Figures and data
Differentiation of the E12.5 mouse rV2 GABAergic and glutamatergic neurons based on transcriptome and chromatin accessibility
A. UMAP projection of rV2 lineage cells based on scRNA profiles.
B. Pseudotime (VIA) scores shown on the scRNA UMAP projection.
C. Expression of marker genes of progenitors and postmitotic precursors of GABAergic and glutamatergic neurons shown on the scRNA UMAP projection.
D. UMAP projection of rV2 lineage cells based on scATAC profiles.
E. Pseudotime (VIA) scores shown on the scATAC UMAP projection.
F. Expression of the marker genes of progenitors and postmitotic precursors of GABAergic and glutamatergic neurons shown on the scATAC UMAP projection (interpolated expression values).
G. Heatmap of scATAC and scRNA cluster similarity scores.
Accessibility of chromatin features in the Tal1 and Vsx2 loci during GABAergic and glutamatergic development.
A. Chromatin accessibility per cell group (normalized signal), Ensembl gene track (Genes), scATACseq features (Feat.), feature linkage to gene (Links with the LinkPeaks abs(zscore) >2) and nucleotide conservation (Cons.) within +/-50 kbp region around Tal1 TSS. Violin plots on the right show the expression levels of Tal1 and Pdzk1ip1 per cell group.
B. Spline-smoothed z-score transformed heatmaps of chromatin accessibility at Tal1-linked scATACseq features (Tal1 cCREs) in the single cells of rV2 GABAergic and glutamatergic lineages with RNA expression (sliding window mean(width=6) smoothed) of Tal1, Gata2, Gad1 and Slc17a6 as column covariable (top). Cells on the x-axis are first grouped per cell group and then ordered by the pseudotime (bottom) within each group.
C. Same as in A, but for the Vsx2 locus.
D. Accessibility of Vsx2 cCREs in the rV2 GABAergic and glutamatergic lineages, shown as in (B). The expression levels of Vsx2, Tal1, Gad1, and Slc17a6 are shown above the heatmaps.
TF binding in putative regulatory elements of Tal1 and the overlap between TFs interacting with other GABAergic fate selector genes.
A. ATAC features within +/-50 kb of the Tal1 gene. Tal1 cCREs are shown in blue. CUT&Tag, CUT&Tag consensus peaks indicating Tal1, Gata2, Gata3, Vsx2, Ebf1, Insm1, and Tead2 binding in the E12.5 mouse r1.
B-D. Footprinting based TF binding activity in the features at +1 kb, +23 kb and +40 kb of the Tal1 TSS. Feature linkage p to Tal1 is indicated by asterisks. Footprint scores at conserved TFBSs are shown for progenitors (PRO1-2), common precursors (CO1-2), GABAergic precursors (GA1-2) and glutamatergic precursors (GL1-2). In each dotplot, the strength of footprint at TFBS in the feature is shown as colour (Footprint score, average of cell group) and the expression of the TF gene in dot size (log1p). Average feature accessibility in cell groups is shown at the right. TFBS names (Hocomoco v12) are shown at the top and the TF gene names (mouse) are shown at the bottom of the dotplot.
The red arrowhead in (B) indicates the conserved Gata2 TFBS at –37 bp position required for the neural expression of Tal1 (see also Supplementary Table 4).
E. Overlap of the TFs with footprints in the cCREs of Tal1, Gata2, and Gata3 in the common precursors of rV2 lineages (CO1-2) and in the rV2 GABAergic precursors (GA1-2). Venn diagrams show the number of TFs with a footprint detected in Tal1, Gata2, or Gata3 -linked features (Fp in cCRE = 1) and with their gene expression > 1.2 (log1p) in the analysed cell group. The TFs associated with the cCREs of all three selector genes in common precursors (CO1-2) and GABAergic precursors (GA1-2) are listed. Blue text: 19 TFs interact with cCREs of Tal1, Gata2 and Gata3 in the CO1-2 cells. Some of these TFs continue to be expressed and interact with the Tal1, Gata2 and Gata3 genes in the GA1-2 cells. 22 TFs interact with Tal1, Gata2 and Gata3 in GA1-2 cells. Green text, TFs found associated with two selector genes in CO1-2 (green in the Venn diagram of CO1-2) and associated with all three selector genes in the GA1-2. Black text, TFs co-regulating the selector TFs in GA1-2 and not expressed in the CO1-2 cells.
§ The probability of finding n overlapping genes considering all mouse genes equally is p<1e-6.
*,** The collective minimum statistical significance of feature to gene links for selector genes Tal1, Gata2 and Gata3 cCREs for the given TF is shown as: *p-value<0.05; **p-value<0.01 (with LinkPeaks z-score above 2 or below -2).
