Cardiac fibroblasts regulate myocardium and coronary vasculature development in the murine heart via the collagen signaling pathway

Figure 1.. Anatomical location of cardiac fibroblasts (FBs) and their spatial relationship with cardiomyocytes and endothelial cells at different stages. (A) RNA staining of Col1a1 revealed the spatial pattern of cardiac FBs at different developmental stages. Scale bar = 500 µm. (B) The development of cardiac FBs can be grouped into four phases. (C) RNA staining analysis of Col1a1, Actn2, and Cd31 revealed the spatial proximity of FB, CM, and EC in E17.5 hearts. (D) Quantification of the number of CMs and ECs that contact with each FB (n=100). Scale bar = 500 µm and 100 µm in the whole heart sections and enlarged sections, respectively.

Figure 1—figure supplement 1.. The anatomical patterns of fibroblasts at different stages. (A) Analysis of fibroblast anatomical pattern by staining Col1a1 at different stages. Scale bar = 500 µm.

Figure 1—figure supplement 2.. The anatomical patterns of fibroblasts, cardiomyocytes, and endothelial cells. (A, B) RNA staining of Col1a1, Actn2, and Cd31 expression at E13.5 and P3 heart sections. Scale bar = 500 µm and 100 µm in the whole heart sections and enlarged sections, respectively.

Figure 1—figure supplement 3.. ScRNA-seq identified distinct fibroblast populations. (A) UMAP plot of Col1a1 expression in cardiac cells. (Bi-iv) UMAP plots of Col1a1 and representative cluster-specific gene expression in cardiac fibroblasts. (C) UMAP plot of fibroblasts labeled by cell cycle phases. (D) Diagram of the four types of cardiac fibroblasts. (E) The top 10 genes expressed in each group of cardiac fibroblasts. (Fi-iii) Identification of the anatomical location of each fibroblast population through RNA staining. Scale bar = 500 µm and 250 µm in the whole heart images and the images with enlarged areas, respectively.

Figure 2.. Molecular analysis of fibroblast (FB) communications with other cardiac cell types in developing hearts. (A) The number and strength of interactions among different cardiac cell types identified from scRNA-seq data. (B) The top signaling pathways derived from FB that impact Ven_CM and Vas_EC development. (C) The detailed ligand-receptor interactions in the collagen pathway between FB and Ven_CM or Vas_EC. (D) Collagen was visualized using CHP staining in mouse hearts at E11.5, E14.5, E16.5, and P3. Scale bar = 500 µm and 150 µm in the whole heart sections and enlarged sections, respectively. (E) The signaling interactions and top pathways between FB and Ven_CM or Vas_EC in primary human hearts.

Figure 2—figure supplement 1.. Heterogeneity analysis of cardiac fibroblasts. (A, B) The cell types and main_fb population in CD1 scRNA-seq dataset. (C, D) The cell types and main_fb population in C57BL/6 scRNA-seq dataset. (E, F) The main_fb cells labeled by stage and chamber.

Figure 2—figure supplement 2.. The expression pattern of extracellular matrix genes in the main population of cardiac fibroblasts. (A) Clustering analysis of the ECM gene expression enrichments. (B) The group of genes (G1) that were highly expressed at early staged fibroblasts in all four zones. (C) The group of genes (G2) that were highly expressed in LV and RV at late embryonic and neonatal stages. (D) The genes (G3) that were preferentially expressed in LA and RA. (E) The genes (G4) that were highly expressed in LA, LV, and RV at neonatal stage.

Figure 2—figure supplement 3.. The expression pattern of group G5 genes in fibroblasts. (A, B) The expression pattern of a group of genes (G5) that displayed expression in all four chambers of FBs (CD1 mice) at late embryonic and neonatal stages.

Figure 2—figure supplement 4.. The expression pattern analysis of extracellular matrix genes in C57BL/6 FBs. (A) Unsupervised clustering analysis of extracellular matrix genes expression in C57BL/6 FBs. (B) The groups of genes that display stage or zone-specific expression patterns.

Figure 2—figure supplement 5.. The molecular interactions between FBs and other cell types. (A, B) The ligand-receptor interactions between FBs and ventricular CM or Vas_EC at each stage. (C) The ligand-receptor interactions between FB and Vas_EC, as well as between FB and Ven_CM, in the human embryonic heart.

Figure 3.. Functional analysis of FB in embryonic heart development. (A) Diagram of the lineage-tracing experiments and the tracing results. Scale bar = 500 µm. (B) The ablated embryos treated with tamoxifen at E13.5 were smaller than control embryos, but the ablated hearts were not obviously different from the control hearts. Scale bar = 1 mm. (C) Representative CD31 staining images in control and ablated hearts. The zoomed-in images showed a reduction of CD31-positive area in the ablated hearts compared to controls. (D) Quantification of the compact and trabecular myocardium thickness (19 sections from 7 ablated hearts and 20 sections from 7 control hearts), and CD31-positive areas in control and ablated hearts (15 sections from 5 ablated and 15 sections from 5 control hearts). The littermate control mice include both double-negative and Pdgfra-CreER +/−mice. (E) The ablated mice and hearts treated with tamoxifen at E15.5 were smaller than controls. (F) CD31 staining analysis of control and ablated hearts. (G) Quantification of the compact and trabecular myocardium thickness, and CD31-positive areas in control and ablated hearts (12 sections from 4 ablated hearts and 12 sections from 4…

Figure 3—figure supplement 1.. Comparative analysis of the expression patterns and lineage tracing results between the genes Postn and Pdgfra. (Ai, ii) UMAP plots of Postn and Pdgfra expression in cardiac cells and cardiac fibroblasts. (B) (i) The workflow to lineage trace the Postn-CreER;mTmG labeled cells. (ii) A few cells in valves and septum were labeled in Postn-CreER: Rosa26-mTmG mouse hearts. Scale bar = 500 µm.