Illuminating cell-cycle progression in the developing zebrafish embryo
Illuminating cell-cycle progression in the developing zebrafish embryo
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DOI：
10.1073/pnas.0906464106
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发表时间：
2009-12-08
影响因子：
11.1
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
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作者：
Sugiyama, Mayu;Sakaue-Sawano, Asako;Miyawaki, Atsushi
通过利用细胞周期依赖的蛋白水解的两个泛素化振荡器，人Cdt 1和geminin，这是SCFSkp 2和APC（Cdh 1）复合物的直接底物，分别，Fucci技术标记哺乳动物细胞核在G（1）和S/G（2）/M期不同的颜色。转基因小鼠表达这些G（1）和S/G2/M标记提供了一个强大的手段来研究细胞周期与形态发生过程的协调。我们试图将这些标记引入斑马鱼胚胎中，以利用它们有利的光学特性。然而，尽管细胞周期控制的基本机制似乎在物种之间很好地保守，但基于SCFSkp 2介导的人类Cdt 1降解的G（1）标记在鱼类细胞中不起作用，可能是因为该标记没有被鱼类E3连接酶复合物适当地泛素化。在这里，我们描述了一代的Fucci衍生物使用斑马鱼同源Cdt 1和双生蛋白，它提供了全面的看法，在整个鱼胚胎的细胞增殖。值得注意的是，我们在分化中的脊索中发现了两个细胞周期转换的前后波，G（1）/S和M/G（1）。我们的研究证明了使用Cul 4（Ddb 1）介导的Cdt 1降解途径的有效性，共同所有后生动物的G（1）标记，在非哺乳动物模型的发展。
By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCFSkp2 and APC(Cdh1) complexes, respectively, Fucci technique labels mammalian cell nuclei in G(1) and S/G(2)/M phases with different colors. Transgenic mice expressing these G(1) and S/G2/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G(1) marker based on the SCFSkp2-mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-to-posterior waves of cell-cycle transitions, G(1)/S and M/G(1), in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4(Ddb1)-mediated Cdt1 degradation pathway common to all metazoans for the development of a G(1) marker that works in the nonmammalian animal model.