Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development

  1. Ragini Phansalkar
  2. Josephine Krieger
  3. Mingming Zhao
  4. Sai Saroja Kolluru
  5. Robert C Jones
  6. Stephen R Quake
  7. Irving Weissman
  8. Daniel Bernstein
  9. Virginia D Winn
  10. Gaetano D'Amato
  11. Kristy Red-Horse

  • Curated by eLife

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    Evaluation Summary:

    This study investigates the relationships of endothelial cells that comprise the coronary vessels of the heart in mouse and humans. Starting from the knowledge that two sources of progenitor cells contribute to the coronary vessels, the work shows that adult coronary endothelial cells do not retain expression memory of their source, nor do they respond differently to cardiac injury. Finally, human datasets were generated and compared to mouse to show overall strong similarity between the species in coronary endothelial cell subtypes, suggesting that mouse is a relevant model for translation to human treatments and therapies.

    (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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells ‘remember’ their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources—the endocardium (Endo) and sinus venosus (SV)—but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary endothelial cells (ECs) transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well established but still remodeling, capillary ECs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

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  1. Version published to 10.7554/elife.70246 on eLife
  2. Version published to 10.1101/2021.04.25.441380v2 on bioRxiv
  3. eLife

    Evaluation Summary:

    This study investigates the relationships of endothelial cells that comprise the coronary vessels of the heart in mouse and humans. Starting from the knowledge that two sources of progenitor cells contribute to the coronary vessels, the work shows that adult coronary endothelial cells do not retain expression memory of their source, nor do they respond differently to cardiac injury. Finally, human datasets were generated and compared to mouse to show overall strong similarity between the species in coronary endothelial cell subtypes, suggesting that mouse is a relevant model for translation to human treatments and therapies.

    (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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Phansalkar et al., have dissected the endothelial cell (EC) heterogeneity of cardiac blood vessels across development in mouse and human. They describe that the EC heterogeneities of cardiac blood vessels are sequentially governed by the progenitor sources and environmental cues, during initial and late development, respectively. Moreover, authors show that these ECs become homogeneous in adult. They also claim that human fetal hearts take on generally a similar path for establishment of cardiac blood vessels. Overall, although this study is descriptive and lacks mechanical insight, it is novel and intriguing. However, despite the thorough analysis of vast single-cell transcriptomic data, there are a number of interpretations that could be misleading and confusing. Therefore, they need more precise and detailed analysis on the sc-RNA data.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript by Phansalkar et al. investigates the relationships of endothelial cells (EC) that comprise the coronary vessels of the heart in mouse and humans using state-of-the-art analysis of new scRNA seq data from both mouse and humans, complemented by spatial localization studies. scRNA seq datasets from e12.5, e17.5, and adult mice with lineage markers for endocardial or sinus venosus (SV)-derived EC were analyzed, and the lineage-tagged mice were also used to investigate the relative contributions of EC from the lineages in a cardiac injury model. These studies revealed that initial transcriptional signatures followed the source of EC, but as development proceeded and in adults these signatures were lost, and the transcriptomes reflected the location of the EC, and that EC from both embryonic sources contributed to revascularization after cardiac injury. Finally, new scRNA seq human fetal coronary EC datasets were analyzed and cross-referenced to the mouse data, and the conclusion was that there was substantial overlap in the data.

    Overall, the study has many strengths. The experiments were very well-controlled, alternate hypotheses were considered, and the interpretation of the results was appropriate. The study used a variety of techniques to support conclusions, and the scRNA seq data was analyzed using multiple platforms and analytical tools. The impact of this work is substantial, as it indicates that coronary EC exhibit significant plasticity developmentally, a phenomenon that was observed in earlier studies but without the direct comparison of the expression profiles provided here, and it suggests that in this population there is little "memory" of source with time, rather that location and environmental inputs become the drivers of expression profiles. The analysis of a cardiac injury model reveals lack of functional diversification, since both lineages contributed to the healing. The cross-referencing to human scRNA seq data made good use of the mouse data to show overall concordance, with some minor differences. Minor issues with this version of the work center around presentation of the data, which is sometimes confusing, and that the second lineage was not labelled in the injury model but deduced from lack of labeling (although the labeling was rigorously described early in the study).

  6. eLife

    Reviewer #3 (Public Review):

    To vascularize heart, endothelial cells (ECs) originating from both endocardium (Endo) and sinus venosus (SV) form capillary plexus and become subsequent mature vessels from the outside-in (SV) or the inside-out (Endo). It has been unclear how ECs exhibit either Endo-specific or SV-specific gene expression during coronary vessel growth.

    Phansalkar R. et al. demonstrate that Endo- or SV-characteristic transcription has been retained in the early coronary capillary formation, while in the later stage Endo-derived and SV -derived cells converge to form capillary ECs. They utilized scRNAseq of lineage-traced mouse ECs during development and those of ECs of the mice with ischemia/reperfusion. They further investigated human coronary vessel development using human fetal hearts and found that Endo-derived ECs and SV-derived EC of fetal human heart converge and differentiate similarly to mice.

    The gene expression in Endo-derived ECs and SV-derived ECs is well characterized by scRNAseq and immunohistochemistry using sections of e12, e17.5, and adult mouse hearts. At 17.5e and later, the gene expression of capillary ECs is determined by the localization-dependent character rather than lineage (Endo or SV). Therefore, the authors suggest that localization-driven heterogeneity is observed in plexus ECs. Spatial gene expression implies that it depends on blood flow; less in septum (Cap1 cluster) and more in outer layer (Cap2 cluster).

    They demonstrate that during cardiac development and maturation, even after pathological conditions, coronary ECs exhibit convergent differentiation of two distinct lineages (Endo- and SV-derived cells).

  8. Version published to 10.1101/2021.04.25.441380v1 on bioRxiv