A. J. Barlow-Anacker1, P. A. Trainor3, M. L. Epstein2, A. Gosain1,2 1University Of Wisconsin,Pediatric Surgery,Madison, WI, USA 2University Of Wisconsin,Neuroscience,Madison, WI, USA 3Stowers Institute For Medical Research,Kansas City, MO, USA
Introduction:
Hirschsprung’s disease (HSCR) is a congenital defect that results from the absence of ganglia along variable lengths of the bowel. These ganglia form from neural crest cells (NCC), a multipotent progenitor population that migrates over incredible distances to colonize the bowel. During migration NCC must survive, proliferate and differentiate to establish a functional enteric nervous system (ENS). Mutations in >10 different genes have been identified in HSCR patients; Endothelin receptor B (EdnrB) is one of the key players and NCC-specific deletion of EdnrB in mice (EdnrB-null) results in colonic aganglionosis, recapitulating the HSCR phenotype. The inability of NCC to colonize the entire colon in these mice has been demonstrated to result from temporal changes within the gut microenvironment. Identification of the molecular profile of the non-permissive colonic microenvironment in EdnrB-null animals may delineate how NCC interact with and subsequently colonize the gut microenvironment.
Methods:
RNA was isolated from the terminal colon of E14 EdnrB-null and wild type (WT) embryos and used to probe Affymetrix GeneChip mouse genome microarrays. A nine-way independent pair-wise comparison between EdnrB-null and WT tissue was performed and log2 mean values equivalent to a gross fold change of ± 1.5 used as a threshold to identify differentially expressed genes. Quantitative PCR was then performed to validate the differential gene expression. E13 colonic explants were cultured in the presence of a blocking peptide to Laminin and the effect on NCC advancement was determined.
Results:
Microarray analysis of the molecular profile of WT and EdnrB-null terminal colonic tissue identified a total of 131 differentially expressed genes. 57 of these genes were up-regulated while 74 were down-regulated. Quantitative PCR confirmed the microarray results. Laminin, a component of the extracellular matrix, was markedly up-regulated in EdnrB-null colon. Ex vivo culture of E13 colon in the presence of a laminin blocking peptide resulted in almost complete NCC colonization of the colon.
Conclusion:
We have identified the molecular profile of EdnrB-null colonic tissue that is non-permissive to NCC advancement along the colon beyond E14. Competitive blockade of laminin, shown to be up-regulated in EdnrB-null vs WT colonic tissue, results in the increased advancement of NCC along the gut ex vivo. These results are the first demonstration of increased colonization by NCC of the non-permissive colon in a mouse model of HSCR. Future approaches directed at decreasing the length of aganglionosis may reduce long-term morbidity in HSCR patients.