, 2007). We wanted to determine whether the excess meningeal tissue or the increased BMP signaling leads to defects in the structural elements required for corpus callosum formation. We examined whether the midline glial structures critical for development of the corpus callosum (Shu and Richards, 2001 and Smith et al., 2006) were affected in the mutants. Staining the E16.5 cortex with BLBP and GFAP, markers for glial wedge cells, did not show alterations (Figure 3A). We also used BLBP to examine the glial wedge both before (E15.5) and after (E17.5) the callosum forms and found that before the callosum forms, there is no clear defect Y 27632 in the organization

of this structure (Figure 3B). The glial wedge is made up of radial glial cells that function both as progenitor cells for the Calretinin-expressing medial cingulate neurons that produce the pioneer axons crossing the callosum and as part of the scaffolding for these crossing axons (Rash and Richards, 2001).

Anti-Calretinin antibody labeled these cingulate neurons and their axons (Figure 3A), and the mutants did not show any difference in the numbers of these cingulate cortical neurons, although there were apparent defects in their axon projections across the midline (Figure 3A). We also dated the birth of Calretinin+ neurons in the cingulate cortex with BrdU injections and found that Calretinin+ neurons were born around BAY 73-4506 molecular weight E12.5 (data not shown). Staining for BrdU at E14.5 also did not show any apparent difference in the number of neurons born at E12.5. In addition, the subcallosal sling is made up of a group of migratory neurons, frequently visualized with NeuN

staining (Shu and Richards, 2001), that make up the ventral limit of callosal axon projection. In both control and experimental acallosal mice, NeuN clearly labeled this group of glial sling neurons (Figure 3A). All of these results suggest that neither a mechanical effect of the excess meninges nor the ectopic BMP7 expressed by the meninges affected the cingulate pathfinding neuron generation, the midline glial wedge, or the glial sling neurons in the mutant medial cortex but that, nevertheless, the axons failed to cross the midline. Although the Calretinin+ cingulate others neurons were generated, their axons failed to cross the midline in the mutant cortex (Figure 3A). Given that the glial wedge and sling are apparently minimally affected, we hypothesized that increased BMP7 released by the meninges might affect axon outgrowth of cingulate cortical neurons. To test this, we introduced a BMP7-expression construct into the E13.5 embryonic cortical midline by in utero electroporation. We examined the electroporated brains at E15.5, a day before the initial pioneer axons should cross the midline.