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In vivo dissection of Rhoa function in vascular development using zebrafish

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Abstract

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

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Abbreviations

EC:

Endothelial cell

RHOA:

Ras homolog gene family, member A

ROCK:

Rho-associated protein kinase

CCM:

Cerebral cavernous malformation

Hpf:

Hours post fertilization

ENU:

N-Ethyl-N-nitrosourea

SSLP:

Simple sequence length polymorphism

LDA:

Lateral dorsal aortae

PHBC:

Primordial hindbrain channel

CtA:

Cranial central artery

DA:

Dorsal aorta

CV:

Cardinal vein

ISV:

Intersegmental vessel

DN:

Dominant negative

CA:

Constitutively active

BBB:

Blood brain barrier

HUVEC:

Human umbilical vein endothelial cells

GFP:

Green fluorescent protein

EGFP:

Enhanced green fluorescent protein

UAS:

Upstream activating sequence

2A:

P2A viral cleavage peptide

Tg:

Transgenic

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Acknowledgements

The authors would like to thank members of the Weinstein laboratory for their critical comments on this manuscript. Schematics of larval zebrafish were created with BioRender.com.

Funding

This work was supported by the intramural program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (ZIA-HD001011 and ZIA-HD008915, to BMW).

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  1. Joseph J. Yano

    Present address: Department of Cell and Molecular Biology, University of Pennsylvania, 440 Curie Blvd, Philadelphia, PA, 19104, USA

  2. Amber N. Stratman

    Present address: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA

Authors and Affiliations

  1. Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Dr. Bethesda, Bethesda, MD, 20892, USA

    Laura M. Pillay, Joseph J. Yano, Andrew E. Davis, Matthew G. Butler, Megan O. Ezeude, Jong S. Park, Keith A. Barnes, Vanessa L. Reyes, Daniel Castranova, Aniket V. Gore, Matthew R. Swift, James R. Iben, Madeleine I. Kenton, Amber N. Stratman & Brant M. Weinstein

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Pillay, L.M., Yano, J.J., Davis, A.E. et al. In vivo dissection of Rhoa function in vascular development using zebrafish. Angiogenesis 25, 411–434 (2022). https://doi.org/10.1007/s10456-022-09834-9

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