We further examined the cell type and temporal specificity of Boc expression by performing immunofluorescent staining in P4 and P14 Boc heterozygous mutant mice. We found LacZ expression at both P4 and P14, with the level of expression markedly increased at P14 (Figures 4A and 4C). We also looked at

coexpression with the neuron projection subtype markers CTIP2 and SATB2. We found that the majority of LacZ positive cells were also positive for the callosal projection subtype marker SATB2, while very few neurons colabelled with the corticofugal projection marker CTIP2. Bcl-2 inhibitor We also stained sagittal brain sections for placental alkaline phosphatase (PLAP) activity, which preferentially labels axonal projections in the Boc heterozygous mutant mice (Friedel check details et al., 2005, Leighton et al., 2001 and Okada et al., 2006). We found that there was an absence of PLAP labeled descending axonal projections in the internal capsule, where

corticofugal projections normally exit the cortex (Figure S5B). Fluorogold labeling of ipsilateral local projection neurons in layer III and Va also colocalized with LacZ positive cells (Figure S5A). Taken together these findings reveal that Boc is expressed predominantly by callosal and local projection neurons, many of which are known to form synaptic connections onto deep-layer corticofugal projection neurons (Petreanu et al., 2007 and Wise and Jones, 1976). Boc is known to be highly expressed in the nervous system both in embryonic and adult tissues (Mulieri et al., 2002), and has previously described roles in attracting commissural axons across the midline in the developing spinal cord (Okada et al., 2006), and in repulsion of ipsilateral retinal ganglion neurons to prevent aberrant crossing of the optic chiasm TCL (Fabre et al., 2010). Thus Boc expression in the embryonic telencephalon, or

early postnatal expression in callosal projection neurons, may regulate aspects of cortical development that precede synaptogenesis, such as neuron migration and/or long-range axon guidance across the corpus callosum. To explore other possible functions of Boc in the development of the cortex, we examined the brains of homozygous Boc mutant mice (BocKO). The brains of BocKO mice appeared grossly normal with no obvious differences in size or cortical thickness. Fluorogold labeling of callosal projection neurons in layer II/III colocalized with LacZ positive cells in the null mutant ( Figure 5A). Examination of the layer Va boundary of LacZ expression with fluorogold labeling of layer VI corticothalamic projections suggested that neuronal migration and layer formation is also normal in Boc null mutants ( Figure 5B). Direct examination of PLAP staining of callosal projections in heterozygous and BocKO mice also did not reveal any change in the number or pattern of callosal axons projecting across the midline ( Figures 5C and 5D).