Primordial cardiomyocytes orchestrate myocardial morphogenesis and vascularization but are dispensable for regeneration

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important question in cardiac biology: whether distinct cardiomyocyte (CM) subpopulations play specialized roles during heart development and regeneration. Using single-cell RNA sequencing and newly generated genetic tools, the authors identify phlda2 as a specific marker of primordial cardiomyocytes in the adult zebrafish heart. They further show that these primordial CMs function are essential for myocardial morphogenesis and coronary vascularization but are dispensable for myocardial regeneration or revascularization after injury. These findings indicate that heart regeneration doesn't simply recapitulate developmental processes.

Strengths:

A major strength of the study is the generation of a phlda2 BAC reporter, which provides a specific and reliable marker for primordial cardiomyocytes. The lack of genetic tools has previously limited functional analysis of this CM population. By using phlda2 regulatory elements to generate reporter and NTR-based ablation lines, the authors can visualize and selectively manipulate primordial CMs in vivo. This enables a direct functional interrogation rather than relying on lineage tracing or correlative evidence. Through genetic ablation, the authors convincingly demonstrate that primordial CMs are essential for myocardial morphogenesis and coronary vascular organization during development but are not necessary for heart regeneration.

Weaknesses:

(1) The manuscript would benefit from clarifying whether the primordial cardiomyocytes ablation affects epicardial cell behaviors during heart development, given that the well-established role of the epicardium in supporting coronary vessel growth, it is possible that the vascular phenotypes observed after primordial CM ablation may be affected, at least in part, by altered epicardial cells.

(2) Because primordial cardiomyocytes form a dense, single-cell-thick layer covering the ventricular surface, it would be informative to determine whether their loss alters the spatial distribution or inward migration of coronary endothelial cells or epicardial cells.

(3) The manuscript carefully examines the relationship between primordial CMs and gata4⁺ cardiomyocytes during regeneration. However, their relationship during heart development should be more fully addressed.

(4) As loss of cardiomyocytes is known to induce gata4:GFP activation during regeneration, it would be important to determine whether ablation of primordial cardiomyocytes alone triggers gata4:GFP expression in neighboring cardiomyocytes. This analysis would further support the conclusion that primordial cardiomyocytes are not required for regenerative responses.

Reviewer #2 (Public review):

Summary:

In the manuscript "Primordial Cardiomyocytes orchestrate myocardial morphogenesis and vascularization but are dispensable for regeneration", Sun et al. identify a novel marker of primordial cardiomyocytes and use it to visualize and ablate the population during development and regeneration. The role of the primordial layer has not been investigated because the tools to manipulate this population have not existed. The manuscript is straightforward, easy to understand, and addresses an important question that has not been explored.

While the manuscript provides important insights into the role of primordial CMs, backed by a convincing methodology, the authors should clarify their requirements for heart development and maturation. Specifically, is the primordial layer required for the fish to survive? Do primordial CMs regenerate when ablated during development, and do the defects observed (in trabecular and compact CMs and coronary vessels) resolve after 10 days post-treatment when they were detected?

Strengths:

The major strengths are the identification of a marker that enables manipulation of primordial cardiomyocytes and the tools generated by the team.

Weaknesses:

The major weakness is not considering the longer-term consequences of primordial layer ablation during development, as it is unclear whether the animals succumb to the acute cardiac defects observed or fully recover.

Reviewer #3 (Public review):

Summary:

The authors performed single-cell RNA sequencing of adult zebrafish hearts and identified markers for distinct cardiomyocyte subpopulations. One marker, phlda2, marks primordial cardiomyocytes. They generated transgenic reporter lines to characterize phlda2 expression patterns and a phlda2-NTR ablation line to determine the functional requirement of primordial cardiomyocytes during heart regeneration. They found that phlda2+ primordial cardiomyocytes are essential for myocardial morphogenesis and coronary vessel development. Interestingly, when phlda2+ primordial cardiomyocytes are ablated during heart regeneration, gata4+ cortical cardiomyocytes, coronary vessel revascularization, and scar tissue formation are not affected.

Strengths:

The authors identified a new primordial cardiomyocyte marker, phlda2. They further demonstrated that primordial cardiomyocytes are important for heart morphogenesis but dispensable for heart regeneration. Their findings reveal a potential difference between heart development and regeneration programs.

Weakness:

Despite the interesting findings, the authors did not provide supplemental data for their scRNAseq to demonstrate the data quality and support their conclusions, and some results are not well described.

Author response:

We thank the reviewer for the thoughtful and constructive evaluation of our work and for recognizing its potential interest to researchers working on cardiac development and regeneration. We are planning to address the specific concerns as noted by the reviewers in the following way:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important question in cardiac biology: whether distinct cardiomyocyte (CM) subpopulations play specialized roles during heart development and regeneration. Using single-cell RNA sequencing and newly generated genetic tools, the authors identify phlda2 as a specific marker of primordial cardiomyocytes in the adult zebrafish heart. They further show that these primordial CMs function are essential for myocardial morphogenesis and coronary vascularization but are dispensable for myocardial regeneration or revascularization after injury. These findings indicate that heart regeneration doesn't simply recapitulate developmental processes.

