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. 2020 Nov 3;9:e61413. doi: 10.7554/eLife.61413

Figure 1. scRNA-sequencing of Pdcd10-wt and Pdcd10-ko endothelial cells.

(A) Experimental scheme (see Materials and methods for details). (B) Representative photographs of Pdcd10-wt (left) and Pdcd10-ko (right) whole brains at P8. (C) Representative confocal microscopy of the vasculature of Pdcd10-wt (left) and Pdcd10-ko (right) cerebella at P8, stained for Podocalyxin (black; see also Figure 1—figure supplement 1). Scale bars: 1 mm. (D) UMAP plot showing detected cell subpopulations in the Pdcd10-wt and Pdcd10-ko integrated analysis. The total numbers of cells within each cluster are shown in brackets in the color legend (bottom panel). (E) Plot of the percentages of Pdcd10-wt (orange) and Pdcd10-ko (blue) cells in each of the cluster. (F) Plot of the numbers of Pdcd10-wt (orange) and Pdcd10-ko (blue) cells in each cluster. The percentages of total cells in each cluster (%) is reported above each bar. (G) Heatmap of the selected pan- (pink) and blood–brain barrier- (BBB; green) endothelial cell markers (normalized expression shown; see Materials and methods) for the Pdcd10-wt and Pdcd10-ko cells in each cluster. (H) Heatmap of the selected endothelial cell subtype markers (from top to bottom, as indicated: arterial, capillary, venous, tip cells, mitotic), to show the normalized expression levels of the Pdcd10-wt (left) and Pdcd10-ko (right) cells in each cluster (see also Supplementary file 1). For each subtype, the key genes are listed accordingly (top-to-bottom). (I) Heatmap of normalized expression of arterial and venous capillary markers in the capillary and mitotic/capillary clusters (C0, C2, C3, C4, C5, C7). The venous (C9; left) and arterial (C8; right) clusters are reported for reference. The Pdcd10-wt (orange) and Pdcd10-ko cells (blue) are shown separately. Key genes are listed according to the top-to-bottom order in the heatmap. (J) Summary of the prevalent identities of the 17 clusters based on endothelial cell subtype marker expression as in (H, I) and Figure 1—figure supplement 1. The dendrogram of the hierarchical clustering is shown on the left. Non/mixed-ECs clusters (C10, C11, C15, C16) show early segregation. Arterial/venous (C8, C9), capillary (C0, C3, C4, C5), tip (C1 and C6) and mitotic/capillary (C2, C7) cells segregate as distinct groups of the final branches. Clusters 12, 13, and 14 show features of both capillaries and tip cells, but only C12 and C14 segregate on neighbor final branches.

Figure 1.

Figure 1—figure supplement 1. Representative confocal microscopy of Pdcd10-wt and Pdcd10-ko brains at P8 immunostained for Podocalyxin.

Figure 1—figure supplement 1.

Higher magnification images of different brain regions are shown on the right (color coded from left panel) for cortex, thalamus, hippocampus, and cerebellum. Scale bars: 1 mm (main panel left), 500 μm (magnifications).
Figure 1—figure supplement 2. Expression of selected markers of endothelial and contaminant cells in the identified clusters.

Figure 1—figure supplement 2.

(A, B) UMAP non-linear dimensional reduction method for the 32,261 cells examined (Pdcd10-wt, 15,057; Pdcd10-ko, 17,204), color coded for expression levels of pan-endothelial markers Cldn5 and Pecam1 (A) and blood–brain barrier (BBB) endothelial markers Mfsd2a, Slc2a1 (also known as Glut1) (B). Considering jointly Pdcd10-wt and Pdcd10-ko cells in the different clusters, the expression of the endothelial markers appears homogenously distributed in the different clusters. (C) Heatmap (for normalized expression; see Materials and methods) of selected contaminant markers (indicated left) for astrocytes, pericytes and microglia in Pdcd10-wt and Pdcd10-ko cells of each cluster (0–16) (see also Supplementary file 1). (D) Heatmap of average logFC (p<0.05, Pdcd10-ko vs. Pdcd10-wt) for Pdcd10 expression in the 17 clusters identified (0–16). (E) UMAP for the 32,261 cells examined (Pdcd10-wt, 15,057; Pdcd10-ko, 17,204), color coded for expression levels of the indicated EC subtype markers, for (left to right) arterial, venous, capillary, tip and mitotic cells. For each UMAP, the corresponding violin plots of Pdcd10-wt (orange) and Pdcd10-ko (blue) cells in each endothelial cluster is shown.
Figure 1—figure supplement 3. Sequencing quality control and clustering tree of examinedPdcd10-wt andPdcd10-ko ECs.

Figure 1—figure supplement 3.

(A) UMAP non-linear dimensional reduction method for the 32,261 cells examined (Pdcd10-wt, 15,057; Pdcd10-ko, 17,204), color coded, from top to bottom, for sample conditions, transcript counts (number of unique molecular identifiers) and sample origin. Top: Homogenous distribution of Pdcd10-wt (orange dots) and Pdcd10-ko (blue dots) cells shown among the clusters. Middle: Number of unique transcript sequences shown for each cell. Bottom: Homogenous distribution of cells isolated from the four mice used in the analysis (blue and green; Pdcd10-wt, gray and orange: Pdcd10-ko). (B) Clustering tree of examined Pdcd10-wt and Pdcd10-ko endothelial cells. Results from clustering using Seurat with resolution parameters from 0.01 to 1. At a resolution of 0.01, we see the formation of four clusters, one of which continues to split up to form new clusters as the resolution increased. Resolution 0.09–0.1 and resolution 0.4–0.5 are two stable regions in this tree where no additional sub-branching happened in between and the number of clusters stayed the same. After resolution 0.5, many low in-proportion (in_prop) edges ad new clusters with multiple parent clusters showing up, indicating cluster instability. Red box highlights the resolution (0.4) selected for further analysis.
Figure 1—figure supplement 4. Clustering tree of examined Pdcd10-wt and Pdcd10-ko endothelial cells with overlaid prevalent cluster identity.

Figure 1—figure supplement 4.

Resolutions from 0.01 to 0.4, which is part of the clustering tree shown in Figure 1—figure supplement 3. Box A (solid black line): at resolution 0.01, two of the four contaminant cell clusters (C10 and C15) are formed and remain unchanged to resolution 0.4. Box B (solid red line): at resolution 0.03, two major lineages are branched. It is clear at resolution 0.4 that arterial and venous clusters (C8 and C9, respectively) are derive from these two distinct lineages, so are the two major tip cell clusters (C1 and C6). It suggests that C1 and C6 are not the result of over-clustering, but rather represent two distinct tip cell sub-populations. Box (C & C’) (dashed black line): at resolution 0.05 (C) and 0.2 (C’), the remaining two contaminant cell clusters (C11 and C16) are formed and remain unchanged to resolution 0.04. Box D (dashed green line): at resolution 0.4, the mitotic/venous capillary and the venous/venous capillary clusters (C7 and C9, respectively) were formed from the same parent cluster.