The Rac1 Regulator ELMO1 Controls Vascular Morphogenesis in Zebrafish

Abstract

Key Words: Rac1 Ⅲ ELMO1 Ⅲ DOCK180 Ⅲ vascular morphogenesis Ⅲ zebrafish A ngiogenesis, the formation of blood vessels from preexisting ones, is a crucial process during embryonic development as well as in several pathological conditions such as during tumor growth and in ischemic diseases. 1 Angiogenesis is activated by angiogenic growth factors, such as vascular endothelial growth factor (VEGF), leading to the formation of endothelial sprouts. 1 During the last few years it has been shown that angiogenic sprouts consist of 3 types of functionally different endothelial cell types, namely the tip cells, the stalk cells and the phalanx cells. 2 The tip cells are migratory cells at the leading front of the forming angiogenic sprouts; stalk cells are primarily formed by cell proliferation, and phalanx cells represent a more quiescent endothelium. Endothelial cell proliferation and migration have been intensively studied, and several important major signaling cascades have been identified. Based on the recognition of novel molecules regulating these major pathways 3 it is becoming increasingly important to identify and functional characterize the regulators for blood vessel formation. The small GTPases RhoA, Rac1, and Cdc42 have been shown to regulate migration of endothelial cells during angiogenesis. 4 -8 Rac1 is an essential factor during embryonic development as its endothelial specific deletion leads to an early embryonic vascular lethal phenotype. 7 Rac1 is ubiquitously expressed in 20 hpf zebrafish embryos 9 at the start phase of intersomitic vessel formation. 10 Therefore, a specific temporal and spatial regulation of Rac1 activation in the vascular system implies the existence of additional regulators for Rac1. Several important questions regarding the regulation and function of Rac1 in the embryonic and adult vasculature remain unanswered. Little is known about (1) which intracellular proteins modulate the activation of Rac1; (2) the upstream regulators of Rac1 in vivo are; and (3) the functions of these regulators in the vasculature in vivo are.The ELMO1/DOCK180 (engulfment and cell motility 1/dedicator of cytokinesis 180; also known as DOCK1) complex is an unusual GEF for Rac1 regulating cell migration in Caenorhabditis elegans, Drosophila melanogaster, and glioblastomas. 11,12 ELMO1 contains a pleckstrin homology domain for interaction with DOCK180. 13 DOCK180 uses a conserved "Docker" or "CZH2" domain to mediate the nucleotide exchange on Rac1. 11 Binding of ELMO1 to DOCK180 induces a conformational change releasing the DOCK180 self inhibitory loop. Consequently, the activated ELMO1/DOCK180 complex stabilizes Rac1 in its nucleotide free transition state and regulates its localization. 11 has not yet been reported. The aim of this study was to analyze the function of the ELMO1/DOCK180 complex in vascular development and to identify the upstream regulators of the bipartite GEF ELMO1/ DOCK180 in the vascular system. We report a strong, temporally controlled vascular expression of elmo1 in zebrafis...

High Tissue Glucose Alters Intersomitic Blood Vessels in Zebrafish via Methylglyoxal Targeting the VEGF Receptor Signaling Cascade

“…At 48 hpf, when the major trunk vessel in zebrafish have already been formed (37) and where VEGF receptor 2 is strongly activated (Fig. 4A and B), a low dose (0.2 mmol/L) of VEGF receptor 2 antagonist PTK787/Vatalanib was added to tg(fli:EGFP) high tissue MG and high tissue glucose zebrafish embryos and, in this concentration, did not affect physiological blood vessel development in zebrafish (38).…”

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

“…Embryos were kept in E3 solution at 28.5°C with or without 0.003 % 1-phenyl-2-thiourea (Sigma) to suppress pigmentation and staged according to somite number or hours post-fertilization [16]. Morpholinos were diluted in 0.1 M KCl and 1 nL of morpholino dilution was injected through the chorion of one-cell or two-cell stage embryos.…”

“…To attenuate possible off-target effects, a p53 Mo was co-injected 1.5-fold to the other morpholinos used. The following splice-blocking morpholinos (Gene Tools) were used: Quantification of vessel defects in tg(fli1:EGFP) embryos of intersomitic vessels (ISV) and the dorsal longitudinal anastomotic vessel (DLAV) was performed as recently described [16]. Parachordal vessel/parachordal lymphangioblasts (PL) were quantified as recently described [16].…”

“…The following splice-blocking morpholinos (Gene Tools) were used: Quantification of vessel defects in tg(fli1:EGFP) embryos of intersomitic vessels (ISV) and the dorsal longitudinal anastomotic vessel (DLAV) was performed as recently described [16]. Parachordal vessel/parachordal lymphangioblasts (PL) were quantified as recently described [16]. In brief, embryos were divided in three groups according to the presence (2), partial (1) or complete absence (0) of the parachordal lymphangioblasts.…”

Histone Deacetylase 9 Promotes Angiogenesis by Targeting the Antiangiogenic MicroRNA-17–92 Cluster in Endothelial Cells

“…40 Embryos were kept in E3 solution at 28.5°C with or without 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to suppress pigmentation and staged according to somite number or hours postfertilization. 41 …”