Fig. 3: Spry genes regulate the number of fibroblasts that express Fgf ligands.

A, B Comparison of transcriptional expression of target genes in the EGF (A) and VEGF (B) signaling pathways based on the scRNA-seq data from control and Spry-TKO glands. Note that changes of color intensity indicate the levels of increased or decreased mRNA expression, whereas the area of each circle correlates with the percentage of fibroblasts that express the target gene of interest. Also note that, although the average expression of these genes was changed in the mutant glands, the percentage of the fibroblasts expressing each gene remained like the control glands. C GO analysis of the main pathway changes in fibroblasts of control and Spry-TKO mammary glands. D, E Comparison of transcriptional expression of several key ECM remodeling enzymes based on scRNA-seq data (D). Validation of the gene expression changes, using several Mmps as an example, by qPCR experiments (E). F, G Collagen contraction assay. F Photographs of a representative experiment. G Graph shows size change of collagen gels, expressed as a percentage of the original area ± SD (n = 3). *P < 0.05. H, I Comparison of transcriptional expression of Fgf2, Fgf7, and Fgf10 based on the scRNA-seq data (H). Note that both the average expression levels and the percentages of fibroblasts expressing these Fgf genes were changed in the Spry-TKO fibroblasts. Specifically, unlike in (A, B), the percentages of cells that express the Fgf genes changed in mutant fibroblasts. Validation of the mRNA expression changes of the Fgf ligands by qPCR experiments (I). J–L t-SNE plots showing the combined single cell transcriptomes of mammary stromal fibroblasts with regards to Fgf2 (J), Fgf7 (K), and Fgf10 expression (L). Note that these Fgfs are expressed by distinct, though overlapping clusters of fibroblasts. Abbreviations: Avg. average; Exp. Expression. Data were from three independent experiments and were presented as mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.