The expression of Shh and its receptor Boc by two complementary nonoverlapping populations CP-673451 of neurons during synaptogenesis suggests a mechanism for achieving specificity of circuitry, where the target cell expresses the ligand (Shh) and the presynaptic cell expresses the corresponding receptor (Boc) (Sanes and Yamagata, 2009 and Williams et al., 2010). To examine whether the Boc mutant animals have a similar cortical phenotype to ShhcKO mutants, we performed Golgi analysis on P20 brains of BocKO mice and wild-type littermate controls ( Figures 6A–6D). We observed significant reductions

in spine density, and growth and complexity of basal dendrites located in layer V neurons of BocKO animals ( Figures 6E and 6F), while there was no difference in branch growth and spine density in layer II/III ( Figures 6G and 6H). These findings suggest that the non-cell-autonomous decrease in dendritic growth of layer V neurons may be due to a loss of synaptic activity from presynaptic Boc expressing neurons ( McAllister et al., 1996). To test for the loss of presynaptic input from Boc expressing neurons we utilized in utero electroporation to introduce synaptophysin-GFP,

a marker for active presynaptic terminals ( Kelsch et al., 2008, Li and Murthy, 2001, Meyer and Smith, 2006 and Nakata et al., 1998), into lower layer II/III cortical neurons at embryonic day 14 (E14) ( Figures S6A–S6C). We targeted neurons in lower isothipendyl layer II/III because find more of the extensive number of these cells exhibiting LacZ reporter gene expression observed in the Boc gene trap reporter mice, and also because of the preference for neurons located in this layer to make synaptic connections onto layer V pyramidal neurons ( Anderson et al., 2010 and Petreanu

et al., 2007). In addition to synaptophysin-GFP, we coelectroporated a plasmid for a pCAG-mTdTom-2A-H2BGFP plasmid ( Trichas et al., 2008) that labels electroporated cells with a nuclear GFP, and a membrane TdTomato, in order to label axonal projections ( Figures 7A–7C). In BocKO mice we coelectroporated the synaptophysin-GFP along with the mTdTomato axonal marker. We also coelectroporated a short hairpin RNA targeted against Boc (Boc-shRNA) into the brains of wild-type non mutant mice that should have normal levels of Boc and Shh, except for the population of electroporated cells. When we compared the density of synaptophysin-GFP puncta located on the axons of Boc-shRNA expressing versus control-shRNA expressing cells, we found a significant reduction in the density of puncta on axons located in layer V ( Figures 7C–7F). Notably, this reduction was observed in both ipsilateral and contralateral layer V axons, while there was no significant difference in the puncta density in layers II/III ( Figure 7J). We found a similar pattern of reduced puncta when we compared BocKO and control animals ( Figure 7K).