Fig 2: miR-34a shares similar targets with miR-449a, and they can induce each other mutually(A) Schematic depiction of miR-34a and miR-449a and matching targets, namely, SATB2, HDAC1, and Sirt1. (B) SATB2, HDAC1, Sirt1 and DNMT3A protein levels after treatment of HCT116 cells with 100 nmol/L miR-NC or miR-34a mimics. (C) Luciferase activity of the SATB2, HDAC1, and Sirt1 3'UTR reporters in 293T cells transfected with 100 nmol/L miR-NC or miR-34a mimics and 200 ng of reporter plasmid for 24 hours. (D) miR-34a mRNA levels after transfection with siSirt1, siHDAC1, siDNMT3A or siSATB2. (E) miR-449a mRNA levels after treatment of HCT116 cells with 100 nmol/L miR-NC or miR-34a mimics. (F) miR-34a mRNA levels after treatment of HCT116 cells with 100 nmol/L miR-NC or miR-449a mimics.

Fig 3: Gene activity scores correlate with cell type-specific expression of marker genes.a, UMAP projections of all primary scATAC-seq cells coloured by gene activity score. From top left to bottom right: NKX2.1 (also known as NKX2-1) marking MGE cells, AQP4 marking glia/astrocytes, TBR1 marking excitatory neurons, FEZF2 marking deep layer excitatory neurons, HES1 marking radial glia, HOPX marking outer radial glia, SATB2 marking upper layer excitatory neurons, CCL4 marking microglia, CRYAB marking truncated radial glia, LHX6 marking MGE-derived interneurons, OLIG1 marking oligodendrocyte precursors, and SOX2 marking radial glia. b, Schematic of the CellWalker algorithm17 used to assign cell-type labels to scATAC-seq cells based on integration with scRNA-seq data. c, UMAP projection of cell-type labels assigned by CellWalker. d, UMAP projection of radial glia with cell-type assignments from CellWalker. e, UMAP projections of gene activity scores for PAX6 and GLI3, genes that are ubiquitously expressed in radial glia; HOPX, an oRG-specific gene2; and CRYAB, a tRG-specific gene2. f, UMAP projection of Z-scores of enrichment of oRG- and tRG-specific peaks (Fisher’s exact, two-sided test, P < 0.05). g, UMAP projections of Z-scores of enrichment of area-specific peaks for each area in all primary scATAC-seq cells (Fisher’s exact, two-sided test, P < 0.05).

Fig 4: Mosaic deletion of Foxg1 during early development revealed that FoxG1 cell-autonomously repressed the granule cell fates. (A,B) Mosaic deletion of Foxg1 in the cortical plate (B, arrows), while the adjacent cells were FoxG1+ (B, arrowheads) after an injection of a low dose of TM at E10.5 and analysis at E18.5. (C–D’) CalR expression was confined to Foxg1-deleted cells (arrows), while adjacent FoxG1-positive cells did not co-express CalR (arrowheads).((E–F’) Only Foxg1-deleted cells expressed Prox1 (arrows), while adjacent FoxG1-positive cells did not co-express Prox1 (arrowheads). (G–J’) Normal progenitors, but not adjacent Foxg1-deficient cells, developed into Foxp2+(G–H’) or Satb2+(I–J’) cortical excitatory neurons. (K,L) Double immunostaining for Prox1 with Pax6 after the injection of a low dose of TM at E10.5 and analysis at E14.5. (M–N”) High magnification images of the boxed regions in (K,L), respectively. In the mutants (N–N”), Prox1+ cells exhibited a mosaic distribution pattern. (M–N”’)Foxg1-deficient cells exhibited strong staining for Prox1, indicating that they were postmitotic granule cells located in the upper region of the putative cortical plate. Foxg1-deficient Pax6+ progenitors in the VZ/SVZ expressed Prox1 at low levels (N”’, arrowheads). (M”’,N”’) Show high magnification images of the boxed regions in (M”,N”), respectively. (O,P) Mosaic deletion of Foxg1 was accompanied by a mosaic expression pattern of Lef1. Red arrowheads: Foxg1-deleted regions; black arrows: Foxg1-expressing regions. (Q–R”) Double immunostaining showed that a few of progenitors were Foxg1-ablated cells that expressed Prox1 in the mutant dorsal-lateral cortex after the injection of a lower dose of TM at E10.5 and analysis at E14.5. (Q”,R”) Show high magnification images of the boxed regions in (Q’,R’), respectively. (S–T”) Double immunostaining for Prox1 and Pax6 showed that Foxg1-ablated Prox1+ cells co-expressed Pax6+ in the dorsal-lateral cortical VZ/SVZ (T”, arrowheads). (S”,T”) Show high magnification images of the boxed regions in (S’,T’), respectively. (U–V”) Double immunostaining for FoxG1 and Prox1 showed that Foxg1-ablated cells expressed Prox1 in the mutant dorsal-lateral cortex after the injection of a low dose of TM at E12.5 and analysis at E14.5. (S”,T”) show high magnification images of the boxed regions in (S’,T’), respectively. (W–X”) Double immunostaining showed that mosaically generated Prox1+ cells co-expressed Pax6+ in the dorsal-lateral cortical VZ/SVZ (X”, arrowheads). (W”,X”) Show high magnification images of the boxed regions in (W’,X’), respectively. Scale bars: (M”’–N”’,O,P,Q”–R”,S”–T”,U”–V”,W”–X”): 50 µm; (M–N”,Q–R’,S–T’,U–V’,W–X’): 100 µm; (A–L): 200 µm. )

Fig 5: Single-cell RNA sequencing of E18.5 neocortices from WT and Tcf4KO mice. (A) Dot plot of cell clusters (y-axis) and a selection of markers used to assign the cell type (x-axis). (B) tSNE plot colored by genotype (left) and cluster identity (right). (C) Right: tSNE plot of Satb2-expressing glutamatergic cells used for further analysis. Left: Violin plot of Satb2 expression in the Satb2 cluster. The red line depicts the median. (D) Selection of the first 50 GO terms associated with up- and downregulated genes in Satb2-expressing glutamatergic cells. GO terms for neurogenesis, neuronal differentiation and axon development were downregulated in the Tcf4KO cells. (E) Violin plots of DEGs in the Satb2 cluster that are associated with neurogenesis and neuron and axon development. The red line depicts the median.