We also used anti-Foxc2 antibody, which

predominantly labels the dural meningeal precursors ( Zarbalis et al., 2007); Foxc2-expressing dural cells are also expanded in the mutant embryos ( Figure 2C). Cxcl12, which labels the inner pial layer, was likewise expanded and ectopically expressed in the mutants selleck compound ( Figure 2C). These results suggest that ectodermal Wnt6 induces pia, arachnoid, and dural meningeal cells to overgrow both over the surface of the cortex and between the hemispheres. We suspected that the non-cell-autonomously expanded meninges may be the cause of the corpus callosal agenesis and that perhaps we could use the Msx2-Cre;Ctnnb1lox(ex3) mutants to identify

a normal role for the meninges in corpus callosum development. The Wnt6-expression zone marks the areas of epithelial-mesenchymal transition, and Bmp signaling is known to be involved in this transition ( Kalluri and Weinberg, 2009 and Lavery et al., 2008), and, in the mutant embryos, the development of the skull vault appeared compromised ( Figure 1B′). Because the normal source of BMPs for much of skull development is the meninges ( Kim et al., GSI-IX datasheet 1998 and Rice et al., 2005), we hypothesized that the ectopic meningeal tissue may express BMP-signaling components affecting the skeletogenic mesenchyme for the mouse skull vault. This also raised the possibility that excess Bmps produced by the meninges might interfere with MYO10 corpus callosum development. We examined the expression of several Bmp ligands and found that BMP7 was ectopically expressed in the excess meningeal tissue at E14.5–15.5 and that the overall expression level in the meninges was increased ( Figures

2D; Figure S2C). To determine whether the increased expression of BMP7 alters the underlying Bmp-signaling levels within the cortex, we stained the brain with an anti-pSMAD1/5/8 antibody. This showed that the mutant brains had increased phosphorylation of SMAD1/5/8, confirming that the ectopic BMP7 expression in the meninges is biologically active and increases cortical Bmp signaling ( Figure 2E). Interestingly, the increased phospho-SMAD staining was barely present at E13.5 but dramatically increased by E15.5 ( Figure 2E), in parallel with the onset of recombination in the skin. Phosphorylation of SMAD2, which is a downstream target of canonical TGF-β ligands, rather than Bmp ligands, was not induced (data not shown). An interesting supporting point for the increased Bmp signaling in the developing cortex that we see with these markers is increased Zic1 expression in the cortical ventricular zone ( Figure 2A). Zics are known to be induced by Bmp ligands in the developing central nervous system (CNS) ( Aruga et al., 2002).