Fig. 5: Satb2/Nr4a2 dCKO mice phenocopy what observed in Satb2^Emx1 single CKO mice.

a Double immunostaining of Nr4a2 (red) with Satb2 (green) in layer 6b at P0 and P30. Cell counts show that about half Satb2⁺ neurons are co-labeled with Nr4a2 and almost all Nr4a2⁺ neurons are positive for Satb2 in layer 6b of control mice at P0 and P30. Yellow arrowheads indicate Satb2⁺/Nr4a2⁺ colabeled neurons and white arrowheads indicate single-labeled neurons. b Deletion of Satb2 and Nr4a2 is shown by immunostaining in the cortex of Satb2/Nr4a2 dCKO mice compared with controls at P25. c–f The number of Ctgf⁺ (c), Cplx3⁺ (d), Trh⁺ (e) and Tnmd⁺ (f) neurons is significantly reduced in the layer 6b of Satb2/Nr4a2 dCKO compared to controls. g Diagram of the construction of luciferase report gene plasmids. h–k Luciferase reporter assay shows that the transcriptional activities of the Ctgf, Cplx3, Trh, and Tnmd are significantly increased in the presence of Satb2 overexpression. l, m The transcriptional repression of Trh and Tnmd is detected in the presence of Nr4a2 overexpression. n Proposed model: Satb2 promotes the transcription of Ctgf, Cplx3, Trh, and Tnmd, while Nr4a2 suppresses the transcription of Trh and Tnmd during the differentiation of layer 6b neurons. dCKO, Satb2/Nr4a2 double CKO mice; Ctx, cortex. n = 3 for each group in c–f for statistics, n = 4 for each group in h–m for statistics, *P < 0.05, **P < 0.01,***P < 0.001. Scale bars = 50 μm in (a), 10 μm in enlarged figures in (a), 50 μm in (b), 100 μm in (c–f), 50 μm in inserts in (c–f).