Anatomical location of cardiac fibroblasts and their spatial relationship with cardiomyocytes and endothelial cells at different stages. (A) RNA staining of Col1a1 revealed the spatial pattern of cardiac fibroblasts at different developmental stages. Scale bar=500µm. (B) The development of cardiac fibroblasts 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=500µm and 100µm in the whole heart sections and enlarged sections, respectively.

Molecular analysis of fibroblast 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 expression pattern in CD1 mouse hearts at E11.5, E14.5, E16.5, and P3. Scale bar=500µm and 150µm 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.

Functional analysis of FB in embryonic heart development. (A) Lineage tracing revealed an abundant of Pdgfra-CreER labeled cells were GFP+ in developing mouse hearts. Scale bar = 500µm. (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=1mm. (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 thickness of compact and trabecular myocardium (19 sections from 7 ablated hearts and 20 sections from 7control hearts), and CD31 positive areas in control and ablated hearts (15 sections from 5 ablated and 15 sections from 5 control hearts). (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 control hearts.). Scale bar =500µm in the whole heart sections and 100 µm in the zoomed in images. * represents p<0.05; ** represents p<0.01.

Single cell analysis of the embryonic heart defects after fibroblast ablation. (A) Diagram of the MULTI-seq experiments to profile control and ablated hearts at two developmental stages. (B) The scRNA-seq data from two replicates are highly consistent. (C) UMAP plots of scRNA-seq data labeled by cell type, cell cycle phase, and genotypes. (D) Quantification of the FB and mural cell percentages at each conditions. (E) The percentages of other cardiac cell types under each condition. (F) Detailed analysis of the fibroblast population revealed four groups, including a dying fibroblast subpopulation. (G) UMAP plot of Ven_CMs from different conditions. (H) Quantification of the different cell cycle phased Ven_CMs under each condition. (I, J) Heatmap and pathway enrichment of genes that are differentially expressed in control and ablated Ven_CMs at E16.5. (K) UMAP plot of Vas_EC labeled by conditions. (L, M) Heatmap and pathway enrichment of genes that were upregulated in ablated Vas_ECs compared to control Vas_ECs.

Identification of signaling defects in ablated hearts. (A) The top signaling pathways between FB and Ven_CM or Vas_EC in control and ablated hearts. (B) Collagen staining revealed a significant reduction in collagen deposition in ablated hearts compared to control hearts. (C, D) Regulatory analysis predicted the ligands that regulate the genes differentially expressed in control and ablated Ven_CMs and Vas_EC. (E) Representative signaling pathways that were downregulated or upregulated in the dying FBs.

Short term and long term functional analysis of fibroblasts at neonatal stage. (A) Diagram of the experiment to ablate fibroblasts with three doses of tamoxifen treatment from P1 to P3. Scale bar=1cm. (B) No obvious size difference was observed between control and ablated hearts. (C) Representative images of control and ablated hearts stained with CD31. (D) Quantification of the compact and trabecular myocardium thickness and CD31 positive areas in ventricular at control and ablated hearts (12 sections from 4 ablated hearts and 12 sections from 4 control hearts.). (E) Collagen accumulation in control and ablated hearts at P4. Scale bar=500µm and 150µm in the whole heart sections and enlarged sections, respectively. (F) Tamoxifen was given from P3 to P5 to ablate the fibroblasts and hearts were collected at P17 for analysis. (G) The percentage of mice died at different days. (H) Representative echo images of the control and ablated hearts. (I) Quantification of the heart rate, body weight, and heart function in control and ablated mice at P17. * represents p<0.05; ** represents p<0.01; *** represents p<0.001.