Fig 2: MUC1-C induces expression of BRN2 and NE markers.a,b RNA-seq was performed in triplicate on LNCaP-AI/tet-MUC1shRNA cells treated with vehicle or 500 ng/ml DOX for 7 days. a The datasets were analyzed with GSEA, using the Hallmark gene signature collection. Silencing MUC1 was significantly associated with upregulation of the Androgen Response pathway. b Heatmap depicting the effects of silencing MUC1 on AR pathway genes. c LNCaP, C4-2B and LNCaP-AI cells were analyzed for BRN2 mRNA levels by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative mRNA levels compared to that obtained for LNCaP cells (assigned a value of 1). d Lysates from LNCaP, C4-2B, and LNCaP-AI cells were immunoblotted with antibodies against the indicated proteins. e LNCaP-AI cells stably expressing a tet-CshRNA or tet-MUC1shRNA were treated with 500 ng/ml DOX for 7 days. BRN2 mRNA levels were analyzed by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative mRNA levels compared to that obtained for DOX-treated LNCaP-AI/tet-CshRNA cells (assigned a value of 1). f LNCaP-AI/tet-CshRNA and LNCaP-AI/tet-MUC1shRNA cells were treated with vehicle or 500 ng/ml DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. g LNCaP-AI cells expressing a tet-CshRNA or tet-MUC1shRNA were treated with 500 ng/ml DOX for 7 days. ASCL1 (left), AURKA (middle) and SYP (right) mRNA levels were analyzed by qRT-PCR. The results (mean±SD of five determinations) are expressed as relative mRNA levels compared to that obtained for DOX-treated LNCaP/tet-CshRNA cells (assigned a value of 1). *p < 0.05 (Student’s t-test). Source data are provided as a Source Data file.

Fig 3: BRN2 loss produced abnormities of monkey interneuron development.(A) BRN2 and COUP-TFII staining showed the wide distribution of BRN2 in the telencephalic ganglionic eminences (GEs). PSB, pallial-subpallial boundary. (B) NKX2-1 and COUP-TFII expression in BRN2+/+ and BRN2−/− GEs, showing that COUP-TFII was specifically expressed in BRN2+/+ CGE/LGE, whereas it was obviously activated in the whole BRN2−/− GEs (including both LGE/CGE and MGE). The sections of BRN2+/+ and BRN2−/− were from equivalent coronal levels (details are in Materials and Methods). (C) Visualization of major classes of interneuron and interneuron progenitor (iNP) by UMAP analysis. (D) Developmental trajectory of interneurons and iNPs showing that BRN2 loss changed interneuron development trajectory. Cells in cluster 3 were markedly decreased in BRN2−/− monkeys. (E) DEGs between BRN2+/+ and BRN2−/− interneurons and iNPs (table S4). Representative transcription factors (TFs), GO terms, and KEGG pathways are shown. (F and G) The sections of BRN2+/+ and BRN2−/− telencephalon were from equivalent coronal levels. (F and G) Representative staining images of HOPX and SOX1 in BRN2+/+ (F) and BRN2−/− (G) GEs. F1 and G1 are magnifications of the squares in (F) and (G). (H) PAX6 and SOX2 expression in BRN2+/+ and BRN2−/− GEs. (I) SST and NKX2-1 expression in BRN2+/+ and BRN2−/− GEs, respectively. (J) CALRETININ (CR) expression in BRN2+/+ and BRN2−/− telencephalon. Arrows indicate the migration orientations of CR interneurons. All images of immunofluorescence were from coronal sections spanning the rostral-caudal extent of the telencephalon. Scale bars, 500 μm. Blue, DAPI, nuclear staining.

Fig 4: BRN2 deletion results in abnormal development of monkey cortex.(A to D) The coronal sections from the same region of primary somatosensory cortices were used to perform comparison analysis between E49 BRN2+/+ and BRN2−/− telencephalon (see Materials and Methods). (A) SOX2 and PAX6 expressions in the BRN2−/− and BRN2+/+ cortex, respectively. (B and B′) Representative staining images (B) and quantification (B′) of PVI, KI67, and DAPI in BRN2−/− and BRN2+/+ cortical cells. DAPI staining was used to identify cell division orientation. Quantification data of division orientations of RGCs are presented as means ± SEM (n = 6 representative images; >100 cells per group were counted, *P < 0.01). (C) Representative staining images of TUJ1 and SOX1 in BRN2−/− and BRN2+/+ cortical cells, respectively. (D and D′) HOPX and SOX1 expressions in the BRN2−/− and BRN2+/+ cortex, respectively. Right images are higher magnifications of left images. (D′) Quantification of HOPX cell migration into the SVZ region from VZ region (means ± SEM, n = 4 representative images; >500 cells per group were counted, *P < 0.01). Scale bars, 100 μm. (E and F) Visualization of major classes of neural cells from BRN2+/+ and BRN2−/− single cells across three different stages by Uniform Manifold Approximation and Projection (UMAP) analysis. (G) Plots of classical markers representing RGC (SOX2), intermediate progenitor (IP) (EOMES), and excitatory neuron (ExN) (STMN2), respectively. (H) Proportion dynamic of each cell type over cortical development. (I) Developmental trajectory of cortical cells across three different stages. RG-IP-N (RGC-IP-to-neuron) is the indirect way, while RG-N (RGC-to-neuron) is the direct way. The dotted lines show developmental trajectories of RGCs. The arrowheads show the directions of these trajectories. (J) DEGs between the BRN2−/− and BRN2+/+ cortex (see table S3). Representative transcription factors (TFs), gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are shown. Blue, DAPI, nuclear staining.

Fig 5: Syngap1 levels do not affect the number of neurons and glia cells generated in mice.a, a’, CTIP2 (red) and BRN2 (green) immunolabeling in the somatosensory cortex of Control (CT), heterozygous (+/−) and homozygous (−/−) Syngap1KO mice at P0 (n=5 CT, n=4 −/− and +/− mice; two-sided unpaired t-test: CTIP2 - CT vs −/− p=0.1967; CT vs +/− p=0.0870; BRN2 - CT vs −/− p=0.7649; CT vs +/− p=0.6772). Arrowheads highlight migration defects for BRN2-positive cells. b, b’, SOX9 (red) immunolabeling in the somatosensory cortex of Control (CT), heterozygous (+/−) and homozygous (−/−) Syngap1KO mice at P0 (n=3 CT, n=4 −/− and +/− mice; two-sided unpaired t-test: CT vs −/− p=0.8901; CT vs +/− p=0.5368). c, c’, CTIP2 (red) and CUX1 (grey) immunolabeling in the somatosensory cortex of Control (CT) and Syngap1+/− mice at P13 (n=4 mice/genotype; two-sided unpaired t-test: CTIP2 - CT vs +/− p=0.9317; CUX1 - CT vs +/− p=0.8127). d, d’, EdU (grey) and CUX1 or SOX9 (red) immunolabeling at P13 in the somatosensory cortex of Control (CT) and Syngap1+/− mice after EdU injection at E18.5 (n=4 mice/genotype; two-sided unpaired t-test: EdU+CUX1+ CT vs +/− p=0.7904; EdU+SOX9+ CT vs +/− p=0.0690). DL, Deep Layer; UL, Upper Layer; VZ, Ventricular Zone; WM, White Matter. Values are mean ± SEM; ns, not significant. Scale bars: 50 μm (P0), 100 μm (P13).