Construction of a single-cell RNA expression atlas during mouse cranial neural tube closure.

(A) Images of mouse embryos spanning the stages of cranial neural tube closure. Bars, 100 µm. (B) Schematics of cranial tissues collected for scRNA-sequencing (dashed boxes, left) and neural tube closure in the midbrain (right) at the indicated stages. The heart was removed at later stages. (C and D) UMAP projections of all cranial cell populations analyzed colored by cell type (C) or embryonic stage (D). (E) UMAP projections of cranial neural plate cells colored by embryonic stage. (F-I) UMAP projections of cranial neural plate cells colored by normalized expression of genes primarily associated with the forebrain (Otx2), midbrain and midbrain-hindbrain boundary (MHB) (En1), hindbrain (Gbx2), and ventral midline (Shh). (J) UMAP projection of cranial neural plate cells colored by neural plate region.

Temporal changes in gene expression reveal shared and region-specific transcriptional trajectories.

(A-C) Cells from the forebrain (A), midbrain (B), and hindbrain (C) at progressive stages of neural plate development, plotted relative to the time-correlated diffusion component 0 (DC0) in each region. (D-F) Normalized expression of E-cadherin (Cdh1) and N-cadherin (Cdh2) plotted relative to the normalized time-correlated diffusion component (DC0) in each region. (G) E-cadherin protein (magenta) is lost from cell-cell junctions and N-cadherin protein (green) accumulates at cell-cell junctions between E7.75 and E8.5 in the mouse cranial neural plate. (H) UMAP projections of cranial neural plate cells colored by normalized gene expression, showing examples of genes that are downregulated or upregulated throughout the cranial neural plate (left) and genes that are specifically upregulated in the forebrain, midbrain/MHB, or hindbrain. Bars, 100 μm (left panels in G), 20 μm (middle and right panels in G).

Region-specific patterns of gene expression along the anterior-posterior axis.

(A) UMAP projections of cranial neural plate cells from E8.5-9.0 embryos colored by their value along the anterior-posterior-correlated diffusion component (DC0) (left) or by the normalized expression of markers primarily associated with the forebrain (Otx2), midbrain (En1), and hindbrain (Gbx2). (B and C) Line plot (B) and heatmap (C) showing the normalized expression of known markers of anterior-posterior identity relative to DC0. DC0 correctly orders forebrain, midbrain, and hindbrain markers relative to their known positions along the anterior-posterior axis. (D) Heatmap showing the normalized expression of 483 genes with high information content relative to DC0. Gene expression profiles are grouped into 11 clusters based on similarities in expression along DC0. Examples of genes in each cluster are listed on the right. (E and F) Schematics showing the predicted expression of example transcriptional regulators along the anterior-posterior axis. Anterior (A), posterior (P). (G) Heatmaps showing the normalized expression of example transcriptional regulators relative to DC0. Heatmaps show one gene per row, one cell per column, with the cells in each row ordered by their value along DC0. Colored bars (right) show the cluster identity relative to DC0. Cells assigned to the forebrain, midbrain/MHB, or hindbrain are indicated at the top of each heatmap.

Patterned gene expression along the mediolateral axis of the developing midbrain.

(A) UMAP projections of cranial neural plate cells from E8.5-9.0 embryos colored by their value along the mediolaterally correlated diffusion component (DC2) (left) or by the normalized expression of markers for the ventral midline (Shh), medial cells (Ptch1), or lateral cells (Pax7). (B and C) Line plot (B) and heatmap (C) showing the normalized expression of known markers of mediolateral cell identity relative to DC2 in the midbrain/MHB. DC2 correctly orders midline, medial, and lateral markers relative to their known positions along the mediolateral axis. (D) Heatmap showing the normalized expression of 222 genes with high information content relative to DC2. Gene expression profiles are grouped into 7 clusters based on similarities in expression along DC2. Examples of genes in each cluster are listed on the right. Cluster 3 (not shown) displayed divergent UMAP patterns associated with anterior-posterior patterning and was excluded from further analysis. (E) Heatmaps showing the normalized expression of specific transcriptional regulators relative to DC2. (F) Schematics showing the predicted expression of example transcriptional regulators along the mediolateral axis of the midbrain/MHB. Heatmaps show one gene per row, one cell per column, with the cells in each row ordered by their value along DC0. Colored bars (right) show the cluster identity relative to DC2. Cells assigned to the midline or midbrain/MHB are indicated at the top of each heatmap.

