Figure 2

Ngn2 regulates callosal axon projection and corpus callosum formation in vivo. (A-F) Coronal sections of E18.5 Ngn2+/- embryos (A-C) and Ngn2-/- (D-F) embryos were immunostained with EGFP (A,D) since EGFP was inserted into the Ngn2 locus to create the null allele. The sections were also immunostained with the axonal marker L1 (B,E) to reveal many of the cortical axon tracts. Merged EGFP and L1 images (C,F) show a reduction in callosal axons in Ngn2-/- embryos compared to the heterozygous embryos. The immunostaining also revealed malformations of the corpus callosum, including aberrant axonal projections at the midline and an abnormal corpus callosum (blue arrowhead, E). (G-L) Horizontal sections from E18.5 Ngn2+/- embryos (G-I) and Ngn2-/- (J-L) embryos were immunostained for the axonal markers L1 (G,J) and NF165 (H,K). (I,L) Merged images. The immunostaining of axonal markers shows a reduction of callosal axons and the lack of a corpus callosum (J-L), the formation of probst bundles (red arrowhead, J), and misprojecting axons at the midline (blue arrowhead, J) in the embryos lacking Ngn2 None of these phenotypes were observed in the heterzygous embryos. (M-R) Ngn2+/- cortices (M-O) and Ngn2-/- (P-R) cortices were injected with the anterograde tracer BDA at E18.5 to determine if the axons that prematurely defasciculate orginated from cortical neurons. Coronal sections were immunostained with the axonal marker L1 to reveal callosal axons (M,P). The BDA tracings were revealed with streptavidin conjugated with Alexa 546 (N,Q). (O,R) Merged images. Arrowheads in M-O indicates callosal axons appropriately reaching the midline. Prematurely defasciculating axons were revealed in the Ngn2-/- embryos by both L1 staining and BDA anterograde tracings (P-R, arrowheads). This phenotype was not observed in Ngn2+/- embryos (M-O). BDA anterograde tracings definitively demonstrate that these axons are cortical in orgin.