Contraction-induced endocardial id2b plays a dual role in regulating myocardial contractility and valve formation

Reviewer #1 (Public review):

Summary:
Chen et al. identified the role of endocardial id2b expression in cardiac contraction and valve formation through pharmaceutical, genetic, electrophysiology, calcium imaging, and echocardiography analyses. CRISPR/Cas9 generated id2b mutants demonstrated defective AV valve formation, excitation-contraction coupling, reduced endocardial cell proliferation in AV valve, retrograde blood flow, and lethal effects.

Strengths:
Their methods, data and analyses broadly support their claims.

Weaknesses:
The molecular mechanism is somewhat preliminary.

Reviewer #2 (Public review):

Summary:
Biomechanical forces, such as blood flow, are crucial for organ formation, including heart development. This study by Shuo Chen et al. aims to understand how cardiac cells respond to these forces. They used zebrafish as a model organism due to its unique strengths, such as the ability to survive without heartbeats, and conducted transcriptomic analysis on hearts with impaired contractility. They thereby identified id2b as a gene regulated by blood flow and is crucial for proper heart development, in particular, for the regulation of myocardial contractility and valve formation. Using both in situ hybridization and transgenic fish they showed that id2b is specifically expressed in the endocardium, and its expression is affected by both pharmacological and genetic perturbations of contraction. They further generated a null mutant of id2b to show that loss of id2b results in heart malformation and early lethality in zebrafish. Atrioventricular (AV) and excitation-contraction coupling were also impaired in id2b mutants. Mechanistically, they demonstrate that Id2b interacts with the transcription factor Tcf3b to restrict its activity. When id2b is deleted, the repressor activity of Tcf3b is enhanced, leading to suppression of the expression of nrg1 (neuregulin 1), a key factor for heart development. Importantly, injecting tcf3b morpholino into id2b-/- embryos partially restores the reduced heart rate. Moreover, treatment of zebrafish embryos with the Erbb2 inhibitor AG1478 results in decreased heart rate, in line with a model in which Id2b modulates heart development via the Nrg1/Erbb2 axis. The research identifies id2b as a biomechanical signaling-sensitive gene in endocardial cells that mediates communication between the endocardium and myocardium, which is essential for heart morphogenesis and function.

Strengths:
The study provides novel insights into the molecular mechanisms by which biomechanical forces influence heart development and highlights the importance of id2b in this process.

Weaknesses:
The claims are in general well supported by experimental evidence, but the following aspects may benefit from further investigation:

(1) In Figure 1C, the heatmap demonstrates the up-regulated and down-regulated genes upon tricane-induced cardiac arrest. Aside from the down-regulation of id2b expression, it was also evident that id2a expression was up-regulated. As a predicted paralog of id2b, it would be interesting to see whether the up-regulation of id2a in response to tricane treatment was a compensatory response to the down-regulation of id2b expression.

(2) The study mentioned that id2b is tightly regulated by the flow-sensitive primary cilia-klf2 signaling axis; however aside from showing the reduced expression of id2b in klf2a and klf2b mutants, there was no further evidence to solidify the functional link between id2b and klf2. It would therefore be ideal, in the present study, to demonstrate how Klf2, which is a transcriptional regulator, transduces biomechanical stimuli to Id2b.

(3) The authors showed the physical interaction between ectopically expressed FLAG-Id2b and HA-Tcf3b in HEK293T cells. Although the constructs being expressed are of zebrafish origin, it would be nice to show in vivo that the two proteins interact.

Reviewer #3 (Public review):

Summary:
How mechanical forces transmitted by blood flow contribute to normal cardiac development remains incompletely understood. Using the unique advantages of the zebrafish model system, Chen et al make the fundamental discovery that endocardial expression of id2b is induced by blood flow and required for normal atrioventricular canal (AVC) valve development and myocardial contractility by regulating calcium dynamics. Mechanistically, the authors suggest that Id2b binds to Tcf3b in endocardial cells, which relieves Tcf3b-mediated transcriptional repression of Neuregulin 1 (NRG1). Nrg1 then induces expression of the L-type calcium channel component LRRC1. This study significantly advances our understanding of flow-mediated valve formation and myocardial function.

