The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra)–phosphoinositide 3-kinase (PI3K) signaling cascade to steer toward the midline during zebrafish heart tube formation
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eLife assessment
This is a valuable study that shows the involvement of phosphoinositide 3-kinase (PI3K) signaling downstream of platelet-derived growth factor receptor alpha in latero-medial migration of cardiomyocytes during the formation of the early heart tube during zebrafish development. The authors provide convincing evidence using multiple drugs and expression of a dominant negative PI3K subunit, to inhibit the pathway, approaches that show the strong alignment of phenotypes, and which are quantified using live imaging. The demonstration of cardiomyocyte protrusions biased in the direction of migration, and randomised after PI3K inhibition, is a promising area for future exploration.
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Abstract
Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move toward the midline (cardiac fusion) to form the primitive heart tube. Extrinsic influences such as the adjacent anterior endoderm are known to be required for cardiac fusion. We previously showed however, that the platelet-derived growth factor receptor alpha (Pdgfra) is also required for cardiac fusion (Bloomekatz et al., 2017). Nevertheless, an intrinsic mechanism that regulates myocardial movement has not been elucidated. Here, we show that the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway has an essential intrinsic role in the myocardium directing movement toward the midline. In vivo imaging further reveals midline-oriented dynamic myocardial membrane protrusions that become unpolarized in PI3K-inhibited zebrafish embryos where myocardial movements are misdirected and slower. Moreover, we find that PI3K activity is dependent on and interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.
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eLife assessment
This is a valuable study that shows the involvement of phosphoinositide 3-kinase (PI3K) signaling downstream of platelet-derived growth factor receptor alpha in latero-medial migration of cardiomyocytes during the formation of the early heart tube during zebrafish development. The authors provide convincing evidence using multiple drugs and expression of a dominant negative PI3K subunit, to inhibit the pathway, approaches that show the strong alignment of phenotypes, and which are quantified using live imaging. The demonstration of cardiomyocyte protrusions biased in the direction of migration, and randomised after PI3K inhibition, is a promising area for future exploration.
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Reviewer #1 (Public Review):
This is a valuable study demonstrating convincingly that PI3K signaling lies downstream of Pdgfra signaling in zebrafish cardiomyocyte progenitors as they undergo latero-medial migration and midline fusion, essential for heart tube formation, likely via chemotaxis. Whereas the authors used both multiple inhibitory drugs and dominant negative transgene expression to interrupt PI3K expression, with findings strongly aligning, the manuscript would have been stronger if genetic approaches were used to complement the above approaches. Nonetheless, the impact of dnPI3K inhibition allowed the authors to suggest that the effects were cell autonomous to migrating cardiomyocytes. The authors used contemporary live imaging techniques allowing quantification of key cell behaviors, and this is a strength of the paper. …
Reviewer #1 (Public Review):
This is a valuable study demonstrating convincingly that PI3K signaling lies downstream of Pdgfra signaling in zebrafish cardiomyocyte progenitors as they undergo latero-medial migration and midline fusion, essential for heart tube formation, likely via chemotaxis. Whereas the authors used both multiple inhibitory drugs and dominant negative transgene expression to interrupt PI3K expression, with findings strongly aligning, the manuscript would have been stronger if genetic approaches were used to complement the above approaches. Nonetheless, the impact of dnPI3K inhibition allowed the authors to suggest that the effects were cell autonomous to migrating cardiomyocytes. The authors used contemporary live imaging techniques allowing quantification of key cell behaviors, and this is a strength of the paper. There are some issues about the inter-study alignment of trajectory data that need to be addressed. Perhaps the most conspicuous weakness is that the authors have not advanced the model for cardiomyocyte migration beyond adding the involvement of PI3K downstream of Pdgfra, which is to a significant degree expected. The recording of cardiomyocyte protrusions biased in their orientation towards the direction of migration, which is lost in the mutants, is an interesting advance, although it was not shown whether protrusions are causally related to migration.
