Fig. 4

Prototypic SMC markers Cald1, Tpm1, and Pdgfrb detected in LMCs and VSMCs by scRNAseq. To validate the single-cell RNA-sequencing (scRNAseq) clusters predicted to be lymphatic muscle cells (LMCs) and vascular smooth muscle cells (VSMCs) via UMAP analysis, we mined the transcriptomic data for prototypic markers of smooth muscle cells (SMCs). The results showed that Cald1 (Caldesmon; LMCs 1.77 log₂ fold-change (log2FC) and VSMCs 1.48 log2FC) (A), Tpm1 (Tropomyosin alpha-1; LMC 0.33 log2FC and VSMC 0.42 log2FC) (B), and Pdgfrb (platelet-derived growth factor receptor beta; LMCs 0.57 log2FC and VSMCs 0.74 log2FC) (C), were significantly enriched relative to all the other clusters. Feature plots overlaying the UMAP are provided to visualize the change in expression (grey = low, blue = high gene expression), while violin plots are shown to demonstrate the effect size between muscle cell populations compared to all other cell clusters. Wilcoxon rank sum test was performed between the different cell types for statistical analysis with false discovery rate (FDR) < 1.01E−66 for all comparisons. Sample sizes: LMCs = 1985 cells, VSMCs = 1331 cells, Other = 5563 cells. To validate the expression of Pdgfrb in LMCs in vivo, Pdgfrb-CreER;tdT mice were generated and induced with tamoxifen. The popliteal lymphatic vessels (PLVs) (solid arrow) were harvested from these animals and immunostained for alpha smooth muscle actin (αSMA) (green) to label LMCs (D) and robust tdTomato (tdT) expression (red) was noted in PLVs representing Pdgfrb expression (E), which colocalized (yellow) with the αSMA⁺ LMCs (F)