The mammalian enteric nervous system (ENS) derives from migratory enteric neural crest-derived cells (ENCC) that express the transcription factor Phox2b. Studies of these enteric progenitors have typically relied on immunohistochemical (IHC) detection. To circumvent complicating factors of IHC, we have generated a mouse BAC transgenic line that drives a Histone2BCerulean (H2BCFP) reporter from Phox2b regulatory regions. This construct does not alter the endogenous Phox2b locus and enables studies of normal neural crest (NC) derivatives. The Phox2b-H2BCFP transgene expresses the H2BCFP reporter in patterns that recapitulate expression of endogenous Phox2b. Our studies reveal Phox2b expression in mature enteric glia at levels below that of enteric neurons. Moreover, we also observe differential expression of the transgene reporter within the leading ENCC that traverse the gut. Our findings indicate that the wavefront of migrating enteric progenitors is not homogeneous, and suggest these cells may be fate-specified before expression of mature lineage markers appears.

To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear–localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multi-potency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in post-natal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.

Community cancer centers are capable of conducting EP trials. Infrastructure and collaborations are critical components of success. This assessment provides useful information for implementing EP trials in the community.

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