The alignment of L5a neurons develops synchronously with the formation of cortical barrels. Specifically labeled axons of the VPM in the Fzd10-EGFP transgenic line (A). At E17.5, GFP-positive TCAs from the VPM extend into the deep cortical layer (B) and reach the superficial layer of the barrel cortex at P0 (C). No expression of CR is detected in the barrel cortex except in the CajalRetzius cells under the pia. At P3, the GFP-positive TCAs segregate into two strong GFP-positive bands in layer 4 and layer 6, and CR-positive neurons emerge in L5a (D). At P4, the GFP-positive TCAs of the VPM begin to segregate into clusters but do not completely separate from each other (E, F). The CR-positive processes extend into L4 and show a septa-like pattern (G, H arrowheads). Until P8, the GFP-positive clusters in L4 and the CR-positive L5a neurons display a complementary distribution pattern (M, N). Between L4 and L5a, the CR-positive L5a neurons display a distinct serrated alignment, and their processes extend vertically between the GFP-positive clusters (N, asterisk). At P15, the complementary pattern of the TCAs from the VPM and the CR-positive L5a neurons remain evident, but the expression level of CR is decreased (O, P, arrowhead). After ION transection at P2, no septa-like pattern can be observed at P4 (K, L), and the GFP-positive TCAs are not segregated into clusters (I, J). Only uniform GFP-positive and CR-positive bands remain in L4 and L5a, respectively, and no barrel-like or septa-like pattern can be observed at P8 (Q, R) and P15 (S, T). Scale bar in (A): 500?m, Scale bar in other images: 100?m.