Apelin signaling dependent endocardial protrusions promote cardiac trabeculation in zebrafish
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Curated by eLife
Evaluation Summary:
The manuscript describes the presence and genetic control of endocardial cell protrusions in zebrafish hearts that resemble endocardial sprouts recently described in the mouse heart, and which appear necessary for the process of trabeculation, whereby chamber cardiomyocytes undergo staged morphogenesis to form a spongy inner layer. This manuscript is of broad interest to readers who study cardiogenesis and developmental biology. This first formal dissection of endocardial protrusions in zebrafish hearts describes how they anchor to cardiomyocytes, and how they participate in signaling pathways involved in trabeculation. The work combines elegant zebrafish reporters and high-quality imaging, as well as mutant lines and pathway inhibitors to provide key findings of how mutual regulation between the myocardium and the endocardium contribute to understanding of mechanisms underlying organ development.
(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 agreed to share their name with the authors.)
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Abstract
During cardiac development, endocardial cells (EdCs) produce growth factors to promote myocardial morphogenesis and growth. In particular, EdCs produce neuregulin which is required for ventricular cardiomyocytes (CMs) to seed the multicellular ridges known as trabeculae. Defects in neuregulin signaling, or in endocardial sprouting toward CMs, cause hypotrabeculation. However, the mechanisms underlying endocardial sprouting remain largely unknown. Here, we first show by live imaging in zebrafish embryos that EdCs interact with CMs via dynamic membrane protrusions. After touching CMs, these protrusions remain in close contact with their target despite the vigorous cardiac contractions. Loss of the CM-derived peptide Apelin, or of the Apelin receptor, which is expressed in EdCs, leads to reduced endocardial sprouting and hypotrabeculation. Mechanistically, neuregulin signaling requires endocardial protrusions to induce extracellular signal-regulated kinase (Erk) activity in CMs and trigger their delamination. Altogether, these data show that Apelin signaling-dependent endocardial protrusions modulate CM behavior during trabeculation.
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Evaluation Summary:
The manuscript describes the presence and genetic control of endocardial cell protrusions in zebrafish hearts that resemble endocardial sprouts recently described in the mouse heart, and which appear necessary for the process of trabeculation, whereby chamber cardiomyocytes undergo staged morphogenesis to form a spongy inner layer. This manuscript is of broad interest to readers who study cardiogenesis and developmental biology. This first formal dissection of endocardial protrusions in zebrafish hearts describes how they anchor to cardiomyocytes, and how they participate in signaling pathways involved in trabeculation. The work combines elegant zebrafish reporters and high-quality imaging, as well as mutant lines and pathway inhibitors to provide key findings of how mutual regulation between the myocardium and the …
Evaluation Summary:
The manuscript describes the presence and genetic control of endocardial cell protrusions in zebrafish hearts that resemble endocardial sprouts recently described in the mouse heart, and which appear necessary for the process of trabeculation, whereby chamber cardiomyocytes undergo staged morphogenesis to form a spongy inner layer. This manuscript is of broad interest to readers who study cardiogenesis and developmental biology. This first formal dissection of endocardial protrusions in zebrafish hearts describes how they anchor to cardiomyocytes, and how they participate in signaling pathways involved in trabeculation. The work combines elegant zebrafish reporters and high-quality imaging, as well as mutant lines and pathway inhibitors to provide key findings of how mutual regulation between the myocardium and the endocardium contribute to understanding of mechanisms underlying organ development.
