Fig. 2

Further assays in larvae and adults support an absence of resident neuronal progenitors and enteric glia in the intestine. (A) Lineage tracing with an indelible Sox10-Cre line suggests enteric neurons are the sole fate of enteric vagal neural crest cells. At 5 dpf, fish were fixed and underwent IHC for the Cre reporter, mCherry and the neuronal marker HuC/D. All Cre-labelled cells colocalized with HuC/D, and no Cre+, HuC/D− cells were observed. (B) IHC with GFAP does not demonstrate convincing enteric glial cell bodies. Phox2b-kaede fish were fixed at 5 dpf, and axially sectioned for IHC for GFAP, a glial marker with cytosolic expression. Imaging of the endogenous Phox2b-kaede signal in concert with the GFAP IHC revealed a fibrillary pattern of GFAP closely associating with enteric neurons and other cells, which probably represents projections from extrinsic glia or nonspecific binding to other fibrillary proteins. (C) Whole-mount imaging of adult zebrafish intestine suggests that enteric glia and resident neuronal progenitors are not detectable by classical criteria later in development. Adult intestine from Phox2b-kaede×Sox10-mRFP fish that underwent optical clearing with RIMS revealed numerous enteric neurons, but no cell bodies expressing Sox10. Extrinsic glial projections are suggested by a fibrillary pattern of Sox10 expression. Scale bars: 10 µm.