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An Investigation Into the Function and Specification of Enteroendocrine Cells in Drosophila Melanogaster and Mus Musculus

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An Investigation into the Function and Specification of Enteroendocrine cells in Drosophila melanogaster and Mus musculus Alyssa Bost Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2014 © 2014 Alyssa Bost All Rights Reserved Abstract An Investigation into the Function and Specification of Enteroendocrine cells in Drosophila melanogaster and Mus musculus Alyssa Bost Enteroendocrine cells (EEs) are critical components in our bodies’ ability to maintain homeostasis after eating a meal. Hormones released by EEs mediate processes ranging from triglyceride processing to glucose balance to hydration maintenance. Despite their importance, they remain relatively poorly understood in terms of development as well as function. Drosophila melanogaster is a promising model in which to study EEs. I performed a gene expression assay in Drosophila, and found 19 transcription factors likely to be specific to EEs. I am in the process of analyzing their mutant phenotypes in the fly midgut. Additionally, by a limited screen of the homologs to the fly EE-specific transcription factors, I was able to identify two candidates for novel transcriptional regulators involved in EE specification or functionality. I will be analyzing the mutant phenotypes for these two genes, Lmx1a and Lmx1b, in addition to a third mutant Prox1, chosen because of the strong phenotype of its homologous gene’s knockdown in the fly. I am hoping I will be able to add to the ever-growing body of knowledge in reference to enteroendocrine development. Additionally, several assays were performed on flies lacking EEs. I found that flies without EEs lay significantly fewer eggs, and have apparent defects in oviposition and defecation. I will outline several experiments to continue the phenotype analysis of flies lacking EEs. TABLE OF CONTENTS LIST OF TABLES AND FIGURES____________________________________________________________v CHAPTER 1: GENERAL INTRODUCTION ___________________ 1 REVIEW OF THE MODEL SYSTEM _____________________________________________________________ 1 WHAT IS A GUT? ________________________________________________________________________ 1 THE DROSOPHILA GUT ____________________________________________________________________ 3 DEVELOPMENT OF THE DROSOPHILA MIDGUT ____________________________________________________ 4 DROSOPHILA MIDGUT ARCHITECTURE __________________________________________________________ 8 SPECIFICATION OF DAUGHTER CELLS BY NOTCH __________________________________________________ 12 INTESTINAL STEM CELL NICHE ______________________________________________________________ 16 MULTIPLE SIGNALING PATHWAYS MEDIATE INTESTINAL HOMEOSTASIS __________________________________ 21 SIMILARITIES WITH THE MOUSE INTESTINE ______________________________________________________ 24 FOCUS ON ENTEROENDOCRINE CELLS _________________________________________________________ 31 ENDOCRINE OVERVIEW ___________________________________________________________________ 31 ENTEROENDOCRINE CELLS _________________________________________________________________ 33 CHAPTER 2: ENTEROENDOCRINE FUNCTION _____________ 41 SUMMARY ___________________________________________________________________________ 41 INTRODUCTION ________________________________________________________________________ 41 i RESULTS _____________________________________________________________________________ 44 HORMONE AND SEROTONIN STAINING ________________________________________________________ 44 DISCUSSION __________________________________________________________________________ 53 MATERIALS AND METHODS________________________________________________________________ 56 FLY LINES USED_________________________________________________________________________ 56 MEASUREMENT OF BODY MASS _____________________________________________________________ 56 ANALYSIS OF BODY COMPOSITION ____________________________________________________________ 56 FEEDING MEASUREMENT __________________________________________________________________ 56 EGG LAYING ASSAY ______________________________________________________________________ 57 ACTIVITY MONITORING ___________________________________________________________________ 57 VIABILITY _____________________________________________________________________________ 57 TRANSGENICS _________________________________________________________________________ 57 CHAPTER 3: SPECIFICATION OF ENTEROENDOCRINE CELLS __ 59 SUMMARY: ___________________________________________________________________________ 59 CONTRIBUTORS: ________________________________________________________________________ 59 INTRODUCTION: ________________________________________________________________________ 60 GENERAL NEURONAL AND ENDOCRINE SPECIFICATION PARADIGM _____________________________________ 60 THE CURRENT UNDERSTANDING OF VERTEBRATE GASTROINTESTINAL ENTEROENDOCRINE SPECIFICATION __________ 62 GENERATING A DIVERSITY OF CELL TYPES _______________________________________________________ 64 RESULTS _____________________________________________________________________________ 66 DROSOPHILA GENE EXPRESSION ANALYSIS ______________________________________________________ 66 DESCRIPTION OF DROSOPHILA EE-SPECIFIC TRANSCRIPTION FACTORS ___________________________________ 67 ii INVESTIGATING THE EE-SPECIFIC FACTORS IN DROSOPHILA ___________________________________________ 75 SCREEN OF MOUSE HOMOLOGOUS GENES _____________________________________________________ 77 DISCUSSION: __________________________________________________________________________ 84 DROSOPHILA EXPERIMENTS ________________________________________________________________ 84 LMX1A/LMX1B: ________________________________________________________________________ 85 PROX1: ______________________________________________________________________________ 86 PTF1A: ______________________________________________________________________________ 86 MIST1: ______________________________________________________________________________ 86 SUMMARY ____________________________________________________________________________ 87 MATERIALS AND METHODS_______________________________________________________________ 109 FLY LINES USED: _______________________________________________________________________ 109 MICROARRAYS: _______________________________________________________________________ 109 MICE: ______________________________________________________________________________ 109 QPCR: _____________________________________________________________________________ 109 MOUSE ANTIBODY STAINING: _____________________________________________________________ 110 CONFOCAL MICROSCOPY: ________________________________________________________________ 110 CHAPTER 4: DOES EGFR TRANSMIT A NICHE SIGNAL TO THE INTESTINAL STEM CELL? ____________________________ 111 SUMMARY __________________________________________________________________________ 111 INTRODUCTION _______________________________________________________________________ 111 IS THERE AN ISC NICHE? _________________________________________________________________ 111 EGFR IS KNOWN TO INTERACT WITH NOTCH SIGNALING ____________________________________________ 113 iii RESULTS ____________________________________________________________________________ 114 CANDIDATE NICHE PATHWAYS _____________________________________________________________ 114 FOCUS ON EGFR AND ITS ROLE IN ISC MAINTENANCE _____________________________________________ 117 DISCUSSION _________________________________________________________________________ 123 MATERIALS AND METHODS_______________________________________________________________ 125 FLIES _______________________________________________________________________________ 125 ANTIBODY STAINING ____________________________________________________________________ 125 MUTANT CLONES GENERATION _____________________________________________________________ 125 RNAI EXPERIMENTS ____________________________________________________________________ 125 CHAPTER 5: GENERAL DISCUSSION AND PERSPECTIVE _____ 126 PHENOTYPE OF EE LOSS _________________________________________________________________ 126 SPECIFICATION OF ENTEROENDOCRINE CELLS ___________________________________________________ 128 THE EFFECT OF EGFR ON PROLIFERATION ______________________________________________________ 133 FINAL COMMENTS _____________________________________________________________________ 134 REFERENCES _____________________________________ 135 iv List of Tables and Figures Figure 1 Early gut model organism ........................................................................................................................... 1 Figure 2: The Drosophila gut ..................................................................................................................................... 3 Figure 3: Embryonic development of the midgut ....................................................................................................... 5 Figure 4: Development of the adult Drosophila midgut ............................................................................................. 7 Figure 5: Tissue layers and cell type markers of the adult Drosophila midgut ............................................................ 9 Figure 6: Regions of the adult Drosophila midgut ................................................................................................... 11 Figure 7: Notch mutant clones in the midgut are
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