摘要：脊椎动物的器官形成是一个复杂而又难以看到的发育过程。然而，依赖于转基因技术发展起来的细胞命运追踪和组织特异呈像就像黑暗中的灯光，可以照亮器官发生难以观察到的细节。就肝脏器官发育研究来说，本世纪以来虽已获得过几个标示肝脏发育的转基因斑马鱼品系，但迄今还没有报道过能标示肝脏全程发生特别是前期发育的转基因鱼。
最近，中国科学院水生生物研究所桂建芳研究员主持的实验室在对银鲫和石斑鱼14kDa阿朴脂蛋白（Apo-14）进行了分子和表达特征研究的基础上，进一步分析了Apo-14在斑马鱼胚胎发育中的时空表达图式，分离出斑马鱼Apo-14的启动子序列，构建出由Apo-14启动子序列驱动绿色荧光蛋白报告基因表达的转基因斑马鱼Tg(Apo14: GFP)，并观察到其母源表达和受精后转位。
用该转基因鱼雄鱼与野生型雌鱼交配，在其杂合胚胎中可以追踪绿色荧光蛋白起始表达的细胞及其详细的发育行为。有意思的是，当胚胎发育至10小时尾芽刚刚显现时，Apo-14启动子驱动的绿色荧光蛋白开始在卵黄多核层（YSL）表达，并在16至17小时胚胎发育到14体节时，其典型的盐椒状表达图式在卵黄多核层中明显可见。到20小时时，一个绿色荧光点出现在脊索和卵黄囊之间靠近视泡的区域，该荧光点随后被证实为发育成肝脏的肝原基。
进而，他们以此为标记，详细观察记录了肝脏器官发生的动态过程，因为Apo-14启动子驱动的绿色荧光蛋白持续地表达在肝原基和成鱼肝脏中的成肝细胞、肝祖细胞和肝细胞中。此外，还观察到肝祖细胞和卵黄多核层中的阳性细胞核有相似的形态特征，推测它们可能起源于早期胚胎的同一前体细胞。总之，这一成果为研究肝脏发育及其器官发生提供了一个从早期内胚层细胞就开始表达直到完整的肝脏器官形成都可观察的新的转基因鱼。
该研究得到国家基础研究973计划和国家重点实验室自主研究项目的资助，主要由博士研究生王锐等完成，研究论文7月22日在PLoS ONE在线发表。
生物探索推荐英文论文摘要：
An Apo-14 Promoter-Driven Transgenic Zebrafish That Marks Liver Organogenesis
Abstract
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.
