Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain
- Unit on Cellular and Molecular Neurodevelopment, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
- Bioinformatics and Scientific Programming Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
- Flow and Imaging Cytometry Core, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Figures
Figure 1 with 3 supplements
scRNAseq of distinct E12.5 forebrain regions from WT and Nes-dVenus mice.
(A) Coronal section through the LGE/MGE (left) and CGE (right) of a E12.5 Nes-dVenus telencephalon immunostained for GFP (green), Nestin (Red) and Dcx (Blue). Scale bar = 200 μm. (B) Schematic of telencephalic cell dissection and single-cell dissociation. (C) Uniform manifold approximation and projection (UMAP) plots of all cells labeled by brain region (left), mouse line (middle) or putative cell clusters (right). (D) Representative genes displaying enriched temporal and/or spatial expression patterns. CTX, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; CGE, caudal ganglionic eminence; WT, wild-type.
Figure 1—figure supplement 1
Flow cytometry plots depicting isolation of GFP-positive cells in Nes-dVenus mice.
(A) Flow cytometry gating strategy: gated for potential live cells, then gated for DRAQ5+/DAPI- live cells, then gated by DRAQ5 width vs. height to select singlets, then EGFP vs. DAPI to collect GFP+ live cells. (B) FACS plots depicting GFP-positive cells collected from the CTX, LGE, MGE, and CGE in E12.5 Nes-dVenus mice. (C) FACS plots depicting GFP-positive cells collected from the LGE, MGE, and CGE in E14.5 Nes-dVenus mice.
Figure 1—figure supplement 2
Quality-control of sequenced cells.
(A) Cells that had unique feature counts < 4500 or > 1500 were filtered out using Seurat (black horizontal line in top right plot approximates 1500 gene/cell cutoff). Mitochondrial counts over 0.05% and Hbb gene counts over 0.01% were also filtered out. (B) Scatterplots showing the total number of molecules detected versus the number of genes detected before and after the filtrations (top). Additional set of scatterplots showing the total number of molecules detected versus the percentage of mitochondrial genes before and after the filtrations (bottom).
Figure 1—figure supplement 3
Identification of region-specific genes from each telencephalic region at E12.5.
(A) UMAP plot of all cells annotated by brain region. (B) Heatmap showing expression of top 20 differentially expressed genes (DEGs) in each region. Each column represents averaged expression in cells, color-coded as per the color scale. (C) UMAP plots depicting the top 5 DEGs from each region. Cells are color-coded for levels of gene expression as per the color scales.
Figure 2 with 3 supplements
Common neurogenic lineages in each ganglionic eminence.
(A) UMAP plots displaying all GE-derived cells annotated from left to right by: brain region, putative cell clusters, mouse strain and expression of VZ marker Nestin and SVZ/MZ marker Dcx. (B) UMAP visualizations show transcriptional diversity in each GE region. Several genes are depicted to highlight distinct neurogenic stages: Nestin, Hes5, and Olig2 are indicative of VZ cells (green), Asc1 and Gadd45g label intermediate SVZ cells (blue), and Dcx, Sp9, and Mapt depict postmitotic neuronal precursors (red). (C) UMAP plots of MGE, LGE, and CGE cells annotated via putative cell clusters and including Slingshot analyses depicting developmental progression through neurogenic stages.
Figure 2—figure supplement 1
Differential gene expression between mouse lines and brain regions.
(A) Violin plots showing expression of pan-VZ-enriched genes from Nes-dVenus mice and expression of SVZ/early postmitotic genes enriched in cells from WT mice. (B–C) Violin plots depicting differential gene expression profiles of cells arising from the LGE (yellow), MGE (blue), and CGE (red). Highlighted genes include those that are predominantly expressed within one GE (B) or display intriguing expression profiles based on scRNAseq and/or in situ hybridization data (C).
Figure 2—figure supplement 2
Integration and visualization of multiple embryonic mouse scRNAseq datasets.
