Temporal control of progenitor competence shapes maturation in GABAergic neuron development in mice

Abstract

Diverse types of GABAergic projection neuron and interneurons of the telencephalon derive from progenitors in a ventral germinal zone called the ganglionic eminence. Using single-cell transcriptomics, chromatin accessibility profiling, lineage tracing, birthdating, transplantation across developmental stages and perturbation sequencing in mouse embryos, we investigated how progenitor competence influences the maturation and differentiation of these neurons. We found that the temporal progression of neurogenesis shapes maturation competence in ganglionic eminence progenitors, influencing how their progeny progress toward mature states. By contrast, differentiation competence-defined as the ability of progenitors to produce diverse transcriptomic identities-was maintained throughout neurogenesis. Chromatin remodeling, together with a regulatory module composed of the transcription factor NFIB and its target genes, influenced maturation competence in late-born neurons. These findings reveal how transcriptional programs and chromatin accessibility govern neuronal maturation and the diversification of GABAergic neuron subtypes during neurodevelopment.

Fig. 1. Stable differentiation competence in progenitors of GABAergic neurons.

a, Schematic illustrating the difference between maturation and differentiation. b, Summary of methods used to investigate the competence of progenitors located in the GE. c, UMAP plot showing single cells derived from scRNA-seq, TrackerSeq and FT datasets aligned in Monocle3 (n = 41,460 cells; n = 40 embryos). Colors correspond to dataset type. d, UMAP plot showing single cells derived from scRNA-seq, TrackerSeq and FT datasets, with each color representing a different cluster. e, UMAP plot showing scRNA-seq datasets (n = 25,297 cells; n = 20 embryos), with colors indicating various collection stages. f, UMAP plot showing single cells from ventral (GABAergic lineage) and dorsal (glutamatergic lineage) telencephalon (n = 75,431 cells), with colors indicating various collection stages. g, Pearson’s correlation plot between dorsal and ventral progenitors at different developmental stages (*P < 0.05, **P < 0.01; two-sided t-test). h, Lineplot showing relative cell number of dorsal (left) and ventral (right) postmitotic neuronal states across stages. The annotation of dorsal cell states is derived from the original publication. i, Schematic illustrating whole-cell recording for resting membrane potential. j, Boxplots showing membrane potential in cortical and GE progenitors at e13.5 and e15.5; significance was tested using two-sided, unpaired t-tests (Cortex, n = 14; GE, n = 37) with false discovery rate correction. The median and 25th and 75th percentiles are represented as middle line and border lines of boxplots, with whiskers indicating the minimum and maximum value. k, UMAP plot showing TrackerSeq barcoded cells (n = 9,938), each color representing a stage of IUE; IUE at e12.5 and e16.5; scRNA-seq after 96 h. l, UMAP plot showing cell states at the branches used for clone grouping. m, Upset plot showing clonal intersections in TrackerSeq_e12.5+96h. n, Upset plot showing clonal intersections in TrackerSeq_e16.5+96h. o, Barplot showing the frequency of dispersing and nondispersing clones in TrackerSeq_e12.5+96h and TrackerSeq_e16.5+96h. IN, interneuron; MT, mitotic; PN, projection neuron. Source data

Fig. 2. Timing of neurogenesis influences maturation competence.

a, UMAP plot showing FT datasets (n = 6,225 cells; n = 19 embryos) colored by injection and collection stage; injection at e12.5 and e16.5, scRNA-seq after 6 h or 96 h. b, Barplot showing relative cell number of postmitotic neuronal states in FT_{e12.5 + 6h}, FT_{e16.5 + 6h} and FT_{e12.5 + 96h}. c, Violin plots showing the distribution of FT⁺ cells along the combined pseudotime trajectory, displayed for each condition; two-sided, unpaired Wilcoxon rank-sum test (****P < 0.0001, (P = 3.46 × 10⁻⁸², 2.87 × 10⁻²⁵⁵, 1.06 × 10⁻¹⁰⁷), n = 2,000). The central point within the plot represents the median, hinges represent 25th and 75th percentile and whiskers show hinges ± 1.5 × interquartile range. d, Volcano plot displaying differential gene expression in postmitotic cells of FT_e12.5+6h and FT_e16.5+6h; |log₂FC| > 1, Bonferroni-adjusted P < 0.05 using two-sided Wilcoxon rank-sum test. e, Heatmap showing average scaled expression of differential genes in FT_e12.5+6h and FT_e16.5+6h postmitotic cells; visualized in all FT⁺ conditions. f, UMAP plot showing scATAC-seq datasets (n = 23,647 cells; n = 19 embryos); FT injection at e12.5 and e16.5, followed by scATAC-seq after 6 h. g, Coverage plot displaying scATAC-seq and H3K4me1 signal intensity for peak categories. The x axis is relative position (basepairs) and the y axis shows average counts per million. h, Heatmap displaying the accessibility of CREs across pseudotime for FT_{e12.5 + 6h} and FT_{e16.5 + 6h}. Peaks are divided into ‘early’, ‘intermediate’ and ‘late’ based on accessibility profiles along pseudotime bins. Overlapping peaks are annotated in gray and unique peaks are annotated by stage-specific colors. Gray, overlapping motifs; blue, unique motifs. i, Volcano plot displaying −log₁₀(P value) (y axis) and differential binding score (x axis) of TFs. P values were calculated using the subsampling procedure as proposed in ref. . Each dot represents a motif. j, Aggregate footprint profiles of NFIB in FT_e12.5+6h and FT_e16.5+6h. k, Coverage plot showing chromatin accessibility dynamics at NFIB footprint sites for FT_e12.5+6h and FT_e16.5+6h datasets. Source data

