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. 2020 Nov 3;9:e60266. doi: 10.7554/eLife.60266

Figure 2. Sub-clustering of the trophoblast nuclei identifies the cell types of the labyrinth and junctional zone.

(A) UMAP showing the 16,836 nuclei included in the trophoblast subset, clustered and plotted according to transcriptome similarity. Clusters were annotated according to canonical marker genes and named below. Inset shows the number of nuclei collected at each gestational age. (B) Dot plot showing average expression and percent of nuclei in each cluster expressing canonical and novel marker genes identified for each cluster. Genes listed on the x-axis and clusters on the y-axis. (C–E) Nuclei along the differentiation from LaTP to SynTII, SynTI, and S-TGC were ordered by pseudotime using Slingshot. The nuclei included along each pseudotime axis shown at top. The expression of select genes (y-axis) representing each differentiation are shown in each heatmap. Each column represents a nucleus organized in pseudotime proceeding from left to right along the x-axis. (F) Expression of genes unique to several trophoblast populations projected in UMAP space (top) and localization of corresponding protein in E12.5 mouse placenta sections by immunofluorescence staining (Middle – 20x, Scale Bar = 200 µM; Bottom – high magnification inset area shown by white dashed line). Separation of labyrinth (La) and junctional zone (JZ) shown by the orange dashed line.

Figure 2—source data 1. Marker genes for clusters in subclustered trophoblast nuclei.
Figure 2—source data 2. Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP to SynTII (Figure 2C).
Figure 2—source data 3. Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP2 to SynTI (Figure 2D).
Figure 2—source data 4. Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP2 to S-TGC (Figure 2E).

Figure 2.

Figure 2—figure supplement 1. Quality control metrics for snRNA-seq of subclustered trophoblast nuclei and dissection of LaTP populations by specific markers.

Figure 2—figure supplement 1.

(A) Violin plots showing the number of unique genes (left), number of transcripts (middle), and the percent of reads mapping to mitochondrial genes (right) for each cluster identified in the trophoblast dataset in Figure 2A. (B) Scatter plot of expression of Egfr and Met in LaTP and LaTP2 clusters showing Egfr expression to be highly enriched in LaTP2. (C) Expression of Epcam RNA projected in trophoblast nuclei. LaTP and LaTP two clusters are outlined by the dotted line. (D) Immunofluorescence staining of EPCAM and EGFR (top two rows) or EPCAM and MET (bottom two row). White dashed lines outline EPCAM expression as a marker of canonical LaTPs. MET expression colocalizes with EPCAM and represent the LaTP cluster. EGFR and EPCAM domains are largely non-overlapping, with EGFR expressing cells representing the distinct LaTP2 cluster. EGFR is also expressed in mature SynTII. (E) Immunofluorescence staining of EPCAM and EGFR or EPCAM and MET at E8.5. Arrows denote the allantois (Al) and the chorion (Ch). The white box denotes the inset at right.
Figure 2—figure supplement 2. Additional validation of the identities of Junctional Zone clusters.

Figure 2—figure supplement 2.

(A) Nuclei along the differentiation from JZP1 to GCs were ordered by pseudotime using Slingshot. The nuclei included along each pseudotime axis shown at left. The expression of select genes (y-axis) representing differentiation is shown in heatmap (right). Each column represents a nucleus organized in pseudotime proceeding from left to right along the x-axis. (B) Immunofluorescence staining of sections from E12.5 placentas for Ncam1 (Purple) and Slco2a1 (Yellow). Dashed lines separate boundaries between placental regions (Labyrinth, JZ – Junctional Zone, and Decidua). (C) Immunofluorescence staining of sections from E12.5 placentas for Pcdh12 (Green) and Ncam1 (D) DAPI staining in E12.5 placental sections. Dashed box outlines the magnified region shown at left.
Figure 2—figure supplement 3. Characterization of spongiotrophoblast and glycogen gell clusters by specific expression of prolactin genes.

Figure 2—figure supplement 3.

(A) Table of prolactin gene expression in either spongiotrophoblast or glycogen cells from Simmons et al., 2008a and their expression in the snRNA-seq data. (B) Violin plots showing the expression of the prolactin genes in (A). (C) Violin plots showing the expression of additional canonical markers Pcdh12, Tpbpa, Ascl2, Gjb2, and Igf2r.