Differentiation of the Enteric Nervous System in Danio Rerio Open Access

Abstract

The enteric nervous system (ENS) is an intricate web of neurons and glia that control the digestive functions of the gut. There are 17 different, identified subtypes of neurons in the ENS. The ENS is made from a small population of neural crest cells that migrate to and along the gut to populate it. Because such a small migrating population of cells is responsible for the entirety of the ENS, it is vital that the migrating enteric neuron precursor cells (ENPCs) be maintained in an undifferentiated, proliferative state. Errors in ENS development can lead to gut aganglionosis, as is seen in the human disorder Hirschprung's Disease (HSCR). Here we establish a transgenic line, Tg(-8.3bphox2b:Kaede), to further explore a zebrafish model of HSCR based on the lessen mutant. Using this mutant, we found that the aganglionosis seen is due to a decrease in the proliferative potential of the ENPCs. One gene that is frequently mutated in HSCR patients is EDNRB, a gene responsible for keeping ENPCs in a proliferative undifferentiated state. We identified two functional variants of ednrb in zebrafish that have overlapping function in ENS development, and knockdown of these two genes using morpholinos causes aganglionosis phenotypes. Each initially undifferentiated ENPC matures into neuronal subtypes, which appear at different times in ENS development. Control of this differentiation requires that expression of pro-subtype genes be repressed until the right time. We found that expression of id2a was important for maintaining a pool of neuronal nitric oxide synthase (nNOS) neurons. Taken together these data shows that maintaining ENPCs potential to proliferate and differentiate is required to properly pattern the ENS. Understanding how ENS patterning is regulated will allow us to identify new strategies for treating various gastrointestinal disorders.

Table of Contents

Chapter 1: Introduction to Enteric Nervous System Development and Differentiation 1

Introduction 2

HSCR and Other GI Disorders 2

Neural Crest Formation 3

ENS Differentiation 5

Early ENS Specification of the Neural Crest 7

Post-Neural Crest Differentiation Control 10

Subtype Specification 12

Perspectives 16

Figures 18

Figure 1.1 18

Figure 1.2 20

Chapter 2: Functional Analysis of ENS Development 22

Introduction 23

Results 24

Discussion 27

Methods 27

Construct Generation 27

Tg(-8.3bphox2b:Kaede) Construct Injections 28

Transgenic Screening and Raising 28

Med24 Morpholino Injections 28

Stop Motion Imaging 29

Figures 30

Figure 2.1 30

Figure 2.2 32

Figure 2.3 34

Figure 2.4 36

Figure 2.5 38

Chapter 3: Differentiation Control of ENPCs and Other Neural Crest Derived Populations by EDNRB 40

Introduction 41

Results 43

Ednrb1a and Ednrb1b are Evolutionarily Conserved 43

Functional Analysis of Ednrb1a and Ednrb1b in Zebrafish Development 44

Discussion 46

Methods 50

Bioinformatics Analysis 50

Whole-Mount In situ Hybridization 51

Mopholino Injection and Morphant Analysis 51

Figures 52

Figure 3.1 52

Figure 3.2 54

Figure 3.3 56

Figure 3.4 58

Figure 3.5 60

Figure 3.6 62

Figure 3.7 64

Figure 3.8 66

Figure 3.9 68

Chapter 4: The Function of ID2a in ENS Subtype Differentiation 70

Introduction 71

Results 73

bmp and id2a Expression 73

id2a Promoter Region 74

Interaction of Sip1a and p-Smad at the id2a Promoter 75

Quantitative Real Time PCR of id2a Expression 76

Subtype Specification in id2a Morphant Embryos 76

Discussion 77

Methods 81

Whole-mount In situ Hybridization 81

ID2 Promoter Region 82

Co-IP 82

ChIP 82

QRTPCR 83

Neuronal and Subtype Identification 83

Figures 84

Figure 4.1 84

Figure 4.2 86

Figure 4.3 88

Figure 4.4 90

Figure 4.5 92

Figure 4.6 94

Figure 4.7 96

Figure 4.8 98

Figure 4.9 100

Chapter 5: BMP and a Linkage To HSCR 102

Introduction 103

Results 104

Identification of Zebrafish lrba Orthologue 104

Expression patterns of mab21l2 and lrba 104

Functional analysis of mab21l2 and lrba in ENS development 105

Discussion 106

Methods 107

Cloning and In situ Hybridization 107

mab21l2 and lrba morphant analysis 108

Figures 109

Figure 5.1 109

Figure 5.2 111

Figure 5.3 113

Figure 5.4 115

Chapter 6: Future Directions 117

Figures 124

Figure 6.1 124

References 126

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular
Keyword	Zebrafish Enteric Nervous System Differentiation
Committee Chair / Thesis Advisor	Shepherd, Iain, Emory University
Committee Members	Chen, Ping Helen, Emory University Moberg, Kenneth H, Emory University L'Hernault, Steven, Emory University Fritz, Andreas, Emory University

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