Fig. 3. Nfib regulates a shift in gene regulatory programs.

a, An eGRN graph displaying positive interactions between TFs active in APs. Node color indicates enrichment score by stage and node size indicates the number of direct targets per TF. Select TFs are annotated. Direct interactions originating from NFIB are highlighted. b, An eGRN subgraph highlighting downstream targets of Nfib, Tcf4 and Meis2 at e16.5. Nfib, Tcf4 and Meis2 nodes are indicated by node shape. Interactions between Nfib, Tcf4 and Meis2 are highlighted. Node color reflects the enrichment score by stage. c, Heatmap displaying signal enrichment of NFIB peaks across datasets: NFIB and H3K4me3 CUT&RUN at e16.5 GE, and scATAC-seq at e12.5 and e16.5. d, Heatmap displaying signal enrichment of e12.5- and e16.5-enriched peaks across datasets: NFIB and H3K4me3 CUT&RUN at e16.5 GE, and scATAC-seq at e12.5 and e16.5. e, Genome browser tracks of putative enhancer regions for Tcf4 and Meis2 and gene loci for Dlx2 and Dlx5, featuring NFIB CUT&RUN and scATAC-seq at e12.5 and e16.5. f, Enriched TF motifs in NFIB CUT&RUN peaks. TFs are ordered by their P value (binomial test). For each TF, the motif logo, target- and background percentage and the resulting enrichment are shown. The dendrogram on the left shows the sequence similarity of motif logos. Source data

Fig. 4. Intrinsic and extrinsic factors regulating progenitor competence.

a, Schematic overview of donor and host stages for homo- and heterochronic transplantation experiments. b, Distribution of transplanted cells along pseudotime in AP_{e12.5 → e12.5} and AP_{e12.5 → e16.5}; two-sided Wilcoxon rank-sum test (****P < 0.0001, P = 6.02 × 10⁻¹⁴; n = 1,000). Boxplots indicate median as point, 25th and 75th percentiles as hinges and hinges ± 1.5 × interquartile range as whiskers. c, Differentially expressed genes between AP_{e12.5 → e12.5} and AP_{e12.5 → e16.5}; 1 < log₂FC < −1; Benjamini–Hochberg corrected P < 0.05 (two-sided Wald test). Only genes downstream of NFIB, MEIS2 and TCF4 are labeled. d, UMAP embedding of cells collected in Nfib/x knockout (KO) (n = 47,079 cells; n = 10 embryos). Cells are annotated by broad cell state and the cluster’s top two marker genes. e, UMAP embedding of subsetted inhibitory neuron precursors and their progenitors in Nfib/x KO (n = 32,783). Cells are colored by inferred pseudotime scores. f, Cells from Nfib overexpression (OE) shown in UMAP embedding (n = 30,019 cells; n = 7 embryos). Cell labels were predicted using label transfer. Cells with low prediction score are labeled as not assigned. g, UMAP embedding of cells in Nfib OE. Cells are colored by inferred pseudotime scores. h, Proportion change per cluster in Nfib/x KO. For each biological replicate, the fraction of cells containing sgNfib/x was compared to the fraction of cells containing sgLacZ. i, Proportion change per predicted label in Nfib OE. For each biological replicate, the fraction of cells containing Nfib-GFP plasmid was compared to the fraction of cells containing eGFP control plasmid. j, Distribution of pseudotime scores between conditions across broad cell states in Nfib/x KO (top) and Nfib OE (bottom). Dot shows median of corresponding distribution. Two-sided Wilcoxon rank-sum test, ***P < 0.001, **P < 0.01, *P < 0.05, (P = 1.64 × 10⁻⁰⁸, 2.75 × 10⁻¹¹, 0.02) for Nfib/x KO AP, Nfib OE AP and BP, Nfib/x KO, n = 2,205/5,592/5,350/12,768 for AP/BP/IN/iPN; Nfib OE, n = 2,025/1,452/545/2,354 for AP/BP/IN/iPN. k, Change in gene expression upon perturbation for selected genes. Average gene expression was calculated per cluster and condition. Expression change was calculated by dividing average expression in cells containing sgNfib/x by sgLacZ (for Nfib/x KO) or by dividing cells containing Nfib-GFP plasmid by control plasmid (for Nfib OE). Rows are annotated by broad cell state and experiment, columns are annotated by gene list. Stars indicate differential expression which was inferred using Seurat’s FindMarker-function with default parameters (two-sided Wilcoxon rank-sum test); *P < 0.01 (Bonferroni-adjusted). ePN, excitatory projection neuron precursor; IN, interneuron precursor; iPN, inhibitory projection neuron precursor. Source data