Expression patterns of TFs associated with Tal1 cCREs in the developing anterior brainstem.
A. RNAscope® In Situ Hybridization with Insm1, Sox4, and Tal1 RNA probes on paraffin sections of E12.5 wild-type mouse embryos. Transversal sections of r1 are shown. Dashed lines indicate the ventricular zone (VZ) border. Numbered boxes indicate areas shown on the close-up images in (B), numbering order is from apical to basal. Scale bar: 50 μm.
B. Close-up images showing co-expression of Insm1, Sox4, and Tal1 with DAPI.
C. Signal intensity of Insm1, Sox4, and Tal1 probes along the apical-basal axis. Average intensity line is based on three replicates. The 95% confidence interval of fluorescence intensity is highlighted in gray. The y-axis shows average fluorescence intensity per cell, and the x-axis shows the number of average cell diameters (μm) from the VZ.
D. Violin plots showing Insm1, Sox4, and Tal1 mRNA expression detected by single-cell RNA-seq in the rV2 lineage. Cell clusters are aligned by pseudotime order on x-axis, showing first the differentiated cell clusters of GABAergic lineage, and then glutamatergic lineage.
E. RNAscope® In Situ Hybridization with E2f1, Tead2, and Tal1 RNA probes, images shown and labelled as in (A). Transversal sections of E12.5 r1 are shown. Scale bar: 50 μm.
F. Close-up images showing co-expression of E2f1, Tead2, and Tal1 with DAPI in areas indicated in (E).
G. Signal intensity of E2f1, Tead2, and Tal1 probes along the apical-basal axis, with the lines showing the average of three replicates.
H. Violin plots showing E2f1, Tead2, and Tal1 mRNA expression in rV2 neuronal populations.
I. RNAscope® In Situ Hybridization with Ebf1, Tead2, and Tal1 RNA probes, images shown and labelled as in (A) and (E). Transversal sections of E12.5 r1 are shown. Scale bar: 50 μm.
J. Close-up images showing co-expression of Ebf1, Tead2, and Tal1 with DAPI in areas indicated in (I).
K. Signal intensity of Ebf1, Tead2, and Tal1 probes along the apical-basal axis, with the lines showing the average of three replicates.
L. Violin plots showing Ebf1, Tead2, and Tal1 mRNA expression in rV2 neuronal populations.
Dynamic accessibility of TF binding sites suggests a genome-wide function for Gata2, Tal1 and Gata3 in the selection of the GABAergic fate.
A. Genome-wide enrichment of TF binding sites in the accessible regions in GABAergic and glutamatergic lineages. Heatmap of chromVAR z-scores (avg(TFBS motifs) per TF) for the expressed TFs in rV2 cell groups. All TFs of which a TFBS-motif chromVAR score was among top 10 scores in any cell group are shown.
B. Violin plots of the TF gene expression (average scRNA expression) for Tal1, Gata2, and Gata3; and motif accessibility (chromVAR score) of Tal1, Gata2, and Gata3 TF-motifs in the rV2 cell clusters. HOCOMOCO v12 contains three Tal1 motifs, two Gata2 motifs and two Gata3 motifs. Ordering of cell groups is the same in all violin plots.
Genomic targets of the GABAergic selector TFs.
A. Schematic explaining the strategy of identifying the targets of Tal1, Gata2 and Gata3 selector TFs. Within the TAD containing a gene, a feature overlapping a CUT&Tag peak for the selector TF, and a footprint for the TF at a position with weighted mean conservation score > 0.5 are found. Genes linked to features fulfilling these conditions are considered target genes. For linkage, the Spearman correlation-based LinkPeaks score between the feature targeted by the selector TF and expression of the gene is required to be >2 (positive effect link) or <-2 (negative effect link) with a p-value <0.01.
B. Number of target genes and the overlap between Gata2, Gata3 and Tal1 target genes.
C. GSEA of Tal1 targets. Mouse genes are ranked by the difference in the expression in GA1-2 vs GL1-2 cell groups (log2 avg FC). Tal1 target genes are indicated with black lines. In leading edges, the GA1-2-enriched target genes are highlighted in blue and GL1-2-enriched genes with red. Scatterplots show the expression of the target genes in both edges. D-F. Characterization of Tal1 target genes.
D. Count of target features by the positive and negative effect link.
E. Count of target genes by the nearest linked feature, stratified by the nearest feature distance bins as indicated.
F. The variability of target gene expression in rV2 lineage cell clusters, stratified by the nearest feature distance bins.