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Reviewer #2 (Public Review):
The authors provide comprehensive results showing that pharmacological inhibition of PI3K negatively affects heart tube formation via misoriented and slower cardiac movements. They used several cellular and molecular assays to demonstrate the potential mechanisms involved in PI3K-dependent cardiac fusion defects. Moreover, they use several imaging techniques and quantitative assessments to support their findings. Although the manuscript is well-written and most of their results support their conclusions, the manuscript and its findings heavily rely on high concentrations of PI3K small-molecule inhibitors, which will have off-target effects. The off-targets of PI3K pharmacological inhibition should be interpreted with caution and further evaluated. The authors suggest PI3K inhibition mediates heart tube …
Reviewer #2 (Public Review):
The authors provide comprehensive results showing that pharmacological inhibition of PI3K negatively affects heart tube formation via misoriented and slower cardiac movements. They used several cellular and molecular assays to demonstrate the potential mechanisms involved in PI3K-dependent cardiac fusion defects. Moreover, they use several imaging techniques and quantitative assessments to support their findings. Although the manuscript is well-written and most of their results support their conclusions, the manuscript and its findings heavily rely on high concentrations of PI3K small-molecule inhibitors, which will have off-target effects. The off-targets of PI3K pharmacological inhibition should be interpreted with caution and further evaluated. The authors suggest PI3K inhibition mediates heart tube formation throughout PI3K-mediated migration defects rather than PI3K-mediated proliferative defects. However, the authors did not further evaluate this later point; it should be considered carefully.
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Reviewer #3 (Public Review):
This manuscript provides new insights into an important process during cardiac development that is not well understood. The authors combined chemical inhibition experiments for PI3K as well as a genetic tool to overexpress a dominant negative PI3K specifically in cardiac progenitor cells and found that PI3K is important during cardiac fusion. By incubating embryos with the chemical inhibitor at different stages they concluded that PI3K is required between 12-20 somite stages, which corresponds to the time points that cardiac fusion occurs. They performed live imaging on cardiac progenitors during cardiac fusion and observed that inhibiting Pi3K reduces the velocity at which the cells move and affects their direction. The latter seems consistent with the observation that PI3K is not required for protrusion …
Reviewer #3 (Public Review):
This manuscript provides new insights into an important process during cardiac development that is not well understood. The authors combined chemical inhibition experiments for PI3K as well as a genetic tool to overexpress a dominant negative PI3K specifically in cardiac progenitor cells and found that PI3K is important during cardiac fusion. By incubating embryos with the chemical inhibitor at different stages they concluded that PI3K is required between 12-20 somite stages, which corresponds to the time points that cardiac fusion occurs. They performed live imaging on cardiac progenitors during cardiac fusion and observed that inhibiting Pi3K reduces the velocity at which the cells move and affects their direction. The latter seems consistent with the observation that PI3K is not required for protrusion formation but affects the location of these protrusions. Finally, using a low dose of the PI3K inhibitor together with the previously identified Pdgf mutant suggests that both act in the same pathway to regulate the direction of migration of cardiac progenitor cels towards the midline. Overall, the manuscript is well written and experiments are well controlled providing sufficient evidence to substantiate most of their conclusions. Some open questions remain unanswered such as the mode of migration (individual or collective) that drives cardiac fusion.
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Author Response:
We would like to thank the reviewers for their time, insights, and constructive feedback. We appreciate the recognition by the reviewers of the value and importance of our study. The reviewers also highlighted: the importance of carefully using and interpreting data from small molecule inhibitors due to possible off-target effects, considering inter-study differences in the cardiomyocyte cell trajectories, examining a possible role of PI3K signaling in proliferation and the intriguing yet not fully elucidated role of membrane protrusions in cardiac fusion. We agree with this important feedback. We plan to address these comments and others directly, in detail.
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