Finally, to demonstrate that GPC1 can influence the canonical Shh pathway during neural tube development, we examined the expression of Caspase-independent apoptosis several Shh target genes following GPC1 overexpression. Ptc1, Sfrp1, and Hhip were all expressed ectopically after electroporation of pMES-GPC1 ( Figures 7C and 7D), an effect that was never observed following electroporation of a control (pMES-empty)

plasmid. Thus, GPC1 is an enhancer of canonical Shh signaling in vivo. Taken together, our results demonstrate that GPC1 has a specific function in regulating Hhip expression in commissural neurons, thereby eliciting a Shh-dependent change in axonal responsiveness to Shh at the midline choice point. In addition to identifying Selleck Lonafarnib GPC1 as a regulator of commissural axon guidance, our study establishes the existence of another important Shh signaling pathway in commissural neurons: the GPC1-dependent activation of transcription, which in turn modifies the growth cone’s sensitivity to floorplate-derived cues. Our findings not only highlight the remarkable multifunctionality of Shh during neural development but also delineate a molecular mechanism by which navigating axons can switch their responses to intermediate targets. Together with previous reports, our results provide a complex and highly dynamic picture of Shh signaling in commissural axon guidance (Figure 8).

First, Shh collaborates with Netrin-1 to attract axons toward the floorplate, in a Boc-dependent manner (Charron et al., 2003 and Okada et al., 2006). However, Shh not only signals via a rapid,

noncanonical pathway to elicit growth cone attraction (Yam et al., 2009) but simultaneously activates a slower transcriptional pathway which triggers the upregulation of Shh-induced genes in the neurons, including (but perhaps not limited to) Hhip. Additionally, Shh modulates cyclic AMP (cAMP) levels in commissural growth cones to confer sensitivity to repulsive Semaphorins at the midline ( Parra and Zou, 2010). Shh then acts directly as a repulsive guidance cue to guide postcrossing axons anteriorly, in a Hhip-dependent manner ( Bourikas et al., 2005). below Finally, Shh also shapes a chemoattractive Wnt activity gradient, by inducing the graded expression of the Wnt antagonist Sfrp1 along the anteroposterior axis of the spinal cord ( Domanitskaya et al., 2010). Our study shows that Shh not only guides precrossing axons directly by binding to its receptors on the growth cone (Okada et al., 2006 and Yam et al., 2009) but simultaneously activates the transcription of its own receptor, which is required for a subsequent stage of axon guidance. How could the canonical and noncanonical Shh pathways operate in parallel in precrossing neurons? One intriguing possibility is that Smo (which functions in both pathways) is responsible for eliciting the distinct outputs.