(A–C) Integration of 3 scRNAseq datasets of embryonic mouse ventral telencephalon: This study, E13.5 MGE and E14.5 LGE and CGE dataset acquired via Drop seq from Mayer et al., 2018, and MGE and CGE dataset from two different timepoints (E12.5 and E14.5) acquired via Fluidigm C1 system by Mi et al., 2018. UMAP plots depicting all cells from the three studies annotated by dataset (A), putative cell clusters (B) and expression of Nestin and Dcx (C). (D–E) High Nestin-expressing cells (> 1.5 normalized dataset) were extracted from the combined dataset. UMAP plot annotated by dataset (D) and bar graphs showing the total number of high Nestin-expressing cells present in each study (E).
Figure 2—figure supplement 3
Genes defining common neurogenic cell types between brain regions.
UMAP visualizations highlighting genes that help define VZ (green), SVZ (blue) and MZ (red) cell populations in the MGE, LGE, and CGE. Cells are color-coded according to their levels of expression as per the color scale. Red ovals in LGE and CGE highlight the Ebf1-positive, Mapt-positive, Sp9-negative population of putative immature GABAergic projection neurons.
Figure 3
Transcriptional heterogeneity within the VZ, SVZ and MZ in E12.5 GEs.
(A) High Nestin- and Dcx-expressing cells (> 1.5 fold expression above normalized dataset) were extracted from the GE populations and replotted. UMAP plots are annotated by: expression of Nestin and Dcx (upper left), brain region (lower left), separated into VZ (high-Nestin, red), SVZ (Dcx-, Mki67-, and Ccnd2-expressing, light blue) and MZ (Dcx-positive and Ccnd2- and Mki67-negative, dark blue) (upper right), and putative cell clusters (lower right). (B) Heatmap of genes enriched in VZ, SVZ and MZ cells. Each column represents expression in a single cell, color-coded as per the color scale. (C) UMAP plots depicting the top five differentially expressed genes (DEGs) in the VZ, SVZ, and MZ regions. (D) Heatmap showing expression of top 5 DEGs in each cell cluster from (A), with colored bar depicting whether each cluster contains predominantly VZ (red), SVZ (light blue), or MZ (dark blue) cells.
Figure 4 with 1 supplement
Transcriptional heterogeneity of Nestin-expressing VZ cells in the ganglionic eminences.
(A) High Nestin-expressing (> 1.5 normalized dataset) cells were extracted from GE dataset and replotted as UMAP graphs annotated by brain region and putative cell clusters. (B) Heatmap of top 20 DEGs enriched in VZ cells from the LGE, MGE, and CGE. Each column represents expression in a single cell, color-coded as per the color scale. (C) Dot-plot depicting expression levels of DEGs within each brain region (LGE, MGE, and CGE) and cell cluster. (D) Heatmap showing expression of top 5 DEGs in each cluster from (A), with colored bar depicting whether each cluster contains cells from the LGE (yellow), MGE (blue), or CGE (red). Gray bar indicates cluster containing cells from all three GEs. Each column represents averaged expression in cells, color-coded as per the color scale.
Figure 4—figure supplement 1
Thresholding optimization for subsetting high Nestin- and Dcx-expressing GE cells.
(A–B) Ridgeline plots showing the distribution of Nestin (A, red) and Dcx (B, blue) expression when E12.5 GEs were isolated at different expression values from the log normalized count data. (C–D) Violin plot showing the median and interquartile range of Nestin and Dcx post-subsetting at Nestin > 1.5 (C) and Dcx > 1.5 (D).
Figure 5 with 1 supplement
Expression profiles of spatially-enriched genes in VZ cells in the ganglionic eminences.
(A–E) Left, RNAscope HiPlex and HiPlexUp in situ hybridization assays of differentially expressed VZ genes on E12.5 GE coronal sections. Right, the corresponding UMAP plots of VZ-enriched genes depicted in the in situs. Scale bar in A = 200 μm. (F) UMAP plot of Nes-enriched cells annotated by brain region. (G) Schematic of a whole mount view of the E12.5 ventral telencephalon depicting the approximate spatial location of VZ-enriched genes within the GEs.
Figure 5—figure supplement 1
Expression profiles of genes enriched in VZ cells throughout the GEs.
(A–C) Left, RNAscope HiPlex and HiPlexUp in situ hybridization assays of VZ-enriched genes expressed in all GEs. Right, the corresponding UMAP plots of SVZ/MZ-enriched genes depicted in the in situs. Scale bar in A = 200 μm. (D) UMAP plot of Nes-enriched cells annotated by brain region.
