The Role of etv1 in specifying the Enteric Nervous System (ENS) Neuronal Subtypes in Zebrafish Open Access

Abstract

The enteric nervous system (ENS) is the largest subdivision of the peripheral nervous system that governs the function of the GI tract and is completely derived from the neural crest. Abnormal migration and differentiation of these neural crest cells cause the human clinical condition, Hirschsprung disease, along with other intestinal neuropathies. Although there have been many studies investigating how the ENS arises, our understanding of the molecular basis of this process is far from complete. Recent single-cell RNA sequence analysis of zebrafish ENS has revealed that the transcription factor ETV1 is specifically expressed in subsets of differentiating ENS neurons, though its precise function remains unclear. The goal of this project is to investigate the role of etv1 in enteric neuronal specification during the early stages of gut development, using zebrafish as a model organism.

To accomplish this goal, we generated a tg(phox2bb:egfp);etv1^sa14953 mutant zebrafish line, by crossing etv1^sa14953 mutation onto a tg:(phox2bb:egfp) background. Five days post-fertilization (5dpf) larvae from an in-cross of this line were collected and immuno-stained using three primary antibodies. The first antibody, anti-GFP, was used to label phox2bb+ enteric cells. The second, anti-5HT antibody targeted tph1b+ serotonin-producing cells, while the third, anti-nNOS, specifically labeled nos1+ inhibitory motor neurons. Subsequently, larvae were genotyped, and their guts were dissected for imaging using confocal microscopy. Images were analyzed to determine the number of positive cells for each antibody, in the mid/posterior intestine.

etv1 homozygous mutants displayed a significantly lower number of total phox2bb+ enteric cells than their heterozygous and wildtype counterparts, demonstrating that etv1 affects the total enteric cell count. Mutants also showed a clear aganglionosis at the distal end of their guts, suggesting that ETV1 affects the migration of the neural crest cells along the length of the gut. Furthermore, etv1 mutants showed a lower percentage of nos1+ inhibitory motor neurons as well a higher percentage of tph1b+ serotonergic neurons, suggesting the gene’s involvement in the differentiation of the enteric neuronal subtypes. Overall, our study provides valuable insights into the molecular and cellular dynamics of enteric neuron differentiation and the impact of the ETV1 transcription factor on this process.

Table of Contents

Introduction………………………………………………………………..……………………………………………..…….......... 1

Figure 1…………………………………………………………………………………………………………..……….2

Figure 2……………………………………………………………………………………………………………..…….3

Figure 3…………………………………………………………………………………………………………..……….4

Figure 4…………………………………………………………………………………………………………..……….6

Figure 5…………………………………………………………………………………………………………..……….8

Figure 6…………………………………………………………………………………………………………..……….9

Materials and Methods………………………………………………………………….………………………………….........…10

Table 1…………………………………………………………………………………………………………………….12

Table 2…………………………………………………………………………………………………………………….13

Results……………………………………………………………………………………………………………………………….......15

Figure 7……………………………………………………………………………………………………………….…..16

Figure 8…………………………………………………………………………………………………………………...17

Figure 9……………………………………………………………………………………………………………….…..19

Figure 10…………………………………………………………………………………………………………….…….20

Figure 11……………………………………………………………………………………………………………….…..21

Figure 12……………………………………………………………………………………………………………….…..23

Figure 13……………………………………………………………………………………………………………….…..24

Figure 14……………………………………………………………………………………………………………….…..25

Figure 15…………………………………………………………………………………………………………….……..26

Discussion ………………………………………………………………………………………………………………….…….........27

Figure 16……………………………………………………………………………………………………….……....…..29

Figure 17……………………………………………………………………………………………………….……....…..29

Figure 18……………………………………………………………………………………………………….……....…..30

Citations……………………………………………………………………………………………………………….……....…........31

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Evolution and Development Biology, Neuroscience
Keyword	Enteric Nervous System
Committee Chair / Thesis Advisor	Iain Shepherd, Emory University
Committee Members	Andreas Fritz, Emory University Rogel Deal, Emory University

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