General problem: The ENS is the largest most complicated subdivision of the peripheral nervous system and is completely derived from neural crest stem cells (NCSC). My lab is interested in determining what genes are involved in the specification of the NCSC that form the ENS. We are also interested in determining what molecules are involved patterning the migration of NCSC in the intestine and if these same molecules are involved in patterning the axonal projections of the differentiated ENS neurons in the intestine.
Experimental Approach: We have developed the zebrafish as a model system to study ENS development taking advantage of the systems embryological, cell biological and genetic strengths. We have undertaken a genetic screen to identify zebrafish mutants that lack or have a reduction in the number of enteric neurons in the intestine and have identified 6 ENS mutants to date. We are now screening for more ENS mutants using transgenic zebrafish that express green fluorescent protein (GFP) in all differentiated neurons and by screening for zebrafish that have defective intestinal peristalsis. Current Projects: 1) Characterization and positional cloning of ENS mutants . We have identified several ENS mutants using an immunocytochemical screen of 4-day-old fish. We have recovered 6 of these mutants and are now in the process of characterizing them both cellularly and genetically. We want to know: a) At what stage do these mutants first manifest a visible phenotype and how specific are these mutations. b) Are the mutations cell autonomous or non-autonomous. c) What is the genetic basis of the mutant phenotypes. d) How these identified genes cause their ENS phenotypes. 2) Lineage analysis of the origin of ENS precursors in the zebrafish vagal neural crest. Taking advantage of the optical clarity of the zebrafish embryo and the accessibility of the premigratory neural crest we are specifically labeling subpopulations of the vagal neural crest to determine if there is a distinct subpopulation of crest cells that give rise to the ENS. We are fluorescently labeling individual and small groups of neural crest cells by laser uncaging a caged-fluorescein dextran.
3) The in vivo function of zebrafish orthologues of known HSCR genes. Taking advantage of the zebrafish model system we are investigating how known HSCR linked genes, such as Sip1, cause their ENS phenotypes. We have previously shown that the neurotrophic factor GDNF and its receptor complex are absolutely required for normal ENS development in zebrafish as in mouse and human. Using similar techniques we are investigating the function of other known HSCR genes in zebrafish ENS development. These studies are investigating the many unanswered questions as how these known HSCR genes actually cause their ENS phenotypes when mutated. 4) The relationship between gut endoderm development and ENS development. Our studies of the zebrafish ENS mutants suggest that factors derived from the gut endoderm play a critical role in the normal migration and proliferation of ENS precursors. We are taking a microarray approach to determine which gut endoderm factors are down regulated in these mutants. 5) A forward genetic screen to identify new zebrafish ENS mutants. We are now screening for more ENS mutants using transgenic zebrafish that express GFP in all differentiated neurons and by screening for zebrafish that have abnormal intestinal peristalsis.
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