Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis
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eLife assessment
This study showing how the Circle of Willis acquires smooth muscle coverage during development in the zebrafish model is important, and the evidence provided is solid, with only minor weaknesses. The work is of interest to researchers working on cerebral circulation, angiogenesis, and developmental vascular stabilization.
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Abstract
Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries, playing a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular diseases and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating differentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on the zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that the arterial expression of CoW endothelial cells (ECs) occurs after their migration from the cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors upon recruitment to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity wall shear stress. Furthermore, pulsatile blood flow is required for differentiation of human brain pdgfrb+ mural cells into VSMCs as well as VSMC differentiation on zebrafish CoW arteries. Consistently, the flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight the role of blood flow activation of endothelial klf2a as a mechanism regulating the initial VSMC differentiation on vertebrate brain arteries.
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eLife assessment
This study showing how the Circle of Willis acquires smooth muscle coverage during development in the zebrafish model is important, and the evidence provided is solid, with only minor weaknesses. The work is of interest to researchers working on cerebral circulation, angiogenesis, and developmental vascular stabilization.
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Reviewer #1 (Public Review):
Cheng et al investigated how vascular cells of the zebrafish Circle of Willis arteries differentiate using live imaging of transgenic zebrafish embryos. They find an anterior-to-posterior gradient in the differentiation of pdgfrb+ progenitors into acta2+ smooth muscle cells (SMCs). Computational modeling suggests that blood flow velocity and wall shear stress are higher in the anterior Circle of Willis arteries. Using pharmacological manipulations, they show that blood flow is required for the differentiation of SMCs but not for the short-term maintenance of the SMC differentiation state. They provide evidence that the increased expression of the flow-responsive Klf2 transcription factor in endothelial cells predates SMC differentiation, with the same anterior-to-posterior gradient, and that Klf2 expression …
Reviewer #1 (Public Review):
Cheng et al investigated how vascular cells of the zebrafish Circle of Willis arteries differentiate using live imaging of transgenic zebrafish embryos. They find an anterior-to-posterior gradient in the differentiation of pdgfrb+ progenitors into acta2+ smooth muscle cells (SMCs). Computational modeling suggests that blood flow velocity and wall shear stress are higher in the anterior Circle of Willis arteries. Using pharmacological manipulations, they show that blood flow is required for the differentiation of SMCs but not for the short-term maintenance of the SMC differentiation state. They provide evidence that the increased expression of the flow-responsive Klf2 transcription factor in endothelial cells predates SMC differentiation, with the same anterior-to-posterior gradient, and that Klf2 expression is required for SMC differentiation.
Overall, the study is very well-conducted and the paper is well-written. These important data point to hemodynamics as an important driver of artery muscularization in the Circle of Willis.
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Reviewer #2 (Public Review):
Summary:
Cheng et al. explore the development of the arteries that form the Circle of Willis and investigate how blood flow pulsatility influences vascular smooth muscle cell (VSMC) differentiation. Using live confocal imaging of the developing zebrafish, the authors show that endothelial cells in the Circle of Willis arteries transition from venous to arterial identity between 54 hours post-fertilization (hpf) and 3 days post-fertilization (dpf), and that this coincides with pdgfrb+ mural cell progenitor differentiation into acta2+ arterial VSMCs. They find that the anterior portions of the Circle of Willis, including the internal carotid arteries (CaDI), establish acta2 expression earlier than posterior aspects, likely due to faster flow rate and increased pulsatility through the CaDI. Then, using …Reviewer #2 (Public Review):
Summary:
Cheng et al. explore the development of the arteries that form the Circle of Willis and investigate how blood flow pulsatility influences vascular smooth muscle cell (VSMC) differentiation. Using live confocal imaging of the developing zebrafish, the authors show that endothelial cells in the Circle of Willis arteries transition from venous to arterial identity between 54 hours post-fertilization (hpf) and 3 days post-fertilization (dpf), and that this coincides with pdgfrb+ mural cell progenitor differentiation into acta2+ arterial VSMCs. They find that the anterior portions of the Circle of Willis, including the internal carotid arteries (CaDI), establish acta2 expression earlier than posterior aspects, likely due to faster flow rate and increased pulsatility through the CaDI. Then, using computational fluid dynamics, an in vitro co-culture assay, and genetic and drug manipulations of blood flow, the authors provide evidence that pdgfrb+ differentiation is dependent upon pulsatile blood flow and klf2a activation. The results add to our understanding of vascular development and suggest that deficits in pulsatile flow could be potential drivers of arteriopathies.Strengths:
Longitudinal confocal imaging of live developing zebrafish makes the timeline of arterial development in the Circle of Willis easy to understand. This is a strong approach to studying how vascular networks are altered with genetic and pharmacological manipulations.
Rigorous use of multiple techniques to test the hypothesis that pulsatile blood flow is required for smooth muscle cell differentiation. The microangiography experiment, in vitro co-culture assay, and genetic and drug manipulations of heart rate at various developmental time points yield outcomes that are consistent with the hypothesis.
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Reviewer #3 (Public Review):
Cheng et al. studied if and how blood flow regulates the differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire an arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.
Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations …
Reviewer #3 (Public Review):
Cheng et al. studied if and how blood flow regulates the differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire an arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.
Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations regarding the role of blood flow and klf2 involvement have been previously made in other systems and other vascular beds, for example, mouse klf2 mutants, which have deficient VSMC coverage of the dorsal aorta (Wu et al., 2008, JBC 283: 3942-50). The results convincingly show that VSMC differentiation in CW depends on the blood flow and that klf2a flow-dependent function regulates VSMC differentiation.
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