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.
Highlights
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 a microarray transcriptional profile study comparing lymphatic endothelial cells (ECs) isolated from ragged-opossum (RaOp) mutant mice (RaOpϪ/Ϫ mice) [18, 19], which carries a mutation in Sox18 to lymphatic ECs obtained from WT mice, we observed an 8.5fold decrease in Robo4 transcript levels with no detectable change in Robo1 and Robo2 transcript levels detectable
We investigated whether robo4, sox7, and sox18 transcripts were co-expressed in the vasculature during embryonic zebrafish development stages (19 –24 som) where robo4 is expressed in intersomitic vessels (ISVs) [3]
Summary
Human roundabout; robo, zebrafish roundabout; DA, dorsal aorta; DIG, digoxigenin; fli, friend leukemia virus integration; GOF, gain-of-function; hpf, hours postfertilization; ISH, in situ hybridization; ISV, intersomitic vessel; HUVEC, human umbilical vein endothelial cell; LOF, loss-of-function; MO, morpholino; NT, neural tube; Op, opossum; som, somite. Sox and Sox Regulation of Robo Expression in Vessels in vivo, and this study provides evidence that suggests robo may serve as one candidate
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