Strengths:
Strengths of the study are the significance of the question being addressed, use of the zebrafish model, and data quality (mostly very nice imaging). The text is also well-written and easy to understand.

Weaknesses:
Weaknesses include a lack of rigor for key experimental approaches, which led to skepticism surrounding the main findings. Specific issues were the use of morpholinos instead of genetic mutants for the bmp ligands, cilia gene ift88, and tcf3b, lack of an explicit model surrounding BMP versus blood flow induced endocardial id2b expression, use of bar graphs without dots, the artificial nature of assessing the physical interaction of Tcf3b and Id2b in transfected HEK293 cells, and artificial nature of examining the function of the tcf3b binding sites upstream of nrg1.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Chen et al. identified the role of endocardial id2b expression in cardiac contraction and valve formation through pharmaceutical, genetic, electrophysiology, calcium imaging, and echocardiography analyses. CRISPR/Cas9 generated id2b mutants demonstrated defective AV valve formation, excitation-contraction coupling, reduced endocardial cell proliferation in AV valve, retrograde blood flow, and lethal effects.

Strengths:

Their methods, data and analyses broadly support their claims.

Weaknesses:

The molecular mechanism is somewhat preliminary.

We thank the reviewer for the constructive comments. To further elucidate the molecular mechanisms underlying the observed phenotypes, we will conduct the following experiments: (1) perform qRT-PCR to analyze the expression of id2a in hearts isolated from tricane-treated embryos and in id2b-deleted embryos; (2) use RNAscope to detect the expression of id2b in developing embryos; (3) validate the interaction between Id2b and Tcf3b in vivo; and (4) conduct CUT&Tag experiments in developing zebrafish embryos to further validate the Tcf3b binding sites upstream of nrg1.

Reviewer #2 (Public review):

Summary:

Biomechanical forces, such as blood flow, are crucial for organ formation, including heart development. This study by Shuo Chen et al. aims to understand how cardiac cells respond to these forces. They used zebrafish as a model organism due to its unique strengths, such as the ability to survive without heartbeats, and conducted transcriptomic analysis on hearts with impaired contractility. They thereby identified id2b as a gene regulated by blood flow and is crucial for proper heart development, in particular, for the regulation of myocardial contractility and valve formation. Using both in situ hybridization and transgenic fish they showed that id2b is specifically expressed in the endocardium, and its expression is affected by both pharmacological and genetic perturbations of contraction. They further generated a null mutant of id2b to show that loss of id2b results in heart malformation and early lethality in zebrafish. Atrioventricular (AV) and excitation-contraction coupling were also impaired in id2b mutants. Mechanistically, they demonstrate that Id2b interacts with the transcription factor Tcf3b to restrict its activity. When id2b is deleted, the repressor activity of Tcf3b is enhanced, leading to suppression of the expression of nrg1 (neuregulin 1), a key factor for heart development. Importantly, injecting tcf3b morpholino into id2b-/- embryos partially restores the reduced heart rate. Moreover, treatment of zebrafish embryos with the Erbb2 inhibitor AG1478 results in decreased heart rate, in line with a model in which Id2b modulates heart development via the Nrg1/Erbb2 axis. The research identifies id2b as a biomechanical signaling-sensitive gene in endocardial cells that mediates communication between the endocardium and myocardium, which is essential for heart morphogenesis and function.

Strengths:

The study provides novel insights into the molecular mechanisms by which biomechanical forces influence heart development and highlights the importance of id2b in this process.

Weaknesses:

The claims are in general well supported by experimental evidence, but the following aspects may benefit from further investigation:

(1) In Figure 1C, the heatmap demonstrates the up-regulated and down-regulated genes upon tricane-induced cardiac arrest. Aside from the down-regulation of id2b expression, it was also evident that id2a expression was up-regulated. As a predicted paralog of id2b, it would be interesting to see whether the up-regulation of id2a in response to tricaine treatment was a compensatory response to the down-regulation of id2b expression.