Extended Data Fig. 1. Experimental workflow and dataset characteristics.

a, Table with dataset type, collection stage, number of replicates, and number of collected embryos per replicate. b, Violin plot illustrating the number of genes per cell, counts per cell and mitochondrial gene fraction per cell for each replicate. c, Schematic representation of the experimental procedure to generate the scRNAseq datasets. d, Schematic representation of the experimental procedure to generate the TrackerSeq datasets. e, Schematic representation of the experimental procedure to generate FlashTag datasets. f, FT⁺ cells, injected with CFSE. Injection at e12.5 and e16.5, collection after six hours; coronal sections of ganglionic eminences (GE) (n=3). g, FACS plot showing high-intensity FT⁺ cells. h, Coronal sections of the GE at e12.5 (left) and e16.5 (right). Cells are labelled with CFSE (in green), RNAscope hybridization probes for Ascl1 (in yellow), and for Gad2 (in red) (n=1). Source data

Extended Data Fig. 2. Cell state characterization in the GABAergic lineage.

a, Heatmap of top five differentially expressed genes in GABAergic cell clusters. b, UMAP plot of combined datasets with cells colored by cluster identity. Clusters are annotated by one or two top marker genes. c, UMAP plot of combined datasets with inferred Monocle 3 trajectory. Cells are colored by pseudotime. d, Expression of marker genes in the combined dataset. Nes and Fabp7 label APs, Ascl1 and Ccnd2 are markers for BPs. Post-mitotic inhibitory neurons express Gad2 and Dlx6os1. Meis2 labels PNs and Tcf4 labels INs. e, UMAP plot of combined datasets, with cells colored by broad cell states. f, UMAP plot of combined datasets colored by cell cycle phases. Source data

Extended Data Fig. 3. Comparison of early- and late-born cohorts.

a, Barplot showing the cell ratio in different cell-cycle states for the FT_{e12.5 + 6h}, FT_{e16.5 + 6h}, and FT_{e12.5 + 96h} datasets. b, Post-mitotic cells of FT_{e12.5 + 6h}, FT_{e16.5 + 6h}, and FT_{e12.5 + 96h} highlighted in the UMAP-embedding of the merged dataset. Color indicates whether cells are part of branch-clusters or in post-mitotic trunk. Comparison of isochronic cohorts displayed side by side shows that cells from the FT_{e12.5 + 6h} cohort predominantly occupy intermediate positions, indicating progression toward the branch tip (Snhg11), whereas cells from FT_{e16.5 + 6h} and FT_{e12.5 + 96h} cohorts have reached the branch tips. c, Heatmap of marker gene expression for branch tips split by isochronic cohorts demonstrates that low-abundance states exhibit gene-expression profiles consistent with other cells at the branch tips, supporting their correct classification. d, Detailed heatmap of differentially expressed genes between FT_{e12.5 + 6h} and FT_{e16.5 + 6h}; 1<log2FC<–1, Bonferroni-adjusted P<0.05 (two-sided Wilcoxon rank-sum test). Expression is visualized in FT_{e12.5 + 6h}, FT_{e16.5 + 6h} and FT_{e12.5 + 96h} datasets. e, Venn diagram showing the intersection of FT_{e12.5 + 6h}, FT_{e16.5 + 6h}, and FT_{e12.5 + 96h} marker genes; FC > 0.25, Bonferroni-adjusted P < 0.05 (two-sided Wilcoxon rank-sum test). Source data

Extended Data Fig. 4. Transcription factor activity and chromatin remodeling in early and late cohorts.

