Cardiac Fibroblasts regulate myocardium and coronary vasculature development via the collagen signaling pathway

Reviewer #1 (Public review):

Summary:

The study by Deng et al reports single-cell expression analysis of developing mouse hearts and examines the requirements for cardiac fibroblasts in heart maturation. Much of this work is overlapping with previous studies, but the single-cell gene expression data may be useful to investigators in the field. The significance and scope of new findings are limited and major conclusions are largely based on correlative data.

Strengths:

The strengths of the manuscript are the new single-cell datasets and comprehensive approach to ablating cardiac fibroblasts in pre and postnatal development in mice.

Weaknesses:

There are several major weaknesses in the analysis and interpretation of the results.

(1) The major conclusions regarding collagen signaling and heart maturation are based on gene expression patterns and are not functionally validated. The potential downstream signaling pathways were not examined and known structural contributions of fibrillar collagen to heart maturation are not discussed.

(2) The heterogeneity of fibroblast populations and contributions to multiple structures in the developing heart are not well-considered in the analysis. The developmental targeting of fibroblasts will likely affect multiple structures in the embryonic heart and other organs. Lethality is described in some of these studies, but additional analysis is needed to determine the effects on heart morphogenesis or other organs beyond the focus on cardiomyocyte maturation being reported. In particular, the endocardial cushions and developing valves are likely to be affected in the prenatal ablations, but these structures are not included in the analyses.

(3) ECM complexity and extensive previous work on specific ECM proteins in heart development and maturation are not incorporated into the current study. Different types of collagen (basement membrane Col4, filamentous Col6, and fibrillar Col1) are known to be expressed in fibroblast populations in the developing heart and have been studied extensively. Much also has been reported for other ECM components mentioned in the current work.

Reviewer #2 (Public review):

This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. scRNA-seq analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.
Some of the major issues are described below.

(1) The CD31 immunostaining data (Figures 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

(2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Figure 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

(3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Figures 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

(4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

(5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

(6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

(7) What is the genotype of the control animals used in the study?

(8) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

Reviewer #3 (Public review):

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with a focus on the critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cell networks in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated hearts. Lastly, they reported compensatory collagen expression in long-term ablated neonate hearts. Overall, this study provides us with important insight into fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM-focused research.

Strengths:

The authors utilized good analyzing tools to investigate multiple databases of single-cell sequencing and Multi-seq. They identified significant pathways and cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with a human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used a very strident technique to study fibroblast functions by ablating one of the major cell populations of the heart. Considering the importance of the fibroblast population, intriguing in vivo findings were expected. Also, they analyzed the downstream genes in ablated hearts, but did not execute any experimental validation for any of the targets.