Department of Biological Sciences, National Sun Yat-sen University, Taiwan; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan; Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei, Taiwan.
Department of Biological Sciences, National Sun Yat-sen University, Taiwan; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan.
Biochem Biophys Res Commun. 2022 May 14;604:123-129. doi: 10.1016/j.bbrc.2022.03.042. Epub 2022 Mar 9.
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.
许多控制动脉特征的调节因子已经得到很好的描述；然而，促进静脉特征和血管模式形成的转录因子在很大程度上仍然未知。我们之前已经确定了转录因子 Islet2（Isl2）和 Nr2f1b，它们在斑马鱼中通过 Notch 途径介导的静脉和尖端细胞特征的指定中是必需的。然而，Isl2 和 Nr2f1b 之间的相互作用尚不清楚。在这项研究中，我们报告 Nr2f2 在静脉和节间血管（ISV）生长中发挥次要作用，并使用组合敲低策略剖析这三个转录因子 Isl2、Nr2f1b 和 Nr2f2 之间的遗传相互作用。Isl2/nr2f1b、Isl2/nr2f2 和 nr2f1b/nr2f2 的双重敲低显示出血管系统缺陷增强，包括不完全的 ISV、减少的静脉和 ISV 细胞。我们进一步测试了这三个转录因子之间的遗传关系。我们发现 Isl2 可以调节 Nr2f1b 和 Nr2f2 的表达，表明 Isl2 在上游调节 Nr2f1b 和 Nr2f2 的模型。我们假设 Isl2 和 Nr2f1b 可以通过顺式调节结合基序共同发挥作用。在体外荧光素酶测定结果中，我们表明 Isl2 和 Nr2f1b 可以协同增强基因表达。此外，共免疫沉淀结果表明 Isl2 和 Nr2f1b 物理相互作用。综上所述，我们表明 Nr2f1b 和 Nr2f2 转录因子的相互作用与 Islet2 一起在斑马鱼的血管发育中发挥协调作用。