G. Top terms in CellMarker gene set database using the list of Tal1 target genes with exp>0.5 (log1p) in GA1-2 or GL1-2 cell groups or both.
H. GSEA of Gata2 targets, as in (C).
I-L. Characterization of Gata2 target genes, as in (D-G).
M. GSEA of Gata3 targets, as in (C).
N-Q. Characterization of Gata3 target genes, as in (D-G).
R. Venn diagram of selector TF target genes that show specificity to rV2 glutamatergic neurons (defined as avg(exp in GA)<0.5 (log1p) AND avg(exp in GL)>0.5 (log1p), and see Methods). Genes in every overlap category are listed. Vsx2 target genes that show specificity to rV2 glutamatergic neurons (n=3) are separately listed.
S. Venn diagram of selector TF target genes that show specificity to rV2 GABAergic neurons (defined as avg(exp in GL)<0.5 (log1p) AND avg(exp in GA)>0.5 (log1p)). Genes in every overlap category are listed. Vsx2 target genes that show specificity to rV2 GABAergic neurons (n=2) are separately listed.
T. Proposed gene regulatory network guiding the GABA-vs glutamatergic fate selection in the rV2. Arrows represent positive regulation and were drawn when the regulator and the target expression were co-expressed in the same cell group. Blunt arrows represent negative regulation and were drawn when the regulator and the target expression were found in different cell groups (GA1-2 vs GL1-2) and not in the same cell group together.
E12.5 mouse rhombomere 1 single-cell RNAseq.
A. UMAP plot of scRNAseq clusters (left), and the expression level of Nkx6-1, Tal1, Slc17a6 and Gad1 RNA in the clusters (right).
B. The expression of GABA- and glutamatergic neuron markers in the UMAP of the ventrolateral r1 lineage after lineage subsetting (E12.5 rV2 scRNAseq clusters).
rV2 progenitors (rV2 pro), GABAergic neuron (rV2 GABA) and glutamatergic neuron branches (rV2 Glut) are labelled and indicated with arrows.
E12.5 mouse rhombomere 1 single-cell ATACseq.
A. UMAP plot of scATACseq clusters (left), and the inferred expression level of Nkx6-1, Tal1, Slc17a6 and Gad1 RNA in the clusters (right).
B. The expression of GABA- and glutamatergic neuron markers in the UMAP of the ventrolateral R1 lineage clusters after lineage subsetting (E12.5 rV2 scATACseq clusters). Arrows indicate the clusters of rV2 lineage progenitors (rV2 PRO), common precursors (rV2 CO), GABAergic neuron (rV2 GABA) and glutamatergic neuron branches (rV2 Glut).
RNAscope image segmentation and VZ distance calculation schematics
A. Typical dissection image with VZ visible on the upper right corner.
B. Same image with cells segmented with StarDist.
C. Schematics of VZ boundary distance -based ordering of the cells.
Comparison of the previously characterized enhancers of Tal1, Gata2, Gata3 and Vsx2 with the scATAC features identified in this study.
On the genomic loci of the genes, the previously characterised enhancers (details and references in the Supplementary Table 5) are indicated in colour. The features identified in this study are shown on the ATAC features (E12.5 R1) track. The gene-linked features are labelled in blue text.
Genomic features and feature accessibility at Gata2 and Gata3 gene loci.
A. Normalized scATACseq signal in +/-50 kb region of Gata2 (A) TSS, in the rV2 lineage single-cell clusters at top. Below, the Ensembl gene models (Genes), the linkage of features to Gata2 or Gata3 (Links), the scATAC features (Feat.; cCREs are shown in blue), and by-nucleotide conservation of DNA across vertebrate species (Cons.) is shown. Violin plots show the distribution of Gata2 (A) RNA expression (log1p) in single cell clusters.
B. Smoothed heatmaps of the accessibility of the Gata2 cCREs in the rV2 GABAergic and glutamatergic cell lineages (GABA, GLUT). Cluster identities are shown on top of heatmaps. Cells are ordered by the pseudotime value (Pseudotime). cCREs within the TAD bounds are shown on feature heatmaps. RNA expression (log1p) of Gata2, Vsx2, Gad1 and Slc17a6 is shown above the heatmaps.
C. scATACseq signal in the rV2 lineage single-cell clusters and genomic features in +/-50 kb region of Gata3 TSS, similar to (A). Violin plots show the Gata3 RNA expression (log1p) in single cells of rV2 cell clusters. Gata3 gene ATG is located +11 kb from the TSS, the position is indicated in Genes view.
D. Smoothed heatmaps of the accessibility of the Gata3 cCREs in the rV2 GABAergic and glutamatergic cell lineages (GABA, GLUT). RNA expression (log1p) of Gata3, Tal1, Gad1 and Slc17a6 is shown above the heatmaps.
