Increase in upper and lower layer pyramidal neurons in the cortices of Robo1−/− mice at E18.5. (A–B″) Immunohistochemistry in the cortex of Robo1+/+ (A–A″) and Robo1−/− (B–B″) mice for the early- (Ctip2, green; A, A″, B, B″), and late-born (Cux1, red; A′, A″, B′, B″) pyramidal neuron markers. Counts revealed a significant increase in both Ctip2+ and Cux1+ cells in the cortex of Robo1−/− mice. C, D, No distribution defects were observed despite the increase in cell density of either Ctip2+ or Cux1+ cells in Robo1 mutants; **p < 0.004, ***p < 0.0002.