The regenerative response of cardiac interstitial cells
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Curated by eLife
Evaluation Summary:
This paper is of interest to researchers studying cardiac regeneration and regeneration in general. Given the multiple papers characterizing interstitial cell types during mammalian cardiac injury response, this type of characterization in one of the leading regeneration model systems is overdue. The authors utilize single cell sequencing approaches to identify dynamics and key features of interstitial cell populations during zebrafish cardiac regeneration, which largely supports the claims of the paper. The data presented here have the potential to serve as a valuable reference resource for future studies, although some of the conclusions, in particular those on the function of tal1 and mmp14, are not yet sufficiently supported by data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during this process, we performed single-cell RNA sequencing of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition, and novel transgenic animals, we were able to investigate the role of cell type-specific mechanisms during cardiac regeneration. This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations. Here, we provide detailed insight into how interstitial cells behave during cardiac regeneration, which will serve to increase our understanding of how this process could eventually be induced in humans.
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Evaluation Summary:
This paper is of interest to researchers studying cardiac regeneration and regeneration in general. Given the multiple papers characterizing interstitial cell types during mammalian cardiac injury response, this type of characterization in one of the leading regeneration model systems is overdue. The authors utilize single cell sequencing approaches to identify dynamics and key features of interstitial cell populations during zebrafish cardiac regeneration, which largely supports the claims of the paper. The data presented here have the potential to serve as a valuable reference resource for future studies, although some of the conclusions, in particular those on the function of tal1 and mmp14, are not yet sufficiently supported by data.
(This preprint has been reviewed by eLife. We include the public reviews from …
Evaluation Summary:
This paper is of interest to researchers studying cardiac regeneration and regeneration in general. Given the multiple papers characterizing interstitial cell types during mammalian cardiac injury response, this type of characterization in one of the leading regeneration model systems is overdue. The authors utilize single cell sequencing approaches to identify dynamics and key features of interstitial cell populations during zebrafish cardiac regeneration, which largely supports the claims of the paper. The data presented here have the potential to serve as a valuable reference resource for future studies, although some of the conclusions, in particular those on the function of tal1 and mmp14, are not yet sufficiently supported by data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors use single-cell sequencing to identify non-muscle cell populations in the regenerating zebrafish heart and based on this they provide evidence that the transcription factor tal1 is required in endothelial cells and the matrix metalloproteinase mmp14 in macrophages for heart regeneration. While the sequencing data represent a nice resource for future studies and surely will be of interest to a large community of researchers studying heart regeneration and regeneration in general, some of the conclusions, in particular those on the function of tal1 and mmp14, are not yet sufficiently supported by data.
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Reviewer #2 (Public Review):
Rolland et al. analysed the composition and relative quantity of different cardiac cell types, 3, 7 and 14 days after ventricle resection injury in zebrafish and showed mechanistic contributions of these cell types to regeneration. They focused on changes in interstitial cells and characterized cardiac cell populations including macrophages epicardial/epicardial derived, neural crest and endothelial cells. Alongside the identification and marker assignment to different cell types, the authors also provide some novel mechanistic insights to regeneration. First, the lack-of-upregulation of the myofibroblast injury associated gene acta2, which upon inhibition of matrix metalloproteinases (MMPs) and subsequent inhibition of regeneration is upregulated in a similar way to mammals. Second, tal1+ endothelium, which …
Reviewer #2 (Public Review):
Rolland et al. analysed the composition and relative quantity of different cardiac cell types, 3, 7 and 14 days after ventricle resection injury in zebrafish and showed mechanistic contributions of these cell types to regeneration. They focused on changes in interstitial cells and characterized cardiac cell populations including macrophages epicardial/epicardial derived, neural crest and endothelial cells. Alongside the identification and marker assignment to different cell types, the authors also provide some novel mechanistic insights to regeneration. First, the lack-of-upregulation of the myofibroblast injury associated gene acta2, which upon inhibition of matrix metalloproteinases (MMPs) and subsequent inhibition of regeneration is upregulated in a similar way to mammals. Second, tal1+ endothelium, which was previously shown to be important to development was suggested to have dynamic interactions with binding partners as the tal1+ cells express different levels of lmo2/cgnl1 on different days following injury. Tal1+ endothelium was shown to be crucial for proper regeneration as expressing its dominant negative form resulted in fibrosis following injury. Third, mmp14b, which was expressed by macrophages and epicardial derived cells, is crucial for zebrafish cardiac regeneration, possibly by affecting macrophage numbers in the tissue, highlighting the importance of specific MMPs to the regeneration process.
The findings of this paper could be broadly separated to the characterization of the interstitial cells by scRNA-seq and transgenic reporter lines, and the regeneration specific processes facilitated by these cells. For the first part the conclusions of this paper are largely supported by data and will undoubtedly be used as a very valuable reference dataset for future single cell studies. Regarding the mechanistic insights, some questions need to be clarified.
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Reviewer #3 (Public Review):
The zebrafish is an excellent model to study cardiac regeneration as this process is very efficient when compared to injury induced scaring of the mammalian heart. The scRNA-seq data may be a very valuable resource for the research community. The first part of the manuscript largely deals with the description of the different cell types identified by the scRNAseq analysis. Cardiomyocytes seems to be under-represented, which could be due to their large size and shape. All other major cell types were identified based their expression patterns and some cell types (endothelium, fibroblast, macrophages) could be subclustered. This resulted in the identification of a sox10+ cell type within the fibroblast cluster. While interesting, it remains unclear what these cells are and whether these are true neural crest …
Reviewer #3 (Public Review):
The zebrafish is an excellent model to study cardiac regeneration as this process is very efficient when compared to injury induced scaring of the mammalian heart. The scRNA-seq data may be a very valuable resource for the research community. The first part of the manuscript largely deals with the description of the different cell types identified by the scRNAseq analysis. Cardiomyocytes seems to be under-represented, which could be due to their large size and shape. All other major cell types were identified based their expression patterns and some cell types (endothelium, fibroblast, macrophages) could be subclustered. This resulted in the identification of a sox10+ cell type within the fibroblast cluster. While interesting, it remains unclear what these cells are and whether these are true neural crest derived cells as claimed by the authors. In the second part of the manuscript the authors investigate Tal1+ endothelial cells and generate a transgenic line with an endothelial specific and inducible expression of a dominant negative (dn)Tal1 protein. Expression of the dnTal1 in endothelial cells results in impaired regeneration, albeit the role of Tal1 in endothelial cells during regeneration was not studied in more detail. In the last part of this study, the authors focus on the macrophage sub clusters that they identified from the scRNAseq data. The authors claim that these sub clusters represent resident and recruited macrophages, but these claims need experimental validation. Macrophages that appear after injury express mmp14 and an Mmp14 inhibitor is used to reveal a possible role for Mmp14. Treatment of fish with the mmp14 inhibitor resulted in a reduced number of macrophages in the wound area, differences in their transcriptome and impaired heart regeneration. Whether macrophage numbers decline due to reduced proliferation or migration was not addressed nor how Mmp14 inhibition affects heart regeneration.
Overall, this work describes a very interesting scRNAseq dataset of the interstitial cells in the regenerating zebrafish heart. The second part of the manuscript contains new hypothesis which need careful validation to support the authors' conclusions.
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