Functional Analysis of ROCK1 and ROCK2 in Vascular Development Using Zebrafish

Dysregulated Rho-Associated Kinase 1 and 2 (ROCK1/2) protein activity has been implicated in the development of cranial vascular malformations, leading to severe hemorrhage and stroke in human patients. However, the individual roles of ROCK1 versus ROCK2 in regulating blood vessel development have yet to be comprehensively characterized. We use zebrafish as a model organism to determine if and how Rock1 and Rock2 regulate vascular development and integrity. To determine if Rock1 and/or Rock2 are required for blood vessel integrity or patterning, we use CRISPR/Cas9 to mutate rock1 and rock2 protein-encoding genes individually or in combination in transgenic zebrafish embryos with fluorescently labelled blood vessels and imaging these embryos using florescence confocal microscopy. Additionally, to determine whether mutating the rock1 gene leads to transcriptional compensation by rock2 and/or vice versa, we use quantitative real-time PCR (qRT-PCR) to measure relative rock1 and rock2 mRNA expression levels in CRISPR/Cas9-injected zebrafish embryos. We find potential distinct roles of rock1 and rock2a/2b in trunk vascular development and cranial vascular development respectively. Further, we uncover evidence for the occurrence of genetic compensation by rock1 following the mutagenesis of rock2a/2b in combination.