As suggested by the reviewer, we will perform qRT-PCR to analyze the expression of id2a in hearts isolated from tricane-treated embryos, as well as in id2b-deleted embryos.

(2) The study mentioned that id2b is tightly regulated by the flow-sensitive primary cilia-klf2 signaling axis; however aside from showing the reduced expression of id2b in klf2a and klf2b mutants, there was no further evidence to solidify the functional link between id2b and klf2. It would therefore be ideal, in the present study, to demonstrate how Klf2, which is a transcriptional regulator, transduces biomechanical stimuli to Id2b.

We have examined the expression levels of id2b in both klf2a and klf2b mutants. The whole mount in situ results clearly demonstrate a decrease in id2b signal in both mutants. As noted by the reviewer, klf2 is a transcriptional regulator, suggesting that the regulation of id2b may occur at the transcriptional level. However, dissecting the molecular mechanisms underling the crosstalk between klf2 and id2b is beyond the scope of the present study.

(3) The authors showed the physical interaction between ectopically expressed FLAG-Id2b and HA-Tcf3b in HEK293T cells. Although the constructs being expressed are of zebrafish origin, it would be nice to show in vivo that the two proteins interact.

We agree with the reviewer and will perform additional experiments to validate the interaction between Id2b and Tcf3b in vivo. Due to the lack of antibodies targeting these proteins, we will overexpress Flag-id2b and HA-Tcf3b in zebrafish embryos and conduct a co-IP analysis.

Reviewer #3 (Public review):

Summary:

How mechanical forces transmitted by blood flow contribute to normal cardiac development remains incompletely understood. Using the unique advantages of the zebrafish model system, Chen et al make the fundamental discovery that endocardial expression of id2b is induced by blood flow and required for normal atrioventricular canal (AVC) valve development and myocardial contractility by regulating calcium dynamics. Mechanistically, the authors suggest that Id2b binds to Tcf3b in endocardial cells, which relieves Tcf3b-mediated transcriptional repression of Neuregulin 1 (NRG1). Nrg1 then induces expression of the L-type calcium channel component LRRC1. This study significantly advances our understanding of flow-mediated valve formation and myocardial function.

Strengths:

Strengths of the study are the significance of the question being addressed, use of the zebrafish model, and data quality (mostly very nice imaging). The text is also well-written and easy to understand.

Weaknesses:

Weaknesses include a lack of rigor for key experimental approaches, which led to skepticism surrounding the main findings. Specific issues were the use of morpholinos instead of genetic mutants for the bmp ligands, cilia gene ift88, and tcf3b, lack of an explicit model surrounding BMP versus blood flow induced endocardial id2b expression, use of bar graphs without dots, the artificial nature of assessing the physical interaction of Tcf3b and Id2b in transfected HEK293 cells, and artificial nature of examining the function of the tcf3b binding sites upstream of nrg1.

We thank the reviewer for the constructive assessments. Our specific responses are as follows:

(1) As all the morpholinos used in this study, including those targeting bmp ligands, the cilia gene ift88, and tcf3b, have been published and validated using genetic mutants in previous studies, we believe these loss-of-function analyses are sufficient to delineate their role in regulating id2b expression or function.

(2) To assess the role of BMP versus blood flow in regulating endocardial id2b expression, we plan to perform live imaging in the id2b:GFP knockin line prior to the initiation of the heartbeat, with or without of BMP inhibitors.

(3) We will revise the data presentation and use bar graphs with individual data points.

(4) We plan to perform additional Co-IP experiment in zebrafish embryos to assess the interaction between Tcf3b and Id2b.

(5) To further validate the tcf3b binding sites upstream of nrg1, we will conduct CUT&Tag experiments in developing zebrafish embryos.