In contrast

to RET, strong NCAM expression could be seen along precrossing, crossing, and postcrossing commissural axons, using antibodies to PSA, the polysyalic acid carried by the core NCAM protein (Figure 6A, Figure S3B). We also observed that like NCAM, GFRα1 was also selleck inhibitor present along commissural axons. Notably, the GFRα1, but not NCAM, immunolabeling was more intense along crossing axon segments, whereas NCAM, but not GFRα1, distributed more broadly along postcrossing axon segments (Figure 6A). NCAM/GFRα1 colabeling was also performed in cultured commissural neurons. Confocal stack images showed that the proteins were present in the soma, along the axon, and in the growth cone, presenting both colocalization and individual distribution profiles (Figure 6B). Overall, these observations indicated that NCAM and GFRα1, but not RET, had the expected expression to mediate the gdnf effect in commissural axons. Next, we investigated the pattern of commissural projections in mice lacking gdnf receptors by DiI tracing in open books. To further rule out RET contribution, we analyzed a RET floxed mouse line in which RET had been deleted in spinal commissural neurons by crossing the line with a Wnt1-CRE line (Retfwnt1; Charron et al., 2003). Observations of DiI tracing in E13.5 Retfwnt1 open books revealed Bortezomib clinical trial no evidence

of aberrant axon choices during FP crossing. A moderate 10% increase of stalling was observed after ablation of RET but no significant turning defects were detected ( Figure S3C). Non-specific serine/threonine protein kinase Next, commissural axon trajectories were examined in open books from E13.5 NCAM knockout mouse line ( Figures 6C and 6D). In contrast, significant defects of FP crossing and rostrocaudal turning were detected in the NCAM null embryos, compared to the wild-type ones, which were similar to those observed in the gdnf null embryos. These in vivo analyses indicated that NCAM, but not RET, is probably the commissural neuron receptor of gdnf. If so, inactivation of NCAM, but not RET, should compromise the ability

of commissural neurons to be sensitized to Sema3B by gdnf in our neuronal culture model. This was assessed by application of a function blocking NCAM antibody and by the genetic loss of NCAM. In both cases, we found that commissural neurons failed to acquire the collapse response to Sema3B that was normally triggered by gdnf ( Figures 6E and 6F). Moreover, application of function-blocking antibodies to GFRα1 also resulted in a lack of commissural axon sensitization to Sema3B ( Figure 6E). Collapse assays were also performed on RETfwnt1 neurons and revealed that they could acquire responsiveness to Sema3B normally, thus confirming that RET is dispensable for this process ( Figure 6G). Thus, NCAM, but not RET, mediates the gdnf regulation of commissural axon responsiveness to Sema3B.