Burtscher Ingo, Barkey Wenke, Lickert Heiko
Helmholtz Zentrum München, Institute of Diabetes and Regeneration Research, Neuherberg, Germany.
Genesis. 2013 Aug;51(8):596-604. doi: 10.1002/dvg.22404. Epub 2013 Jun 26.
The Foxa2-winged helix/forkhead box transcription factor (TF) is absolutely required for endoderm formation and organogenesis. Foxa2 plays essential roles during lung, liver, pancreas, and gastrointestinal tract development and regulates cell-type specific programs in the adult organism. To specifically address Foxa2 function during organ development and homeostasis, we generated a Foxa2-Venus fusion (FVF) reporter protein by gene targeting in embryonic stem (ES) cells. The FVF knock-in reporter is expressed under endogenous Foxa2 control and the fluorescent protein fusion does not interfere with TF function, as homozygous mice are viable and fertile. Moreover, the FVF protein localizes to the nucleus, associates with chromatin during mitosis, and reflects the endogenous Foxa2 protein distribution pattern in several tissues in heterozygous animals. Importantly, live-cell imaging on single-cell level of the FVF and Sox17-Cherry fusion double knock-in reporter ES cell line reveals the dynamics of endoderm TF accumulation during ES cell differentiation. The FVF reporter also allowed us to identify the endoderm progenitors during gastrulation and to visualize the different branching morphogenesis modes of the lung and pancreas epithelium in ex vivo embryo and organ cultures. In summary, the generation of the FVF reporter line adds an important new tool to visualize and analyse endoderm-derived organ development and homeostasis on the cellular and molecular level.
叉头框转录因子A2（Foxa2）对于内胚层形成和器官发生是绝对必需的。Foxa2在肺、肝、胰腺和胃肠道发育过程中发挥着重要作用，并在成年生物体中调节细胞类型特异性程序。为了具体研究Foxa2在器官发育和内环境稳定中的功能，我们通过对胚胎干细胞（ES细胞）进行基因靶向操作，生成了一种Foxa2-维纳斯融合（FVF）报告蛋白。FVF敲入报告基因在Foxa2内源性控制下表达，荧光蛋白融合不干扰转录因子功能，因为纯合小鼠是可存活且可育的。此外，FVF蛋白定位于细胞核，在有丝分裂期间与染色质结合，并反映杂合动物几种组织中内源性Foxa2蛋白的分布模式。重要的是，对FVF和Sox17-樱桃融合双敲入报告基因ES细胞系进行单细胞水平的活细胞成像，揭示了ES细胞分化过程中内胚层转录因子积累的动态变化。FVF报告基因还使我们能够在原肠胚形成过程中鉴定内胚层祖细胞，并在体外胚胎和器官培养中观察肺和胰腺上皮的不同分支形态发生模式。总之，FVF报告基因系的产生为在细胞和分子水平上可视化和分析内胚层来源器官的发育和内环境稳定增加了一个重要的新工具。
