Our analysis so far has led us to propose that BMP7 helps to block the formation of the corpus callosum by inhibiting callosal pioneer axon outgrowth, thereby inhibiting

formation of the corpus callosum until such time as Wnt3 expression begins in the pioneer BYL719 cell line neurons and antagonizes the effects of BMP7, allowing initial callosal axon outgrowth. This left us with one significant puzzle: why were the Msx2-Cre;Ctnnb1lox(ex3) mice missing the expression of Wnt3? We hypothesized that another secreted factor normally produced by the meninges was also overexpressed in the mutants and that this factor helps to regulate Wnt3 expression in the cingulate cortex. Our thoughts immediately turned to the Gdf5/6/7 inhibitory molecule Dan ( Dionne et al., 2001), which some time ago we showed is expressed by the meninges ( Kim and Pleasure, 2003). In that same study, we also showed that one of the ligands that Dan inhibits, Gdf5, is expressed by the Cajal-Retzius cells, but we were unable at that time to identify any functional significance for this pattern of expression ( Kim and Pleasure, 2003). Interestingly, the Cajal-Retzius cells are the most Obeticholic Acid supplier superficial cortical neurons in layer

1 and lie immediately adjacent to the cingulate very pioneer neurons in the medial cortex. To test the role of these factors, we examined the expression of Gdf5 and Dan in the mutant mice and found that Gdf5 is expressed in Cajal-Retzius cells in both control and mutant mice (Figures 8A and 8C), and the expanded meninges in the mutant express abundant Dan (Figures 8B and 8B′), implying that the levels of this inhibitor were increased in the vicinity. We also performed double labeling in the dorsal neocortex using Calretinin as a Cajal-Retzius cell marker (in the cingulate Calretinin stains, both

pathfinding neurons and Cajal-Retzius cells, but, in the rest of the cortex, Calretinin is a selective marker for Cajal-Retzius cells) and confirmed that Gdf5 was coexpressed by Cajal-Retzius cells, whereas Dan was expressed in the overlying meninges (Figure 8C). We then examined whether electroporation of Gdf5 in the midline of the cortex at E12.5 is sufficient to induce early Wnt3 expression by E14.5 and found that, indeed, Gdf5 electroporation induces low levels of Wnt3 expression in the medial cortex ( Figure 8D). Thus, our model is that Wnt3 expression in the cingulate pioneer neurons is normally positively controlled by GDF5 activity from the adjacent Cajal-Retzius neurons but that the excess meningeally produced Dan in the mutant leads to decreased expression of Wnt3 in the mutant embryos.