Fig 2: Rsp neurons lose their identity in Satb2 CKO mice. FN1 is restricted to the SubC of controls (a) but expressed by neurons in the Rsp region of Satb2 CKO mice at P4 (b). c–h ER81, Citp2, and Nr4a2 are expressed in the SubC and specific layers of the Rsp in control mice (c, e, g). In Satb2 CKO mice, however, they are expressed throughout the Rsp regions while remain largely unchanged in the SubC (d, f, h). Cbln1 shows restricted expression in the Rsp of control mice (i), but its expression is lost in Satb2 CKO mice (j). Zbtb20 is expressed in superficial Rsp and CA1 of control mice (k), but undetectable in the Rsp region while remains unchanged in the CA1 of Satb2 CKO mice (l). Note that ectopic Zbtb20+ cells (arrows) are present in the subicular region of Satb2 CKO mice. Mef2C is intensely expressed in the Rsp and weakly in the SubC of control mice (m), but its expression was largely reduced in the Rsp region to a comparable cellular density of the SubC in Satb2 CKO mice (n). Dkk3 is strongly expressed in upper Rsp of control mice (o) but absent in the Rsp region of Satb2 CKO mice (p). Intense Lmo4 expression is reduced in the Rsp regions with a similar intensity to that in the SubC in Satb2 CKO mice (r) compared with controls (q). s, t Diagram of Rsp fate change in Satb2 CKO mice. DG dentate gyrus. n = 3 mice for each genotype. Scale bar = 500 µm

Fig 3: Distribution of Nurr1 protein expression in the brain based on Nurr1 immunohistochemistry in frozen sections for Q-IHC (A). Nurr1 expression is shown in SNpc and VTA region (B), temporal cortex, CA3 of the hippocampus, and dorsal endopiriform cortex (C). A representation of the procedure used to acquire images is show for the temporal cortex in which the region of interest is outlined (dashed line) and Stereo Investigator software used to acquire images at 40X magnification (green and red box). DEn, dorsal endopiriform cortex; SNpc, substantia nigra pars compacta; Te, temporal cortex; VTA, ventral tegmental area. Scale Bar = 1000 µm.

Fig 4: Difference in Nurr1 protein expression between heterozygous (n = 3) and wild type Nurr1 mice (n = 3) in six different regions of the brain using 20 nM of Nurr1 primary antibody concentration. Based on chromogen intensity, neurons of the +/- mice had significantly lower Nurr1 expression as compared to the +/+ mice but this varied across region. Magnitude of reductions in the +/- mice were 27% for the parietal/temporal cortex, 49% for the claustrum/dorsal endopiriform cortex, 25% for subiculum, 33% for substantia nigra pars compacta, 22% for ventral tegmental area, and 21% for CA1 region of the hippocampus.

Fig 5: Dmrt5 function is explored in various transgenic mouse models (WT, Dmrt5-/-, Dmrt5lox/lox;Emx1CRE+ and Dmrt5lox/lox;NestinCRE+) with different time-points of Cre-recombination. (A, B) Coronal sections from E15.5 (A) and E18.5 (B) WT and Dmrt5-/- brains that were immunostained for Tbr1 (red) and counterstained with Hoechst (blue). At E15.5, Tbr1-immunoreactive cells were present in CP, and Cl in the Dmrt5-/- and Dmrt5lox/lox;Emx1Cre similarly to WT. The enlarged images from the regions indicated with boxes demonstrate that the SP is present in WT, but absent in Dmrt5-/- and Dmrt5lox/lox;Emx1Cre. Moreover, the CP is reduced in thickness in Dmrt5-/- and in Dmrt5lox/lox;Emx1Cre. At E18.5, the SP layer is present in Dmrt5lox/lox;Emx1Cre brains. In Dmrt5-/-, the separation between the CP and SP is not evident with Tbr1 immunohistochemical or Hoechst stainings. (C–F) Coronal sections of developing brains from WT (C), Dmrt5-/- (D), Dmrt5lox/lox; Emx1CRE+ (E), and Dmrt5lox/lox;NestinCRE+ (F) immunostained for Nurr1 at E15.5 (upper row) and E18.5 (lower row). Enlarged images of the regions of dorsal cortex indicated by the boxes are presented for each brain. SP was not visible at E15.5, but sparse Nurr1+ SP cells are detected at E18.5 in Dmrt5-/- brains (white arrows in the high-power image of D). Nurr1 expression is absent at E15.5 in Dmrt5lox/lox;Emx1CRE+ embryonic brain even though the SP layer was later comparable to the WT at E18.5. Nurr1 expression is not affected in SP in Dmrt5lox/lox;NestinCRE+ embryonic brains. Nurr1 immunoreactivity is detected in dLns in the lateral cortex and in the Cl of WT, Dmrt5-/-,Dmrt5lox/lox; Emx1CRE+ and Dmrt5lox/lox;NestinCRE+. Scale bars represent 100 µm for all images.