Wnt3 expression alone had no clear effect on axon growth compared to control. In both the Wnt3 and GFP conditions, many Forskolin manufacturer axons (about 60%) freely grew across the COS7 cells ( Figure 6C). To test the ability of Wnt3 to antagonize the negative effects of BMP7 in this assay, we coexpressed the ligands and found that Calretinin+ axons now quite readily crossed BMP7 + Wnt3-expressing COS7

cells ( Figure 6C, p < 0.001). Thus, Wnt3 apparently has minimal, if any, stimulatory effect on axon growth in this assay unless BMP7 is present, in which case it apparently counteracts the negative effects of BMP7. To examine this interaction in vivo, we introduced BMP7 along with Wnt3 in utero. Strikingly, we observed formation of the corpus callosum when we expressed Wnt3 expression along with BMP7 ( Figure 7A). Thus, it appears that Wnt3 is able to counteract the negative

effects of BMP7 on callosal pathfinding axon outgrowth. This is consistent with the onset and spatial distribution of Wnt3 at E14.5 being a critical regulator of callosum formation by allowing the pioneer axons to cross the BMP7-expressing midline meninges. Screening Library Because the mutant cortex lost Wnt3 expression before the initial pioneer axons crossed the midline, we wondered whether adding back Wnt3 would rescue the failure of the pioneer axons crossing the midline in the mutants with excess meninges (the Msx2-Cre;Ctnnb1lox(ex3) mice). To test this, we electroporated a Wnt3-expression construct into the midline cortex of Msx2-Cre;Ctnnb1lox(ex3) mice at E13.5 and examined E17.5 embryos and found that TAG1- and L1-positive corpus callosal axons are obvious in the Wnt3-electroporated brain, but GFP-electroporated brains failed to form the midline callosal trajectories ( Figure 7B). To further address

our hypothesis that Wnt3 signaling MTMR9 antagonizes BMP7 signaling, thereby allowing the corpus callosal axons to cross the midline, we examined staining for pSMAD1/5/8 in the medial cortex of BMP7-electroporated mice either with GFP or Wnt3 coelectroporation. In mice that were electroporated with BMP7 and GFP, as expected, the level of pSMAD1/5/8 immunoreactivity was markedly increased in the BMP7-electroporated medial cortex (Figure 7C). However, when Wnt3 was coelectroporated with BMP7, and the brains were examined 3 days later, the pSMAD1/5/8/ levels were blunted and were perhaps even lower than those seen in the opposite unelectroporated hemisphere (Figure 7C). To quantify these effects, we performed western blotting for pSMAD1/5/8 and normalized the signal to antibodies for GAPDH or all forms of SMAD1. In these experiments, we found that BMP7 + eGFP-electroporated cortex had a 40% higher level of pSMAD1/5/8 compared to cortex electroporated with Wnt3 + BMP7 (Figures S5A and S5B).