State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
PLoS One. 2011;6(7):e22555. doi: 10.1371/journal.pone.0022555. Epub 2011 Jul 22.
Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.
本世纪初，人们获得了几种用于肝脏发育研究的转基因斑马鱼系，但尚未报道任何标记整个肝脏发育和器官发生过程的转基因 GFP 斑马鱼系。在这项研究中，我们通过整体原位杂交分析了内源性 Apo-14 在斑马鱼胚胎发生过程中的表达模式，并揭示了其在肝脏原基和随后的肝脏发育中的表达。随后，我们分离了 1763bp5'-侧翼序列的斑马鱼 Apo-14 启动子，并开发了一种 Apo-14 启动子驱动的转基因斑马鱼 Tg(Apo14: GFP)。并且，在转基因斑马鱼系中观察到了 Apo-14 启动子驱动 GFP 的母体表达和受精后易位。此外，我们通过将 Tg(Apo14: GFP)雄性与野生型雌性杂交，追踪早期杂合胚胎中 Apo-14 启动子驱动 GFP 的起始表达和表达细胞的发育行为。显著的是，在胚胎发育到尾芽突出的 10 hpf 时，Apo-14 启动子驱动 GFP 首先在胚胎体下的 YSL 周围表达。在 16-17 hpf 的约 14- somite 胚胎中，在卵黄囊周围的 YSL 中清楚地观察到典型的“盐和胡椒”表达模式。然后，在大约 20 hpf 时，在靠近耳囊的脊索和卵黄囊之间开始出现一个绿色荧光点，后来证明这是肝脏原基，它产生肝脏。此外，我们研究了 Tg(Apo14: GFP)斑马鱼中肝脏器官发生的动态进展，因为 Apo-14 启动子驱动 GFP 从肝脏原基中的肝母细胞和肝祖细胞持续表达到幼虫和成年肝脏中的肝细胞。此外，我们在 YSL 上观察到与肝祖细胞和 GFP 阳性核类似的形态，表明它们可能起源于早期胚胎中的同一祖细胞。总的来说，本研究提供了一种标记整个肝脏器官发生过程的转基因斑马鱼系。
