Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance

Abstract

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63⁺ basal and SMA⁺ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Figure 1

Identification of salivary gland epithelial cell clusters by single-cell RNA sequencing. (A) Salivary gland epithelial cell transcriptomes visualized with t-distributed stochastic neighbor embedded (t-SNE), colored according to unsupervised clustering. (B) Hierarchical clustering analysis based on the log-transformed mean expression values of the 7 clusters. The tree was computed based on Spearman’s rank correlation with Ward linkage. (C) t-SNE plot based on the hierarchical clustering analysis performed in panel B above. (D) Epithelial cell clusters were interrogated for the expression of known basal, myoepithelial, ductal and acinar genes to determine their patterning amongst the cell clusters. C- cluster.

Figure 2

Spatial expression pattern of ΔNp63 and SMA proteins during the early stages of salivary gland morphogenesis. (A) ΔNp63 is expressed in the epithelial cells of the placode as well as the developing buds and end buds during the early stages of salivary gland development. (B) Expression of the basal and myoepithelial cell markers SMA, K14 and ΔNp63 at E14.5. (C) At E15.5 ΔNp63 and SMA expression is observed in the basal and myoepithelial cells surrounding the developing ducts and acini. Scale bar 37 μm.

Figure 3

Expression pattern of ΔNp63 and SMA during the later stages of salivary gland development and adults. (A,B) Expression of ΔNp63 and SMA at E16.5 and E18.5. (C) Expression and quantification analyses of SMA and ΔNp63 in 8-week old adult glands. Arrow- SMA⁺/∆Np63⁺ cells, white arrowhead- SMA⁻/∆Np63⁺ cells, orange arrowhead-SMA⁺/∆Np63⁻ cells. (D) Expression and quantification analyses of SMA and K14 in 8-week old adult glands. Arrow- SMA⁺/K14⁺ cells, white arrowhead- SMA⁻/K14⁺ cells, orange arrowhead-SMA⁺/K14⁻ cells. Data are represented as mean ± standard deviation (S.D.). Scale bar 37 μm. n = 3.

Figure 4

Contribution of p63⁺ cells during salivary gland morphogenesis. (A) Schematic of the experimental timeline used for the embryonic genetic lineage tracing experiments in the Trp63^CreERT2;Rosa26-tdTomato mice. RFP expression was induced at E12.5 by injection of TAM to pregnant females and cells were traced for 6 days and glands were dissected at E18.5. (B) p63⁺ cells contribute to all epithelial cell lineages during submandibular gland morphogenesis. (C) Quantification of the percentage of RFP⁺ cells which co-express the various cell lineage markers as indicated. Data are represented as the mean ± standard deviation (S.D.). Arrows highlight double positive cells as indicated. Scale bar 37 μm. TAM- tamoxifen, E-embryonic. n = 4.

Figure 5

Contribution of SMA⁺ cells during salivary gland development. (A) Schematic depicting the experimental timeline for the genetic lineage tracing studies performed in the Acta2^CreERT2;Rosa26-tdTomato mice. (B) SMA⁺ cells contribute exclusively to the basal and myoepithelial cell lineages. (C) Quantification of the percentage of RFP⁺ cells which co-express the various cell lineage markers as indicated. Data are represented as mean ± standard deviation (S.D.). Arrows highlight double positive cells as indicated. Scale bar 37 μm. TAM- tamoxifen, E-embryonic. n = 4.

Figure 6

p63⁺ cells maintain the mature cell lineages of the submandibular gland. (A) Experimental timeline used for the genetic lineage tracing experiments in adult Trp63^CreERT2;Rosa26-tdTomato mice. RFP expression was induced by TAM injection in 6-week old adult mice and cells were traced for 1 week and 1, 2 and 6 months. (B) RFP expression was detected in all the epithelial cell lineages which comprise the salivary gland in Trp63^CreERT2;Rosa26-tdTomato mice at 6 months (left panel). Quantification of the percentage of RFP⁺ cells which co-express the various cell lineage markers as indicated (right panel). (C) Co-staining of RFP and the proliferative marker Ki67 in adult submandibular glands 1 week after TAM administration (left panel). Quantification of RFP⁺/Ki67⁺ cells in submandibular glands, expressed as a percentage of total single Ki67⁺ cells (right panel). (D) Submandibular glands were evaluated for RFP expression 1 week and 1, 2 and 6 months following TAM administration (left panel). Quantification of RFP⁺ cells per total number of cells (nuclei) counted in glands isolated at the indicated time points after TAM administration (right panel). Data are represented as mean ± standard deviation (S.D.). Arrows highlight double positive cells as indicated. W- week, M- month. Scale bar 37 μm. n = 4.

Figure 7

SMA⁺ cells maintain the myoepithelial and ductal cell lineages in the adult submandibular gland. (A) Experimental timeline used for the genetic lineage tracing experiments in adult Acta2^CreERT2;Rosa26-tdTomato mice. RFP expression was induced by TAM injection in 6-week old adult mice and cells were traced for 1 day, 1, 2 and 6 months. (B) RFP expression was detected in the myoepithelial and ductal cell lineages (left panel). Quantification of the percentage of RFP⁺ cells which co-express the various cell lineage markers as indicated (right panel). (C) Submandibular glands were evaluated for RFP expression 1 day (D) and 1, 2 and 6 months (M) following TAM administration (left panel). Quantification of RFP⁺ cells per total number of cells (nuclei) counted in glands isolated at the indicated time points after TAM administration (right panel). (D) Co-staining of RFP and the proliferative marker Ki67 in adult submandibular glands 1 day after TAM administration (left panel). Quantification of RFP⁺/Ki67⁺ cells in submandibular glands, expressed as a percentage of total Ki67⁺ cells (right panel). Data are represented as mean ± standard deviation (S.D.). Arrows highlight double positive cells as indicated. D-day, M-month. Scale bar 37 μm. n = 4.

Figure 8

Model outlining the function of p63 and SMA stem/progenitor cells along the salivary gland epithelial cell hierarchy. (A) A schematic representation of the SMG. In adult glands, p63⁺ stem/progenitor cells maintain all the mature epithelial cell lineages (left panel). Conversely, SMA⁺ progenitor cells maintain the myoepithelial and ductal cell lineages in adult glands (right panel). (B) Proposed position of p63 and SMA in the salivary gland stem/progenitor cell hierarchy.