Nature smiles—still unconfessed The secret thought she thinks— Inscrutable she guards unguessed The Riddle of the Sphinx. Hilfield, Dorset 1929 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Abstract p. i Acknowledgments p. iii List of Figures p. viii List of Tables p. x Note on Nomenclature p. xi Chapter I. Introduction Nature of Positional Information p. 1 Determination of Embryonic Axis inDrosophila Melanogaster p. 4 Anterior-Posterior Axis of theDrosophila Embryo p. 10 Dorsal-Ventral Axis of theDrosophila Embryo p. 18 Chapter H. The nudel Gene Encodes an Unusual Mosaic Protein with a Protease Domain Involved in Defining Embryonic Dorsal-Ventral Polarity inDrosophila Introduction p. 36 Materials and Methods p. 39 Results p. 45 Discussion p. 51 Chapter III. Dorsal-Ventral Patterning of theDrosophila Embryo Is Only One of Two Distinct Roles of the Maternal Nudel Protein. Introduction p. 80 Materials and Methods p. 83 Results p. 85 Discussion p. 91 Chapter IV. Conclusions p. 107 References p. 117 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Chapter I. Figure 1.1. Model of spatial regulation of serine proteases involved in dorsal-ventral axis formation. p. 32 Figure 1.2. Model of transmission of the positional information that defines the embryonic dorsal-ventral axis. p. 34 Chapter II. Figure 2.1. Dorsalized cuticle phenotype produced bynuclei mutant. p. 60 Figure 2.2. Temperature-sensitive period for nudel function. A) Shift from non-permissive to permissive temperature. p. 61 B) Shift from permissive to non-permissive temperature p. 62 Figure 2.3. Cytogenetic map of thenudel locus on the left arm of the third chromosome. p. 63 Figure 2.4. Molecular map of thenudel region. p. 64 Figure 2.5.

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