Role of Mab21l2 expression in the development and specification of neurons in the Enteric Nervous System Restricted; Files Only

Mukhopadhyay, Anoushka (Fall 2024)

Permanent URL: https://etd.library.emory.edu/concern/etds/8g84mn90t?locale=en
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Abstract

The enteric nervous system (ENS) is the largest subdivision of the peripheral nervous system. The ENS innervates the gut and regulates its function, though the mechanisms behind these process are still not fully understood. ENS cells are derived from neural crest cells, and abnormal migration and specification of these cells manifest in pathologies of the ENS like Hirschsprung’s disease (HSCR). Recently, single-cell-RNA sequencing studies in larval zebrafish have identified several previously uncharacterized cell populations, and regional specialization of the intestinal nervous system. Through a study of a multigenerational family with HSCR, a putative splice variant in the LRBA gene was identified within the linkage region previously associated with HSCR was identified. MAB21L2 is a gene embedded within the LRBA gene. This project investigated the role of mab21l2 in enteric neuronal development and specification during early stages of gut development in zebrafish.

 

To accomplish this goal, we generated a tg(phox2bb:egfp);mab21l2au12 mutant zebrafish line, done by crossing a mab21l2au12 mutation onto a tg:(phox2bb:egfp) background. First generation larva were collected and fixed at 5 days post fertilization and then immunohistochemical stained with one of  three different combinations of antibodies. The antibodies were anti-GFP used to label phox2bb+ enteric cells, anti-5HT used to target serotonin producing cells, anti-HuC/D used to label all differentiated neurons in the gut (including those which are phox2bb-) and finally anti-nnos used to label inhibitory motor neurons. The larva were then blindly genotyped and guts dissected for confocal microscopic imaging. Images were processed to quantify the number of immune positively stained cells for each antibody.

 

mab21l2 homozygous mutants displayed a significantly lower number of total phox2bb+ enteric cells than their heterozygous and wildtype counterparts, suggesting that mab21l2 expression affects the total all phox2bb+ enteric cells. Moreover, Furthermore, mab21l2 homozygous mutants showed significantly lower percentage of nos1+ (nnos+) inhibitory motor neurons and phox2bb- HuC/D + enteric neurons. However, there was no significant difference on the percentage of serotonergic cells in mab21l2 homozygous mutants relative to their heterozygote and wildtype counterparts. These results suggest that mab21l2 potentially plays a role in the general development and specification of certain subtypes of neurons in the ENS subtypes. This study provides further novel insights into the cellular mechanisms that regulate enteric neuron development and differentiation.

Table of Contents

Table of Contents

Introduction ………………………………………………………………………………………………………………………………1

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

Figure 2 …………………………………………………………………………………………………………………….…..4

Figure 3 ………………………………………………………………………………………………………………….……..7

Figure 4 …………………………………………………………………………………………………………………………8

Figure 5 ……………………………………………………………………………………………………………….………12

Figure 6 ………………………………………………………………………………………………………………….……16

Materials and Methods …………………………………………………………………………………………………………..17

Table 1 …………………………………………………………………………………………………………………………20

Figure 7 ……………………………………………………………………………………………………………………….22

Results …………………………………………………………………………………………………………………………………….25

Figure 8 ……………………………………………………………………………………………………………………….25

Figure 9 …………………………………………………………………………………………………….…………………26

Figure 10 …..…………………………………………………………………………………………………………………29

Figure 11 ………………………………………………………………………………………..……………………………31

Figure 12 …………………………………………………………………………………………………………..…………32

Figure 13 …………………………………………………………………………………………………………………..…34

Figure 14 ………………………………………………………………………………………………………………..……36

Figure 15 ………………………………………………………………………………………………………………..……37

Figure 16 ………………………………………………………………………………………………………………..……39

Discussion ..………………………………………………………………………………………………………………………..……41

Conclusion ………………………………………………………………………………………………………………………………45

Bibliography……………………………………………………………………………………………………….……………………46

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