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 was visualized using CHP staining in 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) Diagram of the lineage tracing experiments and the tracing results. 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). 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 control hearts.). The littermate control mice include both double-negative and Pdgfra-CreER+/− mice. 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 genotype. (D) Quantification of the FB and mural cell percentages at each conditions. (E) Detailed analysis of the fibroblast population revealed four groups, including a dying fibroblast subpopulation. (F) UMAP plot of Ven_CMs from different conditions. (G) Quantification of the different cell cycle phased Ven_CMs under each condition. (H, I) Heatmap and pathway enrichment of genes that are differentially expressed in control and ablated Ven_CMs at E16.5. (J) UMAP plot of Vas_EC labeled by conditions. (K, L) Heatmap and pathway enrichment of genes that were upregulated in ablated Vas_ECs compared to control Vas_ECs. The littermate control mice at both stages were Pdgfra-CreER+/−.

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) CHP 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. Collagen was visualized using CHP staining. The littermate control mice were Pdgfra-CreER+/−. 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. The littermate control mice were Pdgfra-CreER+/−. (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.

(A) Analysis of fibroblast anatomical pattern by staining Col1a1 at different stages.

Scale bar=500µm.

>(A, B) RNA staining of Col1a1, Actn2, and Cd31 expression at E13.5 and P3 heart sections.

Scale bar=500µm and 100µm in the whole heart sections and enlarged sections, respectively.

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 genes 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=500µm and 250µm in the whole heart images and the images with enlarged areas, respectively.

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.

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.

(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.

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 pattern.

(A, B) The ligand-receptor interactions between FBs and ventricular CM or Vas_EC at each stage.

(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=500µm.

(A) Representative images of TUNEL staining in control and Pdgfra-CreER; Rosa-DTA hearts. (B) Quantification of TUNEL (7 sections from 2 ablated hearts and 8 sections from 2 control hearts) and pHH3 signals (5 sections from 2 ablated hearts and 4 sections from 2 control hearts). (C) Representative images illustrating the quantification of compact and trabecular myocardium thickness in control and ablated hearts at E16.5. Scale bar=500µm and 100µm in the whole heart images and the images with enlarged areas, respectively.

Comparative analysis of valve development in control and fibroblast-ablated hearts at E18.5.

(A) Scale bar=500µm and 100µm in the whole heart images and the images with enlarged areas, respectively.

Quantification of the defects in control and ablated hearts with one dose of tamoxifen treatment at E15.5.

(A) Diagram of the experimental procedure with one dose of tamoxifen being administered at E15.5. (B) No obvious size differences were observed in the ablated embryos and hearts compared to controls. (C) CD31 staining analysis of control and ablated hearts. The littermate control mice were Pdgfra-CreER+/−. (D) Quantification of the compact and trabecular myocardium thickness in LV and RV (6 sections from 2 ablated hearts and 5 sections from 2 control hearts). (E) Quantification of the ratio of trabecular to compact myocardium in LV and RV. (F) Quantification of pHH3 positive cells in control and ablated ventricles (6 sections from 2 ablated hearts and 5 sections from 2 control hearts for one dose of tamoxifen treated group, while 12 sections from 4 ablated hearts and 12 sections from 4 control hearts for three doses of tamoxifen treated group). (G) RNA staining analysis of Col1a1 in control and ablated hearts with three doses of tamoxifen treatments. The Col1a1 signal was dramatically reduced in the ablated hearts. * represents p<0.05; ** represents p<0.01. Scale bar=500µm.

(A) QC plots of the filtered single cells including the total number of genes, total number of mRNA, and percentage of mitochondria genes. (B) The expression pattern of representative genes to identify fibroblast subpopulations. (C) The genes highly and specifically expressing in the dying_fb cells. (D, E) UMAP plots of Ven_CM and Vas_EC labeled by cell cycle phases.

The predicted ligand-receptor interaction potentials between main_fb and Ven_CM or Vas_EC.

The signaling pathways that were found to reduce or increase their strengths in the dying_Fb compared to the main_fb and valve_like_fb.

Quantification of the defects in control and ablated hearts at neonatal stage with one dose of tamoxifen treatment.

(A) Diagram of the experimental procedure with tamoxifen treatment at P1. (B) No obvious size differences were observed in the ablated hearts compared to control hearts. (C) Representative images of control and ablated hearts stained with CD31. The defects in the right atrium from ablated hearts were pointed out by an arrow. The littermate control mice include both double-negative and Pdgfra-CreER+/− mice. Scale bar = 500µm. (D) Quantification of the compact and trabecular myocardium thickness in LV and RV (6 sections from 3 ablated hearts and 4 sections from 2 control hearts). (E) Quantification of the ratio of trabecular to compact myocardium in LV and RV after one dose of tamoxifen treatment (6 sections from 3 ablated hearts and 4 sections from 2 control hearts). (F) Quantification of pHH3 positive cells in ventricular in control and ablated hearts after one dose of tamoxifen treatment (6 sections from 3 ablated hearts and 8 sections from 4 control hearts). (G) Quantification of pHH3 positive cells in ventricular in control and ablated hearts after three doses of tamoxifen treatments (12 sections from 4 ablated hearts and 12 sections from 4 control hearts). * represents p<0.05; ** represents p<0.01.

(A, B) Collagen accumulation in control and ablated hearts at P18. Scale bar=500µm and 150µm in the whole heart sections and enlarged sections, respectively.

(A) Representative images of brain, lung, kidney, and liver from control and Pdgfra-CreER; Rosa-DTA mice at P18 with tamoxifen treatment at P3 to P5. Scale bar = 5mm. (B) Collagen staining of lung from control and ablated mice at P18. The littermate control mice were Pdgfra-CreER+/−. Scale bar=500µm and 150µm in the whole heart sections and enlarged sections, respectively.

Echocardiographic analysis of heart function in anesthetized control and ablated mice at P18.

The littermate control mice were Pdgfra-CreER+/−.