Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish.

In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.

A zebrafish toolbox for biomechanical signaling in cardiovascular development and disease.

PURPOSE OF REVIEW: The zebrafish embryo has emerged as a powerful model organism to investigate the mechanisms by which biophysical forces regulate vascular and cardiac cell biology during development and disease. A versatile arsenal of methods and tools is available to manipulate and analyze biomechanical signaling. This review aims to provide an overview of the experimental strategies and tools that have been utilized to study biomechanical signaling in cardiovascular developmental processes and different vascular disease models in the zebrafish embryo. Within the scope of this review, we focus on work published during the last two years. RECENT FINDINGS: Genetic and pharmacological tools for the manipulation of cardiac function allow alterations of hemodynamic flow patterns in the zebrafish embryo and various types of transgenic lines are available to report endothelial cell responses to biophysical forces. These tools have not only revealed the impact of biophysical forces on cardiovascular development but also helped to establish more accurate models for cardiovascular diseases including cerebral cavernous malformations, hereditary hemorrhagic telangiectasias, arteriovenous malformations, and lymphangiopathies. SUMMARY: The zebrafish embryo is a valuable vertebrate model in which in-vivo manipulations of biophysical forces due to cardiac contractility and blood flow can be performed. These analyses give important insights into biomechanical signaling pathways that control endothelial and endocardial cell behaviors. The technical advances using this vertebrate model will advance our understanding of the impact of biophysical forces in cardiovascular pathologies.

Blood Flow Suppresses Vascular Anomalies in a Zebrafish Model of Cerebral Cavernous Malformations.

RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.

The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity.

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease.