Generation of Zebrafish (Danio rerio) Animal Models for Congenital Short Bowel Syndrome and Hirschsprung's Disease Público

Abstract

Pediatric intestinal disorders range from minor to severe, and have the potential of causing life-long problems. Congenital short bowel syndrome (CSBS) occurs when only the first fifth of the small intestine is formed, leading to malnutrition and high mortality rates. Hirschsprung's disease is the lack of neurons in the distal region of the large intestine, which results in obstruction of the colon requiring surgery, leading to long-term complications and sometimes death. The genetic components of each of these diseases are highly sought after. In conjunction with our collaborators, who genetically screened five patients with CSBS, I confirmed CAR-like membrane protein (CLMP) as the causative gene of CSBS and established a zebrafish animal model to further understand the role of CLMP in intestinal development. By making this zebrafish model, I helped confirm CLMP as the causative gene of CSBS, as well as providing a tool to further our understanding of the role of CLMP in intestinal development. Additionally, I used zebrafish to model Hirschsprung's disease and identified the fibroblast growth factor and Hedgehog signaling pathways as required for the development of the enteric nervous system. These signaling pathways are required for neural crest migration to the developing gut tube, and when perturbed, lead to a Hirschsprung's-like phenotype. These discoveries will improve genetic screens to aide in diagnosis and treatment of patients with developmental disorders of the gut.

Table of Contents

Chapter 1: Introduction to Gut and Intestinal Development in Zebrafish. 1

1.1- Purpose and Central Hypothesis. 2

1.2- Gastrointestinal Tract Architecture. 3

1.2.1- Gastrointestinal Tract Introduction. 3

1.2.2- Small Intestine. 3

1.2.3- Large Intestine. 5

1.2.4- Enteric Nervous System. 8

1.3- Gut and Enteric Nervous System Development. 10

1.3.1- Intestinal Development. 10

1.3.2- Enteric Nervous System Development. 10

1.4- Congenital Disease of the Small and Large Intestine. 12

1.4.1- Congenital Intestinal Diseases. 12

1.4.2- Congenital Short Bowel Syndrome. 12

1.4.3- Hirschsprung's Disease and Enteric Nervous System Disorders. 13

1.5- Gut and Enteric Nervous System Development in Zebrafish. 14

1.5.1- Zebrafish as a Model System. 14

1.5.2- Zebrafish Gut Development. 14

1.5.3- Enteric Nervous System Development in Zebrafish. 16

1.6- Congenital Short Bowel Syndrome in Humans and Zebrafish. 20

1.6.1- Congenital Short Bowel Syndrome in the Literature. 20

1.6.2- Coxsackievirus and adenovirus receptor-like Membrane Protein. 20

1.6.3- Car-like Membrane Protein in Zebrafish. 22

1.7- Fibroblast Growth Factor and Hedgehog Signaling in Enteric Nervous System Development in Zebrafish. 23

1.7.1- Signaling Pathways in ENS Development in Zebrafish. 23

1.7.2- Fibroblast Growth Factor Signaling Pathway. 24

1.7.3- Fibroblast Growth Factor Signaling in Zebrafish. 28

1.7.4- Previous Implications of Fibroblast Growth Factor Signaling in Enteric Nervous System Development. 28

1.7.5- Hedgehog Signaling Pathway. 29

1.7.6- Hedgehog Signaling Pathway in Zebrafish. 31

1.7.7- Previous Implications of Hedgehog Signaling in Enteric Nervous System Development. 31

1.8- Introduction Overview. 33

Chapter 2: CLMP Is Required for Intestinal Development, and Loss-of-Function Mutations Cause Congenital Short-Bowel Syndrome. 34

2.1- Abstract. 37

2.2- Introduction. 40

2.3- Patients and Methods. 41

2.4- Results. 52

2.5- Discussion. 58

2.6- Figures and Figure Legends. 61

Chapter 3: Fibroblast Growth Factor and Hedgehog Signaling are Required for Enteric Nervous System Development in Zebrafish. 77

3.1- Abstract. 79

3.2- Introduction. 80

3.3- Methods and Materials. 83

3.4- Results. 88

3.5- Discussion. 96

3.6- Figures and Figure Legends. 100

Chapter 4: Summary and Conclusions. 123

4.1- Summary of Results.124

4.2- Discussion of Results. 126

4.3- Future Directions. 128

4.4- Conclusion. 130

Appendix A: Figures in support of "CLMP Is Required for Intestinal Development, and Loss-of-Function Mutations Cause Congenital Short-Bowel Syndrome". 131

Appendix B: In Vivo Visualization of the Development of the Enteric Nervous System Using a Tg(-8.3bphox2b:Kaede) Transgenic Zebrafish. 135

References.159

End. 170

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, General
Palavra-chave	neural crest clmp fibroblast growth factor enteric nervous system zebrafish intestinal hedgehog
Committee Chair / Thesis Advisor	Shepherd, Iain, Emory University
Committee Members	Chen, Ping Helen, Emory University Moberg, Kenneth H, Emory University Koval, Michael H, Emory University Fritz, Andreas, Emory University

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