Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo

The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment.

肝脏在新陈代谢、解毒、消化及稳态维持中发挥关键作用。在发育进程中，脊椎动物胚胎肝脏会经历一系列被称为肝发生（hepatogenesis）的形态发生事件。肝发生可分为三个相互关联的阶段：内胚层特化、肝母细胞分化以及肝脏生长。在此过程中，信号分子与转录因子启动并调控细胞增殖、凋亡、分化、细胞间黏附及细胞迁移的协同进程。目前已知缺氧诱导因子（Hypoxia-inducible factor，HIF）在胚胎发育中不可或缺，但其在肝发生中的作用仍不明晰。本研究以斑马鱼胚胎为模式生物，结果显示敲低缺氧诱导因子2α（HIF-2α）而非缺氧诱导因子1α（HIF-1α）会阻断肝脏生长。尽管HIF-2α不参与肝母细胞特化过程，但该转录因子可在肝脏生长阶段调控肝细胞增殖。此外，我们证实敲低HIF-2α会降低肝脏富集基因1（liver-enriched gene 1，leg1）的表达，该基因编码一种对肝脏生长至关重要的分泌蛋白。同时，leg1的外源性mRNA表达可挽救hif2-α敲低胚胎的肝脏过小表型。我们还发现，HIF-2α可直接结合leg1的启动子区域以调控其表达。有趣的是，我们在硬骨鱼leg1的潜在上游调控序列中发现了富集的高密度HIF结合位点，但在陆生哺乳动物中并未发现此类位点。综上，我们认为HIF-2α是肝脏生长所需的关键因子，并可在斑马鱼胚胎中调控leg1的表达。我们还提出，肝脏发育中的hif2-α-leg1信号轴可能是硬骨鱼适应其生存环境的演化结果。