S8.3 Roles of Axon Guidance Molecules in the Development of the Vagal Innervation of the Mouse Gut

The vagal sensory innervation of the gut is established during fetal life. The guidance of vagal sensory axons is, in part, mediated by enteric netrins and by their receptor, deleted in colorectal cancer (DCC), which is expressed on the growth cones of extending vagal fibers. Within the developing bowel, netrin immunoreactivity has been found in the endoderm, outer gut mesenchyme, and primordial ganglia. We tested the hypotheses that the presence of netrin in developing enteric neurons is due to biosynthesis, rather than uptake, and that neuronal netrin expression is essential for the establishment of the close relationship of vagal sensory axons and enteric ganglia. Crest- and non-crest-derived cells were isolated, respectively, by positive and negative immunoselection from the E15 rat gut with antibodies to p75NTR, which bind selectively to crest-derived cells in the fetal bowel. Transcripts encoding netrin-1 and -3 were detected in the non-crest-derived cells but not in the crest-derived precursors. In contrast, transcripts encoding netrin-1 and -3 were detected after the crest-derived precursors were cultured for 6 days to allow neurons to develop in vitro. To determine whether neurons or their precursors take up netrin, crest-derived cells were cultured for 6 days with stably transfected 293-EBNA cells expressing c-Myc-tagged netrin-1. Although cultured neurons displayed netrin-1 immunoreactivity, they could not be immunostained with antibodies to c-Myc. Enteric neurons, but not their precursors thus express, but do not take up netrin-1. To evaluate the role of neuronal netrin expression in the development of the vagal sensory innervation, we analyzed the intestines of Ret −/− mice, which lack enteric neurons. Vagal sensory axons were traced by applying DiI to fetal nodose ganglia. Vagal axons failed to innervate the bowel distal to the esophagus in the Ret −/− animals. These data support the concept that netrins, produced by intrinsic neurons, are essential for the establishment of the vagal sensory innervation of the gut.