Interaction of transcription factors Islet2 and Nr2f1b to control vascular patterning during zebrafish development.

Many regulators controlling arterial identity are well described; however, transcription factors that promote vein identity and vascular patterning have remained largely unknown. We previously identified the transcription factors Islet2 (Isl2) and Nr2f1b required for specification of the vein and tip cell identity mediated by notch pathway in zebrafish. However, the interaction between Isl2 and Nr2f1b is not known. In this study, we report that Nr2f2 plays minor roles on vein and intersegmental vessels (ISV) growth and dissect the genetic interactions among the three transcription factors Isl2, Nr2f1b, and Nr2f2 using a combinatorial knockdown strategy. The double knockdown of isl2/nr2f1b, isl2/nr2f2, and nr2f1b/nr2f2 showed the enhanced defects in vasculature including less completed ISV, reduced veins, and ISV cells. We further tested the genetic relationship among these three transcription factors. We found isl2 can regulate the expression of nr2f1b and nr2f2, suggesting a model where Isl2 functions upstream of Nr2f1b and Nr2f2. We hypothsized that Isl2 and Nr2f1b can function together through cis-regulatory binding motifs. In-vitro luciferase assay results, we showed that Isl2 and Nr2f1b can cooperatively enhance gene expression. Moreover, co-immunoprecipitation results indicated that Isl2 and Nr2f1b interact physically. Together, we showed that the interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish.

Coral-Derived Natural Marine Compound GB9 Impairs Vascular Development in Zebrafish.

Blood vessels in vertebrates are established and genetically controlled in an evolutionarily-conserved manner during embryogenesis. Disruption of vascular growth by chemical compounds or environmental hormones may cause developmental defects. This study analyzed the vascular impacts of marine compound GB9 in zebrafish. GB9 was isolated from the marine soft coral Capnella imbricata and had shown anti-neuroinflammatory and anti-nociceptive activities. However, the role of GB9 on vascular development has not been reported. We first tested the survival rate of embryos under exogenous 5, 7.5, 10, and 15 µM GB9 added to the medium and determined a sub-lethal dosage of 10 µM GB9 for further assay. Using transgenic Tg(fli:eGFP) fish to examine vascular development, we found that GB9 treatment impaired intersegmental vessel (ISV) growth and caudal vein plexus (CVP) patterning at 25 hours post-fertilization (hpf) and 30 hpf. GB9 exposure caused pericardial edema and impaired circulation at 48-52 hpf, which are common secondary effects of vascular defects and suggest the effects of GB9 on vascular development. Apoptic cell death analysis showed that vascular defects were not caused by cell death, but were likely due to the inhibition of migration and/or proliferation by examining ISV cell numbers. To test the molecular mechanisms of vascular defects in GB9-treated embryos, we examined the expression of vascular markers and found the decreased expression of vascular specific markers ephrinb2, flk, mrc1, and stabilin. In addition, we examined whether GB9 treatment impairs vascular growth due to an imbalance of redox homeostasis. We found an enhanced effect of vascular defects during GB9 and H2O2 co-treatment. Moreover, exogenous N-acetyl-cysteine (NAC) treatment rescued the vascular defects in GB9 treated embryos. Our results showed that GB9 exposure causes vascular defects likely mediated by the imbalance of redox homeostasis.