Strengths:

A major strength of the study is the generation of a phlda2 BAC reporter, which provides a specific and reliable marker for primordial cardiomyocytes. The lack of genetic tools has previously limited functional analysis of this CM population. By using phlda2 regulatory elements to generate reporter and NTR-based ablation lines, the authors can visualize and selectively manipulate primordial CMs in vivo. This enables a direct functional interrogation rather than relying on lineage tracing or correlative evidence. Through genetic ablation, the authors convincingly demonstrate that primordial CMs are essential for myocardial morphogenesis and coronary vascular organization during development but are not necessary for heart regeneration.

Weaknesses:

(1) The manuscript would benefit from clarifying whether the primordial cardiomyocytes ablation affects epicardial cell behaviors during heart development, given that the well-established role of the epicardium in supporting coronary vessel growth, it is possible that the vascular phenotypes observed after primordial CM ablation may be affected, at least in part, by altered epicardial cells.

We thank the reviewer for this thoughtful comment and agree that primordial cardiomyocyte ablation may indirectly affect coronary vessel growth through changes in epicardial cell behavior. Therefore, we will perform additional analyses to examine epicardial cell behaviors, including epicardial coverage and migration following primordial cardiomyocyte ablation using the established epicardial reporter line tcf21:nucEGFP during heart development.

(2) Because primordial cardiomyocytes form a dense, single-cell-thick layer covering the ventricular surface, it would be informative to determine whether their loss alters the spatial distribution or inward migration of coronary endothelial cells or epicardial cells.

We thank the reviewer for this important comment. We will analyze the spatial distribution and inward migration of coronary endothelial and epicardial cells after primordial cardiomyocyte ablation using high-resolution imaging and quantitative analysis

(3) The manuscript carefully examines the relationship between primordial CMs and gata4⁺ cardiomyocytes during regeneration. However, their relationship during heart development should be more fully addressed.

We appreciate the suggestion and will carefully investigate the relationship between primordial cardiomyocytes and gata4⁺ cardiomyocytes during heart development.

(4) As loss of cardiomyocytes is known to induce gata4:GFP activation during regeneration, it would be important to determine whether ablation of primordial cardiomyocytes alone triggers gata4:GFP expression in neighboring cardiomyocytes. This analysis would further support the conclusion that primordial cardiomyocytes are not required for regenerative responses.

We acknowledge the reviewer’s comments and will test whether primordial cardiomyocyte ablation induces gata4:GFP activation in neighboring cardiomyocytes in the adult heart.

Reviewer #2 (Public review):

Summary:

In the manuscript "Primordial Cardiomyocytes orchestrate myocardial morphogenesis and vascularization but are dispensable for regeneration", Sun et al. identify a novel marker of primordial cardiomyocytes and use it to visualize and ablate the population during development and regeneration. The role of the primordial layer has not been investigated because the tools to manipulate this population have not existed. The manuscript is straightforward, easy to understand, and addresses an important question that has not been explored.

While the manuscript provides important insights into the role of primordial CMs, backed by a convincing methodology, the authors should clarify their requirements for heart development and maturation. Specifically, is the primordial layer required for the fish to survive?

We thank the reviewer for this important question. We will examine the survival of fish following primordial cardiomyocyte ablation during development.

Do primordial CMs regenerate when ablated during development, and do the defects observed (in trabecular and compact CMs and coronary vessels) resolve after 10 days post-treatment when they were detected?

We thank the reviewer for this valuable comment. We will perform additional analyses to determine whether primordial cardiomyocytes regenerate after ablation during development and to assess the extent and dynamics of their recovery. We will also evaluate whether the defects in trabecular and compact myocardium and coronary vasculature persist or resolve in adult hearts following primordial cardiomyocyte ablation during development.

Reviewer #3 (Public review):

Summary:

The authors performed single-cell RNA sequencing of adult zebrafish hearts and identified markers for distinct cardiomyocyte subpopulations. One marker, phlda2, marks primordial cardiomyocytes. They generated transgenic reporter lines to characterize phlda2 expression patterns and a phlda2-NTR ablation line to determine the functional requirement of primordial cardiomyocytes during heart regeneration. They found that phlda2+ primordial cardiomyocytes are essential for myocardial morphogenesis and coronary vessel development. Interestingly, when phlda2+ primordial cardiomyocytes are ablated during heart regeneration, gata4+ cortical cardiomyocytes, coronary vessel revascularization, and scar tissue formation are not affected.

Strengths:

The authors identified a new primordial cardiomyocyte marker, phlda2. They further demonstrated that primordial cardiomyocytes are important for heart morphogenesis but dispensable for heart regeneration. Their findings reveal a potential difference between heart development and regeneration programs.

Weakness:

Despite the interesting findings, the authors did not provide supplemental data for their scRNAseq to demonstrate the data quality and support their conclusions, and some results are not well described.

We appreciate the reviewer’s comment. We will include supplemental data to demonstrate the quality of our single-cell RNA sequencing. Additionally, we will provide more detailed descriptions of the key results in the main text and figure legends to clearly support our conclusions regarding primordial cardiomyocytes and their roles in heart morphogenesis and regeneration.