Comparison of the previously characterized enhancers of Tal1, Gata2, Gata3 and Vsx2 with the scATAC features identified in this study.
A. Stacked bar chart showing the count (n) of scATAC features overlapping enhancers and enhancers overlapping scATAC features, separated by the linkage to selector gene.
B. Bar chart showing the percentage of gene-linked features from all features in gene TAD, the percentage of scATAC features overlapping enhancers and enhancers overlapping all scATAC features and gene-linked features.
TF signatures found by scATACseq-footprinting.
A. Heatmap of the TF footprint scores (as avg(TFBS footprint scores) per TF) over the cell groups. Y-axis has been hierarchically clustered with Euclidean distance and complete linkage.
Regulatory features of the glutamatergic fate selector Vsx2 and common regulators of Vsx2 and Vsx1.
A. Vsx2 gene with its protein-coding regions and associated chromatin features.
B. CUT&Tag. Consensus peaks of CUT&Tag signal with Gata2, Gata3, Tal1, Vsx2, Ebf1, Insm1 and Tead2 antibodies in E12.5 mouse r1.
C-E. TF binding activity in the Vsx2-associated features at +20.5 kb, -61 kb and –68.8 kb of Vsx2 TSS according to ATAC-footprinting analysis. In each feature, the strength of footprint (averaged over the cell group shown at the side) at TFBS in the feature is shown as colour and the expression of the TF gene in dot size. Feature accessibility in cell groups is shown at the right (Accessibility dotplot). TFBS names are shown at the top and the TF gene names (mouse genes) are shown under the dotplot.
F. Overlap of the TFs interacting with the cCREs of Vsx1 and Vsx2 in the common precursors of rV2 lineages (CO) and in the rV2 glutamatergic precursors (GL). Common regulators Vsx1 and Vsx2 in CO (n=18) and in GL (n=21) are listed next to venn diagrams. The 18 TFs regulating both Vsx1 and Vsx2 in CO cells are listed in blue text.
§ The probability of finding n overlapping genes considering all mouse genes equally is p<1e-6.
*,** The collective minimum statistical significance of feature-to-gene links for selector genes Vsx1 and Vsx2 cCREs for the given TF is shown as: *p-value<0.05; **p-value<0.01 (with LinkPeaks z-score above 2 or below -2).
CUT&Tag of Tal1 and Vsx2 -associated chromatin in E12.5 mouse R1, and positive and negative controls.
A. Left panel, Heatmaps and profile plots of the CUT&Tag signal intensity along the mouse genes in each replicate. Genes are scaled and the areas of 3 kb before transcription start site (TSS) and 3 kb after transcription end site (TES) are shown. Next to heatmaps, genome views of Tal1, Lmo1 and Gata2 gene loci showing the CUT&Tag signal and detected peaks in all replicates (Re1 - Re4) and consensus peaks (Cons peaks).
B. Genome views of the CUT&Tag signal with the anti-H3K4me3 (positive control) and the anti-IgG antibody (negative control, Re1-Re4). Consensus peaks are not shown. Only two consensus peaks were detected between the Re1-Re4 of IgG treated samples.
CUT&Tag of Gata2 and Gata3 -associated chromatin in E12.5 mouse R1.
Heatmaps and profile plots of the CUT&Tag signal intensity along the mouse genes (gene +/- 3kb) are shown for each replicate. Genome views of Tal1, Lmo1 and Gata2 gene loci show the CUT&Tag signal, the detected peaks in all replicates (Re1 - Re4) and consensus peaks (Cons peaks). Correlation heatmaps in the right panel show the CUT&Tag sample correlation by reads, and by peaks. Venn diagrams show the peak count and peak overlap between the replicates.
Overview of the CUT&Tag for the candidate regulators of Tal1 genes in E12.5 mouse R1.
CUT&Tag of Ebf1, Insm1 and Tead2 -associated chromatin in E12.5 mouse R1, and the signal from the negative control (IgG) and positive control (H3K4me3) samples. Heatmaps and profile plots show the CUT&Tag signal intensity along the mouse gwenes (gene +/- 3kb) in each replicate. Genome views show the CUT&Tag signal and the detected peaks in Tal1, Lmo1 and Gata2 (Ebf1, Insm1 CUT&Tag) or Vgll3 (Tead2 CUT&Tag) gene loci from all replicates (Re1 - Re2) and the defined consensus peaks (Cons peaks).
Correlation heatmaps show the CUT&Tag sample correlation by peaks. Venn diagrams show the peak count and peak overlap between the replicates.
