Endothelial Slit2 guides the Robo1-positive sympathetic innervation during heart development
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eLife Assessment
This study presents a valuable finding on the role of Slit-Robo signaling in cardiac innervation. The evidence supporting the main claims of the authors is solid. The use of several mouse models including constitutive and cell type specific knockout models make the findings more robust. The scope of the presented studies is somewhat limited, as they primarily focus on evaluating the phenotypic changes in cardiac innervation following the loss of various Slit or Robo genes.
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Abstract
Abstract
Axon guidance cues direct nerves in the heart during development, disease and regeneration. These cues determine cardiac innervation patterning by regulating the balance between chemo-attraction and chemo-repulsion. However, the role of one of the most crucial ligand-receptor combinations among axon guidance molecules, the Slit chemo-active ligands and their Roundabout (Robo) transmembrane receptors, remains unknown during cardiac innervation patterning. To test if Slit-Robo signalling is important for cardiac innervation guidance, we analysed Slit and Robo mouse knock-outs. Constitutive Slit2-/- ventricles showed significantly reduced innervation, while Slit3-/- hearts showed temporary increased levels of innervation compared to wild-type littermate controls. Whereas innervation was not affected in Robo2-/-mice, the phenotype seen in Slit2-/-ventricles was recapitulated in Robo1-/-mice. Detailed expression analysis identified expression of Slit2 ligand in the endothelium of the coronary vessels, while Slit3 was highly present in the coronary smooth muscle wall and in the innervation. Both the Robo1 and Robo2 receptors were present in the nerves and at low levels in the vessels. Knocking out Slit2 specifically in the endothelium recapitulated the defects seen in the constitutive Slit2-/-hearts. Ex vivo axon guidance cultures showed that attraction of axons extending from the ganglia was strongly reduced in ventricles with absence of endothelial Slit2 compared to wild-type controls. In absence of endothelial Slit2, adult mice showed reduced response to challenging the sympathetic innervation. In conclusion, we have identified an important new chemo-active Slit2-Robo1 pathway required for correct cardiac innervation development.
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eLife Assessment
This study presents a valuable finding on the role of Slit-Robo signaling in cardiac innervation. The evidence supporting the main claims of the authors is solid. The use of several mouse models including constitutive and cell type specific knockout models make the findings more robust. The scope of the presented studies is somewhat limited, as they primarily focus on evaluating the phenotypic changes in cardiac innervation following the loss of various Slit or Robo genes.
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Reviewer #1 (Public review):
The study aims to determine the role of Slit-Robo signaling in the development and patterning of cardiac innervation, a key process in heart development. Despite the well-studied roles of Slit axon guidance molecules in the development of the central nervous system, their roles in the peripheral nervous system are less clear. Thus, the present study addresses an important question. The study uses genetic knockout models to investigate how Slit2, Slit3, Robo1, and Robo2 contribute to cardiac innervation.
Using constitutive and cell type-specific knockout mouse models, they show that the loss of endothelial-derived Slit2 reduces cardiac innervation. Additionally, Robo1 knockout, but not Robo2 knockout, recapitulated the Slit2 knockout effect on cardiac innervation, leading to the conclusion that Slit2-Robo1 …
Reviewer #1 (Public review):
The study aims to determine the role of Slit-Robo signaling in the development and patterning of cardiac innervation, a key process in heart development. Despite the well-studied roles of Slit axon guidance molecules in the development of the central nervous system, their roles in the peripheral nervous system are less clear. Thus, the present study addresses an important question. The study uses genetic knockout models to investigate how Slit2, Slit3, Robo1, and Robo2 contribute to cardiac innervation.
Using constitutive and cell type-specific knockout mouse models, they show that the loss of endothelial-derived Slit2 reduces cardiac innervation. Additionally, Robo1 knockout, but not Robo2 knockout, recapitulated the Slit2 knockout effect on cardiac innervation, leading to the conclusion that Slit2-Robo1 signaling drives sympathetic innervation in the heart. Finally, the authors also show a reduction in isoproterenol-stimulated heart rate but not basal heart rate in the absence of endothelial Slit2.
The conclusions of this paper are mostly well supported by the data, but some should be modified to account for the study's limitations and discussed in the context of previous literature.
(1) It is well established that Slit ligands undergo proteolytic cleavage, generating N- and C-terminal fragments with distinct biological functions. Full-length Slit proteins and their fragments differ in cell association, with the N-terminal fragment typically remaining membrane-bound, while the C-terminal fragment is more diffusible. This distinction is crucial when evaluating the role of Slit proteins secreted by different cell types in the heart. However, this study does not examine or discuss the specific contributions of different Slit2 fragments, limiting its mechanistic insight into how Slit2 regulates cardiac innervation.
(2) The endothelial-specific deletion of Slit2 leads to its loss in endothelial cells across various organs and tissues in the developing embryo. Therefore, the phenotypes observed in the heart may be influenced by defects in other parts of the embryo, such as the CNS or sympathetic ganglia, and this possibility cannot be ruled out.
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Reviewer #2 (Public review):
The aims of investigating Slit-Robo signaling in cardiac innervation were achieved by the experiments designed. While questions remain regarding signal regulation and interplay between established axon guidance signals and further role of other Slit ligands and Robo expression in endothelium, the results strongly support the conclusions drawn.
Writing and presentation are easy to follow and well structured, Appropriate controls are used, statistical analysis applied appropriately, and experiments directly test aims following a logical story.
The authors demonstrate a novel mechanism for Slit-Robo signaling in cardiac sympathetic innervation. The data establishes a framework for future studies.
Recommendations:
Further assessment of interplay between Slit ligands as well as other signaling pathways …
Reviewer #2 (Public review):
The aims of investigating Slit-Robo signaling in cardiac innervation were achieved by the experiments designed. While questions remain regarding signal regulation and interplay between established axon guidance signals and further role of other Slit ligands and Robo expression in endothelium, the results strongly support the conclusions drawn.
Writing and presentation are easy to follow and well structured, Appropriate controls are used, statistical analysis applied appropriately, and experiments directly test aims following a logical story.
The authors demonstrate a novel mechanism for Slit-Robo signaling in cardiac sympathetic innervation. The data establishes a framework for future studies.
Recommendations:
Further assessment of interplay between Slit ligands as well as other signaling pathways (Semaphorin, NGF, etc) could be investigated. Is it possible to rescue the phenotype by modulation of other signaling pathways? Can combined Slit2/Slit3 KO rescue? Additionally, as the authors state, conditional Robo1 knockouts will be important to validate the findings of constitutive knockout.
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