The development of a temporally controllable lineage system for labeling enteric neural crest precursors in D. rerio and assessment of the possible role of ETV1 as a transcriptional regulator of ENS specification Restricted; Files Only

Amarath-Madav, Rushay (Fall 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/8k71nj279?locale=en
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Abstract

The enteric nervous system (ENS) makes up the largest and most complex portion of the peripheral nervous system. It is the intrinsic nervous system of gut musculature and is responsible for maintaining gut homeostasis and motility. Complications involving the specification and differentiation of enteric neural crest cells (ENCCs) can lead to the development of Hirschsprung’s Disease, and Gastric Esophageal Reflux Disease (GERD), and a number of other diseases related to gut immobility. Here, we aimed to further analyze the lineage of ENCCs during their proliferation and migration from the vagal neural crest to the gut to populate the ENS with up to 17 different neuronal subtypes. Our primary aim was to develop a direct system for labeling enteric neural precursors for temporal analysis of ENS specification. Additionally, we also assessed the role of ETS transcription factor ETV1 in the development of the ENS. Labeling utilizing the Brainbow (PriZm) transgenic line was utilized in labeling studies. A β-actin2 driven multicolor construct was induced to recombine when exposed to Tol2phox2b::Cre-ER constructs treated with (Z) 4-hydroxytamoxifen (4-OHT). Limited recombination has been observed; only some expression of green fluorescence protein (GFP) in a mosaic patterning was seen after 4-OHT treatment. Regarding ETV1, mutants obtained from TILLING were sequenced, and heterozygote mutants were crossed to generate homozygous mutants of ETV1. A distinct no-swim bladder phenotype was seen in the one trial performed thus far at a frequency of 17%. Antibody staining with pan-neuronal markers for Hu and 5-HT were performed, and stained embryos were imaged with a confocal microscope. Images will be used to quantify enteric neurons and assess depletion in the ENS within mutants compared to controls. More trials are needed to further assess the significance of the observed morphant phenotype, as well as a method for genetically proving that suspected mutants are heterozygous.

Table of Contents

Chapter 1: Background…………............................................………………………….….……1

Chapter 2: Direct lineage labelling system…………………………………………….….……..9

Chapter 3: Potential role of ETV1 in ENS specification……………………….………………..17

Chapter 4: Conclusion………………………………………………………………..……….….26

References………………….…………………………………………...………………………..28

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