a, Overview of FT⁺ scATAC-seq datasets: collection stage, number of replicates, and number of embryos per replicate. b, Schematic representation of the experimental procedure to generate FT⁺ scATAC-seq datasets. c, UMAP depiction of gene body accessibility for marker genes. Fabp7 for APs, Ccnd2 for BPs, Dlx5 and Gad2 for postmitotic inhibitory neurons, Maf for INs and Ebf1 for PNs. d, UMAP-embedding of cells in FT⁺ scATAC-seq datasets. Cells are grouped and colored by broad cell state and stage. e, Barplot quantifying peak types (distal, exonic, intronic, and promoter) at e12.5, e16.5, and overlapping sites. f, Volcano plot displaying differentially accessible peaks between stages, with x-axis showing fold-change and y-axis showing –log10(p-value). Two-sided Wilcoxon rank-sum test using FDR-correction. Significant TFs are highlighted (P <= 0.1 and 1<log2FC<–1. g, Coverage plots of e12.5-enriched sites (top) and e16.5-enriched sites (bottom). Aggregated coverage was calculated for each stage separately. h, Aggregate footprint profiles of select transcription factors in FT_{e12.5 + 6h} and FT_{e16.5 + 6h}. Source data

Extended Data Fig. 5. Transcription factor interaction analysis in subnetworks.

Subnetwork for APs (a), BPs (b) and precursors (c). Each subnetwork is merged across e12.5 and e16.5, with node color indicating the difference in expression between stages. The size of nodes reflects the number of downstream targets per TF. Subnetworks show only interaction between TFs. d, Dot plot showing cosine score on the y-axis and TF pairs on the x-axis. Color and size indicate the stage and the number of occurrences of TF1-TF2, respectively. e, Genomic view of transcription factor binding sites for NFIB-MEIS2 and NFIB-TCF4. f, TF interaction network of TFs that regulate genes dynamic along the maturation trajectory in e12.5. Nodes are colored by their average expression at e12.5. g, TF interaction network showing transcription factors (TFs) regulating genes with dynamic expression along the maturation trajectory at e16.5. Nodes are colored based on their average expression levels at e16.5. h, Number of bound genes per TF (out-degree) at e12.5 and e16.5; subsetted for genes dynamic along the maturation trajectory and their upstream TFs. TFs with an out-degree higher or equal than eight are labelled. Source data

Extended Data Fig. 6. Homo- and hetero-chronic transplantation datasets.

a, Overview table of homo- and hetero-chronic transplantation experiment datasets: donor, host, and collection stages, number of replicates and number of embryos for each condition. b, Schematic representation of the experimental procedure for AP labeling and homo- and heterochronic transplantation. c, Images of coronal brain sections after FT⁺ APs transplantation. APs labelled with CFSE; GE, ganglionic eminence (n=4). d, UMAP plot of the combined dataset utilized for cluster reference. e, Predicted cell state composition in each replicate. f, Distribution of transplanted cells along pseudotime in AP_{e16.5 → e16.5} and AP_{e16.5 → e12.5}; two-sided Wilcoxon rank sum test (**** adjusted P<0.0001, P = 4.19^*10⁻¹³, n=1000). Median is indicated by points, 25th and 75th quantiles are inidcated by hinges. Whiskers show hinges +/- 1.5*inter-quartile range. g, Differentially expressed genes between AP_{e16.5 → e16.5} and AP_{e16.5 → e12.5}; 1<log2FC<–1, Benjamini-Hochberg adjusted P<0.05 (two-sided Wald test). Only genes downstream of NFIB, MEIS2 and TCF4 are labelled. Source data

Extended Data Fig. 7. Experimental perturbation of Nfib and associated phenotype.

a, Design of sgRNAs for tCROP-seq experiments and overview of experimental procedure. b, Design of plasmids for Nfib OE experiments and overview of experimental procedure. c, Western blot showing increased expression of exogenous NFIB in Neuro2A-cells (n=1). d, UMAP embedding of inhibitory precursors and their progenitors in Nfib/x KO. Cells are colored by clusters, which are annotated based on broad cell states and the top two marker genes; mt: mitotic; IN: interneuron precursor; iPN: inhibitory projection neuron precursor; NA: not assigned. e, Proportion change in Nfib/x KO. Cells were grouped into broad cell states, by aggregating clusters. f, Proportion change in Nfib OE. Cells were grouped into broad cell states by aggregating predicted labels. g, Number of differentially expressed (DE) genes per cluster between conditions in Nfib/x KO. Color indicates positive or negative enrichment. h, Number of DE genes per cluster between conditions in Nfib OE. Color indicates positive or negative enrichment. Source data