Multiscale analysis of spatial gene expression in the mouse cranial neural plate.

(A) Plot of genes by Z-Normalized Moran’s I relative to DC0 and DC2 for all genes expressed in the E8.5-9.0 cranial neural plate. Genes meeting the cutoff for further analysis are colored according to cluster identity at D=10; other genes are shown in gray. (B) Spatially informative genes from (A) replotted by spatial autocorrelation (Moran’s I) along DC0 and DC2, colored by cluster identity at D=10. (C) Dendrogram of spatially informative genes showing gene clusters at different distances corresponding to the average correlation among genes. Left, the number of clusters at each distance (black curve) and the average correlation between expression patterns within clusters (blue curve). Right, dendrogram showing the relationships between clusters, colored by cluster identity at D=10 as in A and B. (D) UMAP projections of E8.5-9.0 neural plate cells colored by normalized gene expression. Examples of genes that mark specific presumptive brain structures (the telencephalon, diencephalon, and midbrain-hindbrain boundary), midline or lateral domains, or subsets of rhombomeres are shown. Brackets and boxes indicate regions of increased gene expression. Spatial cluster identities at a stringency of D=6 (top and middle panels) or D=4 (bottom panels) are indicated.

Patterned expression of secreted and transmembrane proteins in the cranial neural plate.

(A and B) Heatmaps showing the normalized expression of transmembrane and secreted proteins relative to DC0 (A) or DC2 (B). Heatmaps show one gene per row, one cell per column, with the cells in each row ordered by their value along DC0 or DC2. Colored bars (right) show the cluster identity relative to DC0 or DC2. Cells assigned to the forebrain (FB), midbrain/MHB (MB/MHB), hindbrain (HB), or midline are indicated at the top of each heatmap. (C) Schematics showing the predicted expression of example transmembrane and secreted proteins along the anterior-posterior axis. Anterior (A), posterior (P). (D-F) UMAP projections of cranial neural plate cells from E8.5-9.0 embryos colored by the normalized expression of a subset of Wnt ligands (D), Frizzled receptors (E), and Ephrin ligands and Eph receptors (F). Spatial cluster identities at D=10 are indicated.

Single-cell RNA sequencing reveals region-specific transcriptional programs activated by SHH signaling.

(A) Maximum-intensity projections of the midbrain and anterior hindbrain regions of the cranial neural plate of 5-somite mouse embryos treated with vehicle control (left) or with 2 µM Smoothened Agonist (SAG) stained for FOXA2 and NKX6-1. SAG-treated embryos display an expanded floor plate region. Bars, 100 μm. (B and C) UMAP projections of 1,619 and 1,409 cranial neural plate cells from control and SAG-treated embryos at E8.5, respectively, colored by treatment (B) or anterior-posterior domain (C). (D-J) Heatmaps showing the normalized expression of candidate SHH target genes that were upregulated or downregulated throughout the cranial neural plate (D) or in the indicated region(s) (E-J) in SAG-treated embryos. Heatmaps show one gene per row, one cell per column, with the cells in each row grouped by control (green) or SAG treatment (orange), indicated at the top of each heatmap. Genes involved in SHH signaling (red) and genes assigned to medial (purple), lateral (blue), anterior-posterior (green), or other spatial clusters (orange) are indicated. Genes that had a MAST hurdle value >0.24 or <-0.24 and a false discovery rate adjusted p-value of p<0.001 in at least one region were indicated as differentially expressed in all regions in which p<0.001 and the MAST hurdle value was >0.10 or <-0.10.