roundabout4 is essential for angiogenesis in vivo

Abstract

Stereotypical patterns of vascular and neuronal networks suggest that specific genetic programs tightly control path determination and, consequently, angiogenesis and axon-guidance mechanisms. Our study focuses on one member of the roundabout family of receptors, which traditionally mediate repulsion from the midline. Here, we characterize a fourth member of this family, roundabout4 (robo4), which is the predominant roundabout (robo) that is expressed in embryonic zebrafish vasculature. Gene knockdown and overexpression approaches show that robo4 is essential for coordinated symmetric and directed sprouting of intersomitic vessels and provide mechanistic insights into this process. Also, human robo4 gene functionally compensates for loss of robo4 gene function, suggesting evolutionary conservation. This article reports an endothelial-specific function for a robo gene in vertebrates in vivo.axon guidance ͉ endothelial cell ͉ zebrafish V ascular and neural networks established by angiogenesis and neurogenesis are essential for the regulation of physiological processes. Whether the processes of blood-vessel and peripheralnerve formation are intimately associated at a mechanistic level is a subject of active investigation (1). Recently, description of cellsurface molecules that are shared by neuronal and endothelial cells has suggested that this association may be more prevalent than previously thought. Neuropilin (2), ephrin (3), and plexin (4) are a few examples of molecules that are implicated in both processes. Recently, roundabout (robo), a class of neural guidance receptors that bind slit ligands have joined this group (5). slit-robo signaling mediates axonal repulsion (6) and inhibition of leukocyte migration (7). In vertebrate systems, three robo receptor family members were identified, all with prominent neural expression (8, 9). More recently, a fourth member of the robo gene family, roundabout4 (robo4) was identified (10). Compared with the canonical robo structure, robo4 is smaller in that it possesses only two of the five Ig and two of the three fibronectin domains present in the extracellular component of robo1, robo2, and robo3 (11). robo4 has been described as endothelial-specific, and it binds to slit and inhibits the migration of heterologous cells that express robo4 and primary endothelial cells (12). Although studies of robo4 in endothelial cells suggest that robo signaling is important for regulating endothelial cell migration, there are no reports that document the role of robo signaling in vascular development in vivo. Here, we cloned a zebrafish (Danio rerio) ortholog of human robo4 (hrobo4) and studied its expression and role in vascular development by gene knockdown and overexpression approaches. In contrast to the endothelial-specific expression of murine robo4, zebrafish robo4 is expressed in both endothelial and neural tissues with vascular expression seen in angioblasts, the dorsal aorta (DA), the posterior cardinal vein, and intersomitic vessels (ISV). Morpholino (MO) knockdown of robo...

Subconjunctival gene delivery of the transcription factor GA-binding protein delays corneal neovascularization in a mouse model

“…24,25 Also, a GABP-binding motif is necessary for mediating Robo4 expression in the intact endothelium. 35 By contrast, the Robo4 receptor is essential for Slit2-dependent inhibition of VEGF-165-induced pulmonary endothelial cell migration, tube formation, and permeability in vitro and for inhibiting VEGF-165-driven vascular leaking in vivo by blocking Src family kinase activation.…”

“…22 Moreover, SP1 transcription factor and GABP bind to the human roundabout4 (Robo4) promoter in primary endothelial cells from human coronary artery and induce promoter activation. 23 Robo4 is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis, 24,25 and also in stabilizing the vasculature. 26 Therefore, in the present study, we investigated whether in vivo subconjunctival gene delivery of GABP transcription factor affects gene expression pattern of Robo4 and VEGF in the corneal neovascularization induced by chemical and mechanical injuries.…”

Potential role of the Slit/Robo signal pathway in angiogenesis

“…30 However, during vascular development in the zebrafish embryo, both Robo4 overexpression and knockdown experiments showed an inhibitory effect of Robo4 on cell migration. 26 Questions still remain as to how Robo4 disrupts the directed guidance of vascular sprouts on a molecular basis and on an in vivo basis, and why it requires a precise expression level of Robo4 ( Figure 2B).…”

“…Indeed, independent experiments show angiogenic properties of Robo or Slit proteins in vitro and in vivo. So far, Slit/Robo signaling has been approached from four different directions: (1) studies on Robo function; 20,26 (2) studies on Slit function; 7 (3) studies on the unknown ligand for Robo; 27 and (4) studies on molecular signals that guide ECs migration. 28 Partly, the results from these experiments were contradictory, and the ongoing new experiments are expected to establish the specific role of the Slit/Robo signal in the process of angiogenesis.…”

“…In vivo studies by Bedell et al reported the essential role of Robo4 during vascular development in embryonic zebrafish. 26 When morpholino (MO) phosphorodiamidate oligonucleotides against Robo4 mRNA was injected into a one-cell stage zebrafish embryo, MO knockdown zebrafish showed either a lack or abnormal shape of intersomitic vessels (ISV). Interestingly, overexpression of Robo4 did not induce ISV sprouts.…”