Role of Endothelin Receptor B signaling in the genesis of aganglionic megacolon

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DESCRIPTION (provided by applicant): The enteric nervous system (ENS) develops primarily from a cohort of neural crest- derived cells (ENCCs) that enter at the pharynx before advancing caudally through the developing gut. The advance of ENCCs through the developing gut requires their coordination of proliferation, differentiation, and migration. Defects in any of these cellular processes alone or combined are sufficient to stop ENCC advance. When ENCCs do not complete their advance through the colon, a region of the gut is left without intrinsic innervation and is representative of Hirschprung's disease (HD). One set of mutations commonly associated with HD is found in genes encoding the ligand (EDN3), or its receptor endothelin-B (EDNRB). The receptor is expressed in both ENCCs and gut mesenchyme. To date, the precise cellular defect and molecular mechanism responsible for EDNRB-mediated HD remain unknown. To address these issues we have utilized a novel conditional floxed allele that allows us to knockout ednrb expression in neural crest cells only. By mating our floxed ednrb mice with Wnt1-Cre transgenics we delete ednrb specifically in neural crest-derived ENCC. In addition, the ENCCs also express the yellow fluorescent protein, allowing their movement to be recorded. Our studies have revealed two previously unrecognized cellular defects related to defective EDNRB signaling. First, the ENCC advance in null ednrb embryos is delayed shortly after neural crest cells enter the gut. Second, as null ednrb ENCCs reach the colon, they display aberrant trajectories and defective migration. Our data indicate that these two defects are related; the initial delay results in ENCCs entering the colon a day later than normal. We hypothesize that the initial delay is caused by pre- enteric neural crest defect, and the subsequent failure to advance in the hindgut results from changes in the hindgut environment. The first specific aim is to determine the cellular defect responsible for the initial delay of ENCC advance in the null mutant. The second aim is to determine the mechanism for defective ENCC migration in the colon. The third specific aim is to determine whether ENCCs transplanted into the aganglionic postnatal hindgut can restore coordinated motor function to the hindgut. Together, these studies will identify cellular defects responsible for HD resulting from the absence of EDNRB signaling and determine the feasibility of establishin coordinated motor function with transplanted null ednrb ENCCs. PUBLIC HEALTH RELEVANCE: Our goals are to elucidate the mechanisms underlying Hirschsprung's disease and to devise a method to provide functional innervation to the region of aganglionic colon. These studies will benefit patients with Hirchsprung's disease and other gastrointestinal motility disorders because providing functional innervation is a superior alternative to surgical excision, which leaves patient with lifelonggastrointestinal problems. The information gained here will also be relevant to transplantation of other tissues including neurons.