Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

Figure 1 Single cell RNA sequencing analysis of maxillary processes from wild-type and Cd271 knockout mice. We identified 21 clusters of cells, and the single cell RNA sequencing data were characterized. A: Schematic representation of the experimental workflow. The Cd271 knockout and wild-type maxillary processes were collected from mouse embryos at 16.5 d. After genotyping, cells were suspended as single cells, and the cDNA library was constructed and sequenced; B: Cells extracted from the samples were subjected to Uniform Manifold Approximation and Projection hierarchical clustering and color-coded by cluster grouping, predominant cell type, and sample origin; C: Heatmap showing expression levels of differentially expressed genes in each cluster; D: Dot plot showing prominent marker genes for each cell type (mesenchymal cells, epithelial, muscle cell, macrophage, glial, T cell, endothelial, and perivascular); E: Pie chart showing the number of each cell type; F: Histogram showing the number and proportion of cells from wild-type and Cd271 knockout mice. UMAP: Uniform Manifold Approximation and Projection; KO: Knockout; WT: Wild-type.

Figure 2 Gene set enrichment analysis plots for representative signaling pathways upregulated in maxillary process. Gene set enrichment analysis enriched differentially expressed genes into multifunctional gene networks. A and B: Representative gene set enrichment analysis maps for mesenchymal stem cells; C: Endothelial cells; D: Epithelial cells; E: Glia; F: T cells; G: Macrophages; H: Muscle cells; I: Perivascular cells. Normalized enrichment score normalized enrichment score, corrected for multiple comparisons using false discovery rate method, P values shown in plots. NES: Normalized enrichment score.

Figure 3 Further cluster analysis of mesenchymal stem cells. A: Mesenchymal stem cells were reclustered and color-coded according to cluster, sample origin and cell type; B: Feature scores of progenitors, fibroblasts and osteoblasts marker genes were displayed in the Uniform Manifold Approximation and Projection plot; C: Histogram showing the proportion and the number of cells in each cluster of wild-type and Cd271 knockout mice. UMAP: Uniform Manifold Approximation and Projection; KO: Knockout; WT: Wild-type.

Figure 4 Cd271 knockout mesenchymal stem cells showed weaker proliferation and osteogenic differentiation potential. A: Gene Ontology enrichment analysis of differentially expressed genes between Cd271 knockout and wild-type mesenchymal stem cells (MSCs); B: Growth curves of Cd271 knockout and wild-type MSCs; C: Mineralization assay of Cd271 knockout and wild-type MSCs. Alkaline phosphatase (ALP) and Alizarin red staining of Cd271 knockout MSCs was significantly lighter. ALP activity and calcium quantification were significantly lower in Cd271 knockout compared with wild-type MSCs; D: Protein–protein interaction network interaction analysis of wildtype vs Cd271 knockout differentially expressed genes; E: Quantitative real-time polymerase chain reaction assay of some differentially expressed genes in Cd271 knockout and wild-type MSCs. Data are presented as mean values ± SD (n = 3 biologically independent experiments. Two-sided unpaired t-test, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001) BP: Biological Process; CC: Cell component; MF: Molecular function; ARS: Alizarin red S; ALP: Alkaline phosphatase; KO: Knockout; WT: Wild-type.

Figure 5 The potential developmental trajectories of mesenchymal stem cells by Monocle. A: Pseudo-sequential diagram based on the differentiation process of mesenchymal stem cell (MSC) subpopulations; B: Pseudo-sequence plot of MSCs colored by cluster classification; C: Pseudo-sequential diagram of MSCs drawn with timeline coloring; D: Heatmap of differentially expressed genes in pseudotemporal analysis. The differentially expressed genes were divided into three gene clusters and gene ontology biological process enrichment analysis was performed.

Figure 6 Inference, analysis and visualization of cellular communication networks from a single dataset via CellChat. A: Interaction intensity plot between cells in single cell sequence profiling; B: Interaction intensity map between mesenchymal stem cells (MSCs) and other cell types; C: Contribution map of 30 signaling pathways detected by CellChat to intercellular efferent (or afferent) signaling; D: Alluvial plot of cell outgoing signaling patterns, showing the correspondence between cell populations and signaling pathways; E: Important ligand–receptor pairs for MSCs sending signals to other cell types. F and G: Contribution of efferent (or afferent) signals between cells, the MK signaling pathway (F) and PTN signaling pathway (G) were selected for visualization of network centrality scores; H: Expression of Mdk and Ptn in Cd271 knockout and wild-type MSCs was analyzed by quantitative real-time polymerase chain reaction. Data are presented as mean values ± SD (n = 3 biologically independent experiments. Two-sided unpaired t-test, ^aP < 0.05, ^bP < 0.01). KO: Knockout; WT: Wild-type.