Figure 6 with 2 supplements
Expression profiles of spatially-enriched genes in SVZ/MZ cells in the ganglionic eminences.
(A–E) Left, RNAscope HiPlex and HiPlexUp in situ hybridization assays of differentially expressed SVZ/MZ genes on E12.5 GE coronal sections. Right, the corresponding UMAP plots of SVZ/MZ-enriched genes depicted in the in situs. Scale bar in A = 200 μm. (F) UMAP plot of Dcx-enriched cells annotated by brain region. (G) Schematic of a coronal section through the E12.5 ventral telencephalon depicting the approximate spatial location of SVZ/MZ-enriched genes within the GEs.
Figure 6—figure supplement 1
Characterization of genes enriched in high Dcx-expressing cells.
(A) High Dcx-expressing cells (> 1.5 normalized dataset) were extracted from the full GE dataset and replotted, annotated by brain region (middle) and putative cell clusters (right). (B) Heatmap of top 20 SVZ/MZ-enriched genes showing differential expression between LGE, MGE, and CGE. Each column represents expression in a single cell, color-coded as per the color scale. (C) Dot-plot depicting expression levels of SVZ/MZ-enriched DEGs within each brain region (LGE, MGE, and CGE) and cell cluster. (D) Heatmap showing expression of top 5 DEGs in each cluster from (A), with colored bar depicting whether each cluster contains cells from the LGE (yellow), MGE (blue), or CGE (red). Each column represents averaged expression in cells, color-coded as per the color scale.
Figure 6—figure supplement 2
Expression profiles of additional SVZ/MZ-enriched genes in the ganglionic eminences.
(A–D) Left, RNAscope HiPlex and HiPlexUp in situ hybridization assays of SVZ/MZ-enriched genes on E12.5 GE coronal sections. Right, the corresponding UMAP plots of SVZ/MZ-enriched genes depicted in the in situs. Scale bar in A = 200 μm. (E) UMAP plot of Dcx-enriched cells annotated by brain region.
Figure 7 with 2 supplements
Comparison of transcriptional changes between E12.5 and E14.5 ventral telencephalon.
(A–C) UMAP plots of all E12.5 and E14.5 cells annotated by mouse line and embryonic timepoint (A), brain region (B), and putative cell clusters (C). (D) UMAP plot of E12.5 and E14.5 cells depicting expression levels of Nestin and Dcx. Red box = approximate location of high Nestin-expressing cells (> 1.5 normalized dataset), blue box = approximate location of high Dcx-expressing cells (> 1.5 normalized dataset). (E) UMAP plot depicting high Nestin-expressing cells annotated by brain region (top), putative cell clusters (lower left) and timepoint (lower right). Note the clouds that are strongly enriched for E12.5 cells (dark green oval) and E14.5 cells (dark red oval). (F) UMAP plot depicting high Dcx-expressing cells annotated by brain region (top), putative cell clusters (lower right) and timepoint (lower left). Note that E12.5 and E14.5 cells are largely overlapping populations with no clear differential clustering. (G) Heatmap depicting the top 20 DEGs between the E12.5-enriched (dark green oval) and E14.5-enriched (dark red oval) clouds of VZ cells from (E). The E14.5-derived cells were removed from the E12.5-enriched cloud, and vice versa. Each column represents expression in a single cell, color-coded as per the color scale. (H) Heatmap depicting the top 5 DEGs in VZ cells from each GE region at E12.5 and E14.5. Each column represents averaged expression in cells, color-coded as per the color scale.
Figure 7—figure supplement 1
Single-cell transcriptional profiling of E14.5 ganglionic eminences.
(A) Chart describing the number of cells, with mean reads per cell and genes per cell from each E14.5 ganglionic eminence based on CellRanger output. (B–E) UMAP plots of all E14.5 cells annotated by putative cell clusters (B), brain region (C), mouse line (D) and expression of Nestin and Dcx (E). (F) UMAP plot of high Nestin-expressing E14.5 cells (> 1.5 normalized dataset) annotated by brain region (left) and putative cell clusters (right). (G) UMAP plot of high Dcx-expressing E14.5 cells (> 1.5 normalized dataset) annotated by brain region (left) and putative cell clusters (right).
