Figure 5.

Impaired segregation of immature layer 5 neurons in the absence of Ldb2. (A–L) Immunocytochemical analysis shows striking Ldb1/2-dependent changes in Lmo4 expression. Lmo4 expression (green in A–L) is high in the cingulate cortex of controls (arrow in A), extends into motor cortex, and shows lower expression levels in layers 2/3 lateral to the motor/somatosensory boundary (arrowheads in A–C). Lmo4 is also expressed by subsets of neurons of layers 4–6. Ctip2 (red in A–D) was used to mark layer 5 neurons and Ldb1 (blue in A–H) as a reference marker. Low-power magnification reveals a subtle upregulation of Lmo4 in layers 4 and 5 of Ldb2-KO (B,F,J; arrows in J) compared with controls (A,E,I), while Ldb1-KO displays a striking ectopic upregulation of Lmo4 across the cortical wall (C,G,K). Lmo4 is completely lost in the neocortex of Ldb1/2-DKO (D,H,L), but striatal expression of both Ldb1 and Lmo4 (arrow in H) is maintained due to Emx1-Cre's neocortex specificity. (I–L) High-power confocal pictures of M1/M2 motor area of layer 5 (boxed in A–D) show colocalization between Ctip2 (red in I–L) and Lmo4 (green in I–L). The number of Ctip2⁺;Lmo4⁺ double-positive neurons (yellow in I–L) is increased in Ldb2-KO (J) and Ldb1-KO (K) compared with controls (I). Cortical Lmo4 immunoreactivity is abolished in layer 5 of Ldb1/2-DKO (L). (M) Quantification of Ctip2/Lmo4 coexpression reveals a statistically significant increase in coexpression in layer 5 neurons of Ldb2-KO compared with controls, and a dramatic loss in Ldb1/2-DKO. (N–U) Colabeling with a CPN marker Satb2 (red in N–U) reveals partial overlap between Lmo4 (green in N–Q) and Satb2 in controls (N) and Ldb2-KO brains (O). Satb2 expression is qualitatively reduced in Ldb1-KO (P) and Ldb1/2-DKO (Q), but Satb2⁺ CPNs are present in Ldb1/2-DKO (Q), suggesting that these neurons have downregulated Lmo4. (R–U) Colabeling of Satb2 and Ctip2 shows very few Ctip2⁺;Satb2⁺ (yellow in R) double-positive neurons in layer 5 motor cortex of controls (arrows in R). An increased fraction of Ctip2⁺ neurons coexpress Satb2 in Ldb2-KO (arrows in S) and Ldb1-KO (T). A further increase in Ctip2⁺;Satb2⁺ double-positive layer 5 neurons (arrows in U) is found in motor cortex of Ldb1/2-DKO (U). (V) Quantification of Ctip2/Satb2 coexpression shows statistically significant increases in the fraction of double-positive layer 5 neurons in Ldb2-KO and Ldb1/2-DKO compared with controls. *P ≤ 0.05; **P ≤ 0.01; ***P ≤0.001. Scale bars: 500 µm in (H) for (A–H) and in (Q) for (N–Q); 50 µm in (U) for (I–L) and (R–U).