, 2000), Gli1, or Gli2 were electroporated, Hhip expression was expanded ectopically ( Figure 5A). Conversely, unilateral repression of canonical Shh signaling by PtcΔloop2 (a Hedgehog-insensitive dominant repressor of Smo; Briscoe et al., 2001) caused a specific loss of dorsal Hhip expression

( Figure 5B). This effect GSK1210151A molecular weight was identical to that observed following the loss of GPC1 but occurred with even higher penetrance and severity (compare percent values in Figure 5B to Figure 4D; compare Figure 5E to Figure 4G). Thus, as predicted, Hhip induction in the dorsal spinal cord was dependent on Shh transcriptional activity. In line with our hypothesis, which predicted that GPC1 was acting downstream of Shh to MDV3100 induce Hhip in commissural neurons, repression of the canonical Shh pathway phenocopied the effects of GPC1 silencing. To establish a more direct link between Shh and

GPC1 in Hhip induction, we next tested the ability of a Shh-insensitive GPC1 mutant (GPC1ΔmiRΔGAGΔShh) to rescue dorsal Hhip expression following knockdown of endogenous GPC1. The GPC1 mutant was resistant to knockdown, lacked the GAG attachment sites, and was unable to activate Shh signaling due to ablation of ten critical amino acids ( Kim et al., 2011). Unlike GPC1ΔmiR and GPC1ΔmiRΔGAG, this construct was incapable of binding Shh in coimmunoprecipitation assays ( Figure 5C). Consistent with a requirement for Shh-GPC1 interaction in the induction of dorsal Hhip, we found that GPC1ΔmiRΔGAGΔShh was completely unable to rescue Hhip expression ( Figure 5D; compare Figure 5E to Figure 4G). Furthermore, GPC1ΔmiRΔGAGΔShh was incapable of rescuing the axon guidance defects induced by GPC1 knockdown ( Figure 6). Taken Oxalosuccinic acid together, these results demonstrate a functional link between the GPC1/Shh-mediated induction of Hhip expression and commissural axon guidance. To test whether GPC1 was simply required as a

general enhancer of Shh-mediated transcription, we assessed the expression of other known Shh target genes after GPC1 knockdown (Figure 7) (Goodrich et al., 1996, Oliver et al., 2003, Tenzen et al., 2006 and Domanitskaya et al., 2010). Neither Patched1 (Ptc1) nor Boc were affected by GPC1 silencing. Furthermore, there were no effects on the Wnt antagonist (and Shh transcriptional target) Secreted frizzled-related protein1 (Sfrp1) or on the Wnt receptor Frizzled3 (Fzd3), both of which have been implicated in postcrossing axon guidance ( Lyuksyutova et al., 2003 and Domanitskaya et al., 2010). Importantly, these results suggested that the longitudinal guidance defects elicited by the loss of GPC1 were not due to perturbation of the chemoattractive Wnt-Fzd3 pathway (at least not at the transcriptional level). The lack of dependence on GPC1 for transcription of Boc, Ptc1, and Sfrp1 suggested that GPC1 is required specifically for the regulation of Hhip expression in dI1 neurons, rather than as a general component of Shh-mediated transcriptional activation.