Fig 2: In Vivo Validation of Markers of Tri-IPCs and OB-IPCs(A and B) The expression of GSX2, OLIG2, and DLX2 in the cortical VZ/SVZ of wild-type (A) and ShhN-IUE (B) mice at E17. Note that very few GSX2+ cells (green) were present in the cortical SVZ. Arrows indicate GSX2+OLIG2+DLX2 tri-IPCs, and arrowheads indicate GSX2+DLX2+ OB-IPCs.(C and D) The expression of SP9 and SP8 in the cortical VZ/SVZ of wild-type (C) and ShhN-IUE (D) mice at E17.(E and F) More OB interneuron lineage cells (E) and more tri-IPCs and OB-IPCs (F) were observed in the ShhN-IUE cortices than in the controls.(G) The sequential expression of GSX2/DLX2/SP9/SP8 is linked to lineage differentiation from tri-IPCs/OB-IPCs/OB neuroblasts, indicating the core transcriptional network for OB interneuron generation.Data are presented as means ± SEM; n = 3 mice for each condition. ***p < 0.001, **p < 0.01, *p < 0.05; Student’s t test in (E) and (F). Scale bars, 50 mm in (A)–(D) and (G).

Fig 3: Glutamatergic thalamic cell lineages regionalize the thalamus into distinct molecular domains(A) UMAP representation and RNA velocity field of the single-cell RNA-seq data of the glutamatergic cell lineage in control embryos at each developmental stage. For reference, the same UMAP is also colored by the inferred cell differentiation pseudo-time and the gene expression levels of Sox2, Foxp2, and Cadm1. The expression of Sox2 and Foxp2 genes separates differentiation trajectories into Sox2+ Foxp2− (cTh.N1) and Sox2− Foxp2+ (cTh.N2) populations.(B) Proportion of cells belonging to different glutamatergic thalamic nuclei at each developmental stage. Distinct thalamic subpopulations emerge at different time points. Late- and intermediate-emerging cell subpopulations are indicated by double asterisk (**) and single asterisk (*), respectively.(C) Whole-mount RNA in situ hybridization for Sox2, Foxp2, and Cadm1 in sagittal sections of the E13.5 diencephalon. The expression of these markers regionalizes the developing thalamus into distinct domains. Image credit: Allen Institute.(D) Glutamatergic thalamic IPCs are a heterogeneous population of cells with contributions from both cTh.Pro1 and cTh.Pro2 progenitors. The UMAP representation of the single-cell RNA-seq data corresponding to the glutamatergic thalamic IPC population from E14.5 control embryos is colored by the inferred cell differentiation pseudo-time and the gene expression levels of Olig2, Dbx1, Foxp2, and Sox2. CPT, counts per thousand.See Figure S5; Tables S4 and S5.

Fig 4: Genetic Ablation of Dorsally Derived, Emx1+ Cells Leads to a Reduction in the Number of CC1+ Cells at 21 dpl(A–C) Ventral lesioned white matter showing expression of Olig2, GFP and the nuclear marker Hoechst dye (Hst) (A), GFP alone (B), and Olig2 alone (C). The percentage of Olig2+ cells (C) co-expressing GFP (B) (merged in A) was found to be 95.9% ± 2.7%, confirming efficient transgene expression in Emx1-Cre/Sox10-GFP-DTA corpus callosum at 10 dpl (lesion demarcated by the white dashed line).(D–F) Ventral non-lesioned white matter showing expression of GFP, TdTom, and Hst (D) and TdTom alone (E) in triple transgenic Emx1-Cre/Sox10-DTA/Sox10-GFP-TdTom mice, and expression of GFP, TdTom, and Hst in control, non-ablated white matter (F), revealing that the DTA ablation strategy deleted the majority of Emx1+ cells from the CC. The scale bar represents 100 μm.(G) There was no significant difference between the number of GFP+ cells in ablated and control mice at any of the time points studied.(H) No significant difference was detected in the number of Olig2+ GFP+ cells at any time point.(I) No significant difference was detected in the number of Ki67+ GFP+ cells at any time point.(J) There was a significant reduction in the CC1+ cell number at 21 dpl in the ablated mice compared with controls (p = 0.007 and Student’s t test). The data are presented as mean ± SEM (n = 3). The scale bars represent 100 μm.

Fig 5: SHH Regulates the Production of OB Interneurons and Oligodendrocytes in the Cortical SVZ Predominately by Reducing GLI3(A–D) Immunostainings for GSX2, SP8, EOMES, and OLIG2 in wild-type (control) (A), Smo cko (B), Gli3 cko (C), and Smo Gli3 dcko (D) mice at P0.(E) Quantification for the numbers of GSX2+, SP8+, EOMES+, and OLIG2+ cells per 300 mm width in the cortical VZ-SVZ of control and mutant mice at P0. Data are presented as means ± SEM; n = 3 mice per genotype. *p < 0.05; unpaired Student’s t test in (E). Scale bars, 200 mm in (D).