Netrin/DCC‐mediated attraction of vagal sensory axons to the fetal mouse gut

Abstract

Vagal sensory axons and migrating neural crest-derived precursor cells follow similar pathways to reach the gut. The crest-derived cells express the netrin receptor deleted in colorectal cancer (DCC) and migrate toward netrins expressed by the intestinal mucosa and pancreas; this attraction is required for the formation of submucosal and pancreatic ganglia. We tested the hypothesis that enteric netrins also attract vagal sensory fibers. These axons were located as a function of age in fetal mice by applying the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) bilaterally to nodose ganglia. DiI-labeled axons were found in the esophagus and proximal stomach by E12 and, more distally, in the small bowel at E14-E16. Transcripts encoding DCC were expressed in the nodose ganglia of mice from E12 to adulthood but were developmentally regulated. Paraesophageal anterior and posterior vagal trunks were DCC immunoreactive from E12 to E16. Transcripts encoding netrin-1 were expressed in the developing foregut and midgut; netrin-1 immunoreactivity was detected in the outer gut mesenchyme and mucosal epithelium. Neurites from explanted E14 nodose ganglia grew selectively toward cocultured E14 distal foregut explants (P < 0.01). Antibodies to DCC specifically abolished this preferential outgrowth (P < 0.05). Nodose axons also grew selectively toward cocultured netrin-secreting 293-EBNA cells (P < 0.005); antibodies to DCC again blocked this preferential outgrowth (P < 0.05). These data suggest that netrins, which are expressed in the bowel, attract DCC-expressing vagal sensory axons.

Molecular development of the extrinsic sensory innervation of the gastrointestinal tract

“…Research in the late 1980s and 1990s revealed that vagal axons first enter the mouse stomach at E11 (Baetge and Gershon, 1989), the rat stomach at E12 (Rinaman and Levitt, 1993), and were present in the mouse esophagus at E12 (Sang and Young, 1998). These observations were extended by studies in which the liphophilic dye, 1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindocarbocyanine perchlorate (DiI) was applied to the nodose ganglia of embryonic day (E) 12, E14 and E16 fetal mice (Ratcliffe et al, 2006). This work showed that the stomach receives vagal sensory fibers by E12 and that the celiac ganglion and duodenum are innervated by E14.…”

“…This work showed that the stomach receives vagal sensory fibers by E12 and that the celiac ganglion and duodenum are innervated by E14. DiI-labeled vagal axons reach the distal small intestine by E16 and, at the same stage, form a plexus in the esophageal musculature (Ratcliffe et al, 2006). Vagal terminals in the developing mouse stomach were also recently characterized using DiI (Murphy and Fox, 2007).…”

“…In the gut, netrins act through their receptor, deleted in colorectal cancer (DCC), to ensure that neural crest-derived neuronal and glial precursors reach the submucosal and pancreatic plexuses (Jiang et al, 2003). More recent work has established that vagal sensory axons are attracted to the fetal bowel through a mechanism that involves netrin acting on its receptor, DCC (Ratcliffe et al, 2006). These studies showed that DCC was expressed in nodose ganglia and that its expression was developmentally regulated; DCC immuno-reactivity was also demonstrated in descending vagal axons (Fig.…”

“…The outer mesenchyme and mucosa of the developing foregut expressed netrin-1 and vagal axons labeled by applying DiI to the nodose ganglion extended first into a netrin-1 producing zone and then toward a netrin-1 source. Co-cultures of explants of nodose ganglia with either foregut or netrin-1-secreting EBNA cells in vitro revealed that growing nodose neurites were attracted to the foregut and to netrin-1-secreting cells, and that this targeted growth was blocked by the addition of antibodies to DCC to the culture media (Ratcliffe et al, 2006). Not only were vagal sensory axons attracted to the gut and to specific locations within it, but the axons also failed to enter most of the bowel wall.…”

“…Studies that have demonstrated that axon guidance molecules play important roles in routing crest-derived cells to their correct destinations in the bowel (De Bellard et al, 2003;Jiang et al, 2003), therefore, not only support the idea that extrinsic nerves follow similar cues as the migrating crest-derived cells, but raise the possibility that the crest-derived cells themselves might produce some of the guiding cues. Vagal sensory axons reach the stomach and small bowel later in gestation than crest-derived cells in mice (Baetge and Gershon, 1989;Ratcliffe et al, 2006) and, of interest, the developmental pattern of the vagal innervation in zebrafish also seems to parallel that of the enteric innervation (Olsson et al, 2008). It is possible that because enteric neurons synthesize netrins, netrin-secreting neurons could help guide vagal axons to their appropriate innervation targets within the gut wall (Ratcliffe et al, 2007).…”