The development of a temporally controlled lineage system for visualizing enteric neural precursor specification in D. rerio Público

Abstract

Composed of at least 17 different neuronal subtypes, the enteric nervous system (ENS) is the largest part of the peripheral nervous system and responsible for regulating gut motility and homeostasis. Proper differentiation of enteric neuron subtypes is vital for digestive functioning, with improper differentiation of enteric neural crest cells (ENCCs) leading to a number of human gastrointestinal motility disorders. Despite recent advancements, the exact lineage and specification of ENCCs remains unclear. This study aims to develop a system for labeling enteric neural precursors to permit the temporal analysis of ENCCs differentiation during their migration and proliferation from the vagal neural crest to and along the gut. This study utilizes the ubi:Zebrabow transgenic line of D. rerio, which expresses the Brainbow fluorescent cassette under control of the ubiquitin-B promoter. From this line, we created a double transgenic with a Tol2phox2b::CreER construct to allow recombination within the Brainbow multicolor constructed specifically in phox2b expressing cells when embryos are treated with (Z) 4-hydroxytamoxifen (4-OHT). Successful incorporation and expression of CreER via Tol2 transposition was verified by genomic polymerase chain reaction (PCR) and reverse- transcription polymerase chain reaction (RT-PCR). We observed sparse recombination in enteric neurons of embryos treated with 4-OHT, with the majority of recombination occurring ectopically in myocytes. Cells that had undergone recombination expressed a combination of red, yellow, and cyan fluorescence proteins in a mosaic pattern. Future experiments should aim to achieve consistent recombination within enteric neural precursors and alter the timing of tamoxifen treatments to in order to analyze ENCC specification. 

Table of Contents

Table of Contents

Introduction...................................................................................................................................1

Methods.......................................................................................................................................13

Results..........................................................................................................................................16

Discussion.....................................................................................................................................24

Conclusions and Future Directions...............................................................................................31

References....................................................................................................................................33 

Table of Contents for Figures

Figure 1. ENS circuitry and gut architecture in a mammalian model................................................2

Figure 2. The distribution of the enteric nervous system in mammals and fish..............................4

Figure 3. Zebrafish neural crest proliferation and migration visualization.......................................5

Figure 4. Proposed models for ENS neuronal specification within the gut......................................6

Figure 5. Variable expression pattern lineage map of ENCC differentiation...................................7

Figure 6. Genetic incorporation of CreER using Tol2 transposase....................................................9

Figure 7. Brainbow fluorescence cassette and accompanying expression.....................................11

Figure 8. PriZm and ubi:Zebrabow RFP expression throughout development...............................17

Figure 9. PCR results for CreER incorporation using standard Taq PCR and DNA..........................18

isolation protocol vs. PCR using genomic DNA isolated using the Invitrogen

Platinum Kit from 72hpf embryos.

Figure 10. PCR using primers to amplify CreER using genomic DNA isolated.................................19

from 96hpf injected experimental and control embryos and uninjected embryos.

Figure 11. RT-PCR for Cre mRNA in RNA isolated from 48hpf injected and....................................20

control embryos.

Figure 12. YFP recombination in myocytes of a tamoxifen treated.................................................21

Tol2phox2b8.3::CreER injected ubi:Zebrabow embryo at 7dpf.

Figure 13. Observed recombination in enteric neurons and myocytes of a...................................22

tamoxifen treated Tol2phox2b8.3::CreER injected ubi:Zebrabow embryo at 7dpf.

Figure 14. YFP and CFP recombination in enteric neurons of a tamoxifen.....................................23

treated Tol2phox2b8.3::CreER injected ubi:Zebrabow embryo at 7dpf.

Figure 15. Pigmentation differences between adult wild-type D. rerio..........................................25

and the Casper line.

Figure 16. Expected recombination results following the fated model..........................................30

at time-dependent 4-OHT treatments. 

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
Palabra Clave	Zebrafish Enteric Nervous System Brainbow
Committee Chair / Thesis Advisor	Shepherd, Iain, Emory University
Committee Members	Caspary, Tamara, Emory University Fritz, Andreas, Emory University

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