(A) Heatmaps showing marker genes or genes strongly enriched, as identified in the Allen Brain Atlas, for each layer of the neocortex differentially expressed between control and Rorb KO. Log-transformed transcripts per million (TPM) are color scaled in red and blue for each of the four RNA-seq replicates in the left most heatmap and the mean for each time point and genotype in the middle heatmaps. Log fold change (LFC) between control (Ctl) and Rorb KO is color-scaled in orange and purple in the right most heatmaps. (B) Numbers of differentially expressed genes (DEGs) for the three ages examined. (C) Line plots showing LFC for the same genes. The solid black line indicates no change. Negative LFC indicates decreased expression in Rorb KO, and LFC >0 indicates increased expression in Rorb KO. Each colored line is a layer-specific DEG and the dashed black line plots the mean across the group of genes. (D) RNA-seq expression of layer 5 TFs. Lines plot the mean ± SE. P by moderated t-test adjusted for multiple comparisons (Benjamini-Hochberg). (E) Additional L4 and L5 genes were identified using the Allen Brain Atlas differential search contrasting L4 SSp structures to L5 SSp. Genes with >1.5 fold change and expression threshold >1.6 were selected. Genes already shown in A-C were removed. Hence each gene shown does not meet statistical criteria for differential expression in Ctl/KO by RNA-seq. Line plots show RNA-seq LFC for each layer-specific gene. The solid black line is the mean across genes and the solid gray line indicates no change. Negative LFC indicates decreased expression in Rorb KO, and LFC >0 indicates increased expression in Rorb KO. (F) Overall (first bar), 1% of genes were downregulated (blue) and 2% were upregulated (red). Downregulated genes were overrepresented (26%) among the 102 L4-specific genes (middle bar), while upregulated genes were overrepresented (19%) among the 240 L5-specific genes. Both overrepresentations were significant (p<2.2e-16) by fisher exact test.