Both methods indicated that Hhip expression was significantly reduced in the dorsal spinal cord following knockdown of GPC1 and that this effect could be rescued by expressing GPC1ΔmiR. GPC1ΔmiRΔGAG elicited a partial rescue of Hhip expression ( Figures 4F and 4G), consistent with its ability to partially rescue the axon guidance defects arising from GPC1 knockdown ( Figure 1M). We next determined whether the postcrossing axon guidance effects of GPC1 could be attributable to its ability to induce Hhip expression. We coelectroporated selleck βact-EBFP2-mi7GPC1 with pMES-Hhip and found that indeed this treatment significantly rescued the axon guidance effects of GPC1 knockdown ( Figures 4H and 4I; compare

to Figures 1K–1M). Thus, GPC1 was required to induce Hhip expression in commissural neurons, which in turn mediated the guidance response of postcrossing axons along the longitudinal axis. To determine whether GPC1 was required cell autonomously to induce Hhip expression in dI1 neurons, we examined

embryos electroporated with Math1-EGFPF-mi7GPC1 ( Figure 4J). In these embryos, Hhip was again reduced or absent in the dorsal spinal cord on the electroporated side (average PIelect:PIcontrol = 0.56 ± 0.10 SEM), whereas electroporation of the control Math1-EGFPF-mi1Luc Crizotinib chemical structure construct had no effect (average PIelect:PIcontrol = 0.99 ± 0.09 SEM). This result was consistent with the neuron-specific requirement for GPC1 in commissural axon guidance ( Figure 2). We ruled out the possibility that the GPC1-dependent loss of Hhip expression was a result of gross patterning defects of the spinal cord, as the expression of markers, such as

Pax3 and Islet1, were unchanged ( Figure S6). Similarly, we observed no difference in the expression of Cntn2 (which is normally found in dI1 neurons) between the control and electroporated sides ( Figure S6C), showing that the loss of Hhip expression in the dorsal spinal cord was a direct and specific consequence of GPC1 knockdown. Taken together, these experiments demonstrated that the induction of Hhip expression in commissural neurons was dependent on GPC1. Next, we confirmed that Hhip induction occurred downstream of canonical Shh signaling. The highly dynamic expression pattern of Hhip in the dorsal spinal cord ( Figure S5) prevented accurate comparisons Bay 11-7085 of Hhip levels between embryos; hence, we used the nonelectroporated side of the spinal cord as an internal control. Because Shh is diffusible, the unilateral knockdown of Shh would not restrict its effect to the electroporated side. In other words, effective perturbation of Shh expression at the ventral midline would require bilateral electroporation of miShh, thus eliminating our internal control. To overcome this problem, we instead electroporated constructs encoding components of the canonical Shh pathway and assessed Hhip levels afterward.