Figure 7—figure supplement 2
Expression of pan-VZ genes in high Nestin-expressing E12.5 and E14.5 cells.
UMAP visualizations show a high level of pan-VZ gene expressions throughout the high Nestin subset E12.5 and E14.5 dataset.
Figure 8 with 1 supplement
Genes with differential expression profiles between E12.5 and E14.5 ganglionic eminences.
(A–D) RNAscope HiPlexUp assay of genes differentially expressed in VZ and SVZ cells between E12.5 and E14.5 on coronal sections through the GEs. Dashed circles on the UMAP plot (A) indicate the clouds that are strongly enriched for E12.5 cells (dark green) and E14.5 cells (dark red). Scale bar in A = 200 μm. Dotplot in (A) contains high Nestin-expressing cells from E12.5 and E14.5, dotplots in B-D consist of all E12.5 and E14.5 cells.
Figure 8—figure supplement 1
Genes with similar expression profiles between E12.5 and E14.5 ganglionic eminences.
(A–C) Left, RNAscope HiPlexUp assay of genes displaying similar expression profiles in VZ and/or SVZ cells between E12.5 and E14.5. Right, UMAP plots of the genes depicted in the in situ panels. Scale bar in A = 200 μm. (D–E) UMAP plots of all E12.5 and E14.5 cells annotated by brain region (D) and mouse line and embryonic timepoint (E).
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Nes-dVenus | RIKEN BioResource Research Center | C57BL/6-Tg(Nes-d4YFP*)1HOkn,RRID:IMSR_RBRC04058 | Pooled sexes |
| Strain, strain background (Mus musculus) | Wild-Type | The Jackson Laboratory | C57BL/6 J000664,RRID:IMSR_JAX:000664 | Pooled sexes |
| Sequence-based reagent | Chromium Next GEM Single Cell 3' Kit | 10X Genomics | v2 (120267) v3 (1000092) | v2 & v3 |
| Commercial assay or kit | RNAscope HiPlex Assays | Advanced Cell Diagnostics | 324,102 | v1 |
| Antibody | Anti-GFP (Rabbit polyclonal) | Invitrogen | A11122,RRID:AB_221569 | 1:400 |
| Antibody | Anti-Doublecortin (Chicken polyclonal) | Abcam | AB153668,RRID:AB_2728759 | 1:1,000 |
| Antibody | Anti-Nestin (Rat Monoclonal) | Millipore | MAB353,RRID:AB_94911 | 1:100 |
| Software, algorithm | Cell Ranger | 10X Genomics | RRID:SCR_017344 | v3.0.0 |
| Software, algorithm | Seurat | the Satija Lab at the New York Genome Center | RRID:SCR_007322 | v3.0.0 |
| Software, algorithm | Slingshot | Kelly Street and Sandrine Dudoit at UC Berkeley | RRID:SCR_017012 | https://github.com/kstreet13/slingshotv2.2.0 |
| Software, algorithm | DoubletFinder | Christopher S. McGinnis and Zev J. Gartner at UCSF | RRID:SCR_018771 | https://github.com/chris-mcginnis-ucsf/DoubletFinderv3.0 |
| Software, algorithm | RNAscope HiPlexImage Registration | Advanced Cell Diagnostics | v1 and v2 | |
| Software, algorithm | Photoshop CC | Adobe | RRID:SCR_014199 | 20.0.9 |
| Software, algorithm | ImageJ | National Health Institution | RRID:SCR_003070 | http://imagej.nih.gov/ij/ |
Additional files
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Supplementary file 1
Differentially expressed genes in the VZ and SVZ.
Table contains the genes that are enriched in the Nestin-positive VZ cells (1,083 genes), mitotic Dcx-positive SVZ cells (330 genes) and postmitotic Dcx-positive SVZ/MZ cells (1,320 genes), all with a minimum log-fold change = 0.25.
- https://cdn.elifesciences.org/articles/71864/elife-71864-supp1-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/71864/elife-71864-transrepform1-v2.pdf
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Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain
eLife 11:e71864.
https://doi.org/10.7554/eLife.71864
