IntroductionMicroRNAs (miRNAs) are highly conserved, single-stranded noncoding short RNA molecules (18-24 nucleotides) that regulate gene expression at the posttranscriptional level. miRNAs silence gene expression by inhibiting the translation of proteins from mRNAs or by promoting the degradation of mRNAs. After transcription of the primary miRNA transcripts from the genome, their maturation is mediated by the 2 RNase III endonucleases Dicer and Drosha. Then, mature miRNAs are incorporated into the RNA-induced silencing complex, 1 which mediates the binding of the miRNA to the 3Ј-untranslated region (3Ј-UTR) of the target mRNA leading either to translational repression or degradation of the target mRNA. 2 Because miRNAs control specific expression patterns of target genes, miRNAs represent attractive candidates to interfere with neovascularization.Increasing evidence indicates that miRNAs are important regulators of vascular development and angiogenesis. 3,4 In this context, first studies addressed the function of the miRNAprocessing enzymes Dicer and/or Drosha to explore the general role of miRNAs for angiogenesis. Depletion of Dicer in zebrafish or mice revealed an aberrant vessel growth, and silencing of Dicer in endothelial cells reduced in vitro angiogenesis. [5][6][7] To date, several miRs that regulate endothelial cell function and angiogenesis have been identified, 8 including the pro-angiogenic miRs miR-130a, 9 miR-210, 5,10,11 and miR-378. 12 In addition, miR-126 was shown to regulate vascular integrity and angiogenesis during development and in ischemia-induced angiogenesis. [13][14][15] In contrast, miR-221 and miR-222, 7,16 miR-15 and miR-16, 17,18 and members of the miR-17-92 cluster 19,20 inhibit angiogenesis.In our previous study, we found that the members of the miR-23ϳ27ϳ24 cluster, miR-27a and miR-27b, were highly expressed in endothelial cells. 6 In addition, miR-27b was downregulated after Dicer and Drosha silencing, and inhibition of miR-27b significantly reduced endothelial cell sprouting in vitro, 6 indicating that miR-27b exerts pro-angiogenic effects. Recently, Zhou et al demonstrated that the miR-23ϳ27ϳ24 cluster regulates angiogenesis. 21 In muscle stem cells, miR-27b down-regulates Pax3 expression during myogenic differentiation. 22 Moreover, miR-27 down-regulates Runx1 expression during granulocyte differentiation 23 and the nuclear receptor peroxisome proliferatoractivated receptor-␥ (PPAR-␥) in adipocytes. 24 The myocyte enhancer factor 2C (MEF2C) is another important target of miR-27b during heart development. 25 However, the specific functions and targets of miR-27 in endothelial cells are largely unexplored. As the family members miR-27a and miR-27b differ in only one nucleotide and share the same seed sequence, we investigated the specific role of both family members for the angiogenic activity of endothelial cells and determined the effects on neovascularization. Here we identified the angiogenesis inhibitor semaphorin 6A as a The online version of this article contains a ...

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...