| We found neural crest stem cells (NCSCs), which give rise to the entire peripheral nervous system (PNS), persist into late gestation in many regions of the PNS and throughout adulthood in the gut. We developed techniques to prospectively identify these NCSCs by flow cytometry enabling the examination of stem cell properties as they exist in vivo.; Diversity of mature cell types in the nervous system is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. We found that when present in the same environment, NCSCs isolated from different regions of the PNS had differences in their sensitivity to lineage determination factors which directed fate choice. Regional differences in the types of cells generated in the nervous system arise, at least in part, from regional differences between stem cells.; NCSCs also underwent developmental transitions in cell-intrinsic properties. Postnatal NCSCs had reduced cell cycle status and self-renewal potential compared to fetal gut NCSCs and underwent changes in their neuronal subtype potential. These perinatal changes paralleled changes previously observed in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions.; To begin to understand NCSC regulation at the molecular level, we analyzed the gene expression profile of gut NCSCs. Surprisingly, genes associated with Hirschsprung disease, a relatively common congenital disorder in which the neural crest fails to form ganglia in the hindgut, were highly up-regulated in gut NCSCs relative to whole-fetus RNA. We selected two of these genes for functional analysis, the glial cell line-derived neurotrophic factor receptor Ret and the endothelin-3 receptor endothelin receptor B (EDNRB), which together account for the majority of familial cases of Hirschsprung disease. We found primary roles for both Ret and EDNRB-signaling in NCSC migration, and intact signaling was necessary for NCSC migration into the hindgut. The migratory defect could be bypassed by transplanting NCSCs into the aganglionic colon. Gene expression profiling, combined with reverse genetics and analyses of stem cell function, suggests that Hirschsprung disease is caused by defects in neural crest stem cell function. |
