Expression of the GDNF receptors ret and GFR alpha 1 in the developing avian enteric nervous system

The formation of the enteric nervous system (ENS) from neural crest-derived cell precursors requires the growth factor glial cell line-derived neurotr ophic factor (GDNF) and the receptors Ret and GDNF family receptor alpha 1 (GFR alpha 1). We investigated the location(s), the timing, and the extent to which these GDNF receptors appear in the population of crest-derived pre cursors that form the avian ENS using immunohistochemistry and in situ hybr idization. Sections and whole mounts of embryonic chick. gastrointestinal t ract were costained with antibodies to the receptors and to HNK-1, a marker for crest-derived cells. Neural crest-derived precursors migrate through t he primitive esophagus to colonize the gizzard where an extensive cellular network forms. Ret-immunoreactivity (ir) was found in a network of cells in the gizzard at embryonic day (E)3.5. As development proceeded, Ret-immunor eactive cells appeared at progressively more caudal positions and were pres ent in the colon at E7.5. Costaining with Ret and HNK-1 was performed to de termine the number of Ret-immunoreactive cells in the crest-derived populat ion. Ret appeared in some HNK-1 cells in the esophagus and gizzard at embry onic day (E)3.5. During development, the number of crest cells with Ret inc reased in the ganglia of the gizzard and small intestine. GFR alpha 1-ir wa s also found in HNK-1 cells in the esophagus at E3.5 but did not appear in the gizzard until E4.5. Surprisingly, the colonizing vanguard of crest-deri ved cells lacked both Ret- and GFR alpha-ir. Between E4.5 and E6.5, the fra ction of HNK-1-positive cells expressing GFR alpha 1 increased considerably in the foregut. Ret and GFR alpha 1 were coexpressed in many cells at E6.5 , and the number of such cells increased as development progressed. In the adult, GFR alpha 1 and Ret were found in the neuropil of enteric ganglia. W e conclude that the population of cells expressing the receptors increases during development and persists in the adult, findings that support a neuro trophic role for GDNF in the formation and maintenance of the avian ENS. (C ) 1999 Wiley-Liss, Inc.