(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 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Qi J, et l. investigated how trabeculation is regulated during early cardiogenesis of zebrafish. They claim an essential role for protrusion from the endocardial cells (EdCs) in cardiac trabeculation. The protrusions originate from EdCs express Apelin receptor B (Aplnrb) and respond to Apelin released by monolayered myocardium. Conversely, monolayered cardiomyocytes expressing ErbB proliferate to become multilayered cardiomyocytes (trabeculation) in response to Neuregulin 2a (Nrg2a). Nrg2/Erb2 signaling activates Erk in the cardiomyocytes, thereby leading to proliferation as evidenced by the Erk signaling monitoring transgenic zebrafish line. The authors further confirmed that not only Nrg2a-ErbB signaling but also protrusion is necessary for trabeculation by showing that the lack of protrusion in the …
Reviewer #1 (Public Review):
Qi J, et l. investigated how trabeculation is regulated during early cardiogenesis of zebrafish. They claim an essential role for protrusion from the endocardial cells (EdCs) in cardiac trabeculation. The protrusions originate from EdCs express Apelin receptor B (Aplnrb) and respond to Apelin released by monolayered myocardium. Conversely, monolayered cardiomyocytes expressing ErbB proliferate to become multilayered cardiomyocytes (trabeculation) in response to Neuregulin 2a (Nrg2a). Nrg2/Erb2 signaling activates Erk in the cardiomyocytes, thereby leading to proliferation as evidenced by the Erk signaling monitoring transgenic zebrafish line. The authors further confirmed that not only Nrg2a-ErbB signaling but also protrusion is necessary for trabeculation by showing that the lack of protrusion in the endocardium (overexpression of dominant negative form of IRSp53) resulted in failure of trabeculation in the endocardium-specific Nrg2-overexpressingtransgenic fish.
The explanation and interpretation steps overall support their conclusions, because the experiments were well designed and were done by high-quality imaging techniques and by using several important transgenic zebrafish lines. The present manuscript would be improved by some revision to convince readers interested in the role of protrusions in trabeculation. -
Reviewer #2 (Public Review):
This manuscript by Qi and Helker et al. describes the presence of endocardial protrusions in the zebrafish heart that may be functionally analogous to endocardial sprouts recently described in the mouse heart, which appear necessary for determining trabecular architecture. The paper focuses on the necessity for Apelin/Apelin receptor b signaling for formation of protrusions and subsequent activation of the neuregulin signaling pathway via Erbb2/Erk activation, and for cardiomyocyte extrusion to form the trabecular layer. The authors combine elegant zebrafish reporters and imaging, as well as mutant lines and pathway inhibitors to make the case. Endocardial sprouting/protrusion formation appears to be analogous to endothelial sprouting in developmental vascular beds occurring in response to hypoxia and …
Reviewer #2 (Public Review):
This manuscript by Qi and Helker et al. describes the presence of endocardial protrusions in the zebrafish heart that may be functionally analogous to endocardial sprouts recently described in the mouse heart, which appear necessary for determining trabecular architecture. The paper focuses on the necessity for Apelin/Apelin receptor b signaling for formation of protrusions and subsequent activation of the neuregulin signaling pathway via Erbb2/Erk activation, and for cardiomyocyte extrusion to form the trabecular layer. The authors combine elegant zebrafish reporters and imaging, as well as mutant lines and pathway inhibitors to make the case. Endocardial sprouting/protrusion formation appears to be analogous to endothelial sprouting in developmental vascular beds occurring in response to hypoxia and signalling gradients, and which involves metastable Notch-driven cell fate changes. Whereas Apelin signaling has been shown to be involved in endothelial bed sprouting, there are few pathways that have been identified to be involved in endocardial sprouting in mouse hearts. This is the first comprehensive description of endocardial protrusion formation and "touchdown" in zebrafish hearts and the data reveal that elaboration of protrusions is necessary for neuregulin signaling. These are valuable contributions to the field of heart development and chamber formation, and deepen our conviction that trabeculation is a deeply conserved process worthy of detailed study in the zebrafish model. For the most part the authors justify their claims. The high quality imaging and movies allows for quantitative measurement of morphological progressions, and without doubt the range of lineage, signaling and intracellular compartmental reporters and mutations make this a high quality study. Weaknesses in the manuscript in its current form relate to the lack of clear definitions of the individual steps of trabeculation and how this then relates to the interpretation of genetic or over-expression phenotypes, particularly those surrounding nrg2 over-expression. This leads to some uncertainty about conclusions. Insufficient evidence is provided for how protrustions relate spatially to cardiomyocytes "chosen" to extrude from the outer layer to become trabeculae. Notch signaling, one of the key pathways involved in trabeculation, is dealt with at a fairly superficial level.
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