Sox Factors Transcriptionally Regulate ROBO4 Gene Expression in Developing Vasculature in Zebrafish

Abstract

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from ؊1170 bp to ؊1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain-and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.In developing vertebrates, neural and vascular patterning generate intricate branching networks that share several similar features (1). However, this connectivity is governed by a limited toolkit of signaling receptor systems. These systems must therefore be subjected to exquisite control to achieve proper patterning and avoid miscues. Recently, members of the axon guidance family have shown both expression and functionality in the developing vasculature. Of the four distinct families of axon guidance signaling partners, namely Slit-Robo, Ephrin-Eph, Netrin-Unc, and Semaphorin-Plexin, our laboratory has focused on the Slit-Robo family members and their role in the vasculature.Roundabouts (Robos), 4 a class of cell surface receptors that were originally identified to function in axon guidance (2), have recently been implicated in providing critical directional information for migration of endothelial cells (ECs) (3, 4). Four mammalian Robos are known, of which the fourth member robo4 is expressed in the intersomitic vessels (ISVs) and is strikingly regulated with peak expression passing in a "wave" along the trunk axis from 19 -29 somites (3), suggesting a high degree of transcriptional control of this gene product. Recently, a 3-kb human ROBO4 promoter sequence has been identified that directs endothelial cell-specific expression pattern in vivo and in vitro (5). In addition, a guanine and adenine-binding protein-binding element in the ROBO4 promoter is necessary for endothelial expression in vivo (6). However, to date little is known regarding transcription factors that are involved in regulating robo4 expression during embryoni...

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