The floorplate-stalling phenotype, for instance, is suggestive of a possible Robo/Slit modulatory effect (Long et al., 2004). Consistent with this, GPC1 binds Slit2 with high affinity (Ronca et al., 2001), and Slit2-Robo1 signaling strictly requires binding to heparan sulfate (Hu, 2001). The modulation of other axon
guidance pathways by GPC1 is of interest for Trametinib chemical structure future studies. See the Supplemental Experimental Procedures for additional details about the experiments. A detailed video protocol is available online (http://www.jove.com/video/4384) (Wilson and Stoeckli, 2012). In brief, miRNA plasmids were constructed as described previously (Figure S2A; Wilson and Stoeckli, 2011) and electroporated at HH17–HH18 using a BTX ECM830 square-wave electroporator (five pulses of 25 V, 50 ms duration). Bilateral electroporation was performed using five pulses of 18 V,
50 ms duration. The polarity of the electrodes was then switched, and the electroporation was repeated. The resulting axon guidance phenotypes were assessed by axonal tracing with DiI in open-book preparations at HH25–HH26. The cohorts of axons in DiI injection sites were classified as showing an “ipsilateral” phenotype if >30% of the axons stalled at or turned longitudinally along the ipsilateral floorplate border, as a “floorplate-stalling” phenotype if >50% of axons stalled within the floorplate, or as a “postcrossing” phenotype if >50% of axons failed to turn and/or if axons turned caudally on the contralateral
side of the floorplate. A complementary SCH 900776 nmr DNA clone containing the full-length (1,653 bp) open reading frame of chicken GPC1 was amplified by PCR (GenBank accession number KF040585). Myc or HA tags were inserted between the signal sequence and the first conserved cysteine Methisazone residue. GPC1ΔmiR was generated by silent site-directed mutagenesis of five nucleotides in the mi7GPC1 target sequence ( Zheng et al., 2004). In GPC1ΔmiRΔGAG, all three putative GAG attachment sites ( Zhang et al., 2007) were ablated by converting three critical serine residues to tyrosines. In GPC1ΔmiRΔGAGΔShh, ten additional residues ( Kim et al., 2011) were mutated to alanines using the megaprimer PCR mutagenesis method ( Barik, 2002). In situ hybridization and immunolabeling were performed as described (Mauti et al., 2006 and Wilson and Stoeckli, 2011). Staining intensities in the Hhip images were analyzed using ImageJ software (National Institutes of Health). A threshold was applied. Then the integrated density of pixels in the dorsal or medial spinal cord on both the control and electroporated sides was measured. We used the VassarStats Web site for Statistical Computation (Vassar College; Richard Lowry 1998–2013; http://vassarstats.net).