Yamazaki Atsuko, Kawabata Rika, Shiomi Kosuke, Amemiya Shonan, Sawaguchi Masaya, Mitsunaga-Nakatsubo Keiko, Yamaguchi Masaaki
Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan.
Dev Genes Evol. 2005 Sep;215(9):450-59. doi: 10.1007/s00427-005-0006-y. Epub 2005 Aug 3.
In the sea urchin embryo, micromeres have two distinct functions: they differentiate cell autonomously into the skeletogenic mesenchyme cells and act as an organizing center that induces endomesoderm formation. We demonstrated that micro1 controls micromere specification as a transcriptional repressor. Because micro1 is a multicopy gene with at least six polymorphic loci, it has been difficult to consistently block micro1 function by morpholino-mediated knockdown. Here, to block micro1 function, we used an active activator of micro1 consisting of a fusion protein of the VP16 activation domain and the micro1 homeodomain. Embryos injected with mRNA encoding the fusion protein exhibited a phenotype similar to that of micromere-less embryos. To evaluate micro1 function in the micromere, we constructed chimeric embryos composed of animal cap mesomeres and a micromere quartet from embryos injected with the fusion protein mRNA. The chimeras developed into dauerblastulae with no vegetal structures, in which the micromere progeny constituted the blastula wall. We also analyzed the phenotype of chimeras composed of an animal cap and a mesomere expressing micro1. These chimeras developed into pluteus larvae, in which the mesomere descendants ingressed as primary mesenchyme cells and formed a complete set of skeletal rods. The hindgut and a part of the midgut were also generated from host mesomeres. However, the foregut and nonskeletogenic mesoderm were not formed in the larvae. From these observations, we conclude that micro1 is necessary and sufficient for both micromere differentiation and mid/hindgut-inducing activity, and we also suggest that micro1 may not fulfill all micromere functions.
在海胆胚胎中，小分裂球具有两种不同的功能：它们自主分化为骨骼发生间充质细胞，并作为诱导内胚层和中胚层形成的组织中心。我们证明了micro1作为转录抑制因子控制小分裂球的特化。由于micro1是一个具有至少六个多态性位点的多拷贝基因，通过吗啉代介导的敲低来持续阻断micro1的功能一直很困难。在这里，为了阻断micro1的功能，我们使用了一种由VP16激活结构域和micro1同源结构域的融合蛋白组成的micro1活性激活剂。注射编码融合蛋白的mRNA的胚胎表现出与无小分裂球胚胎相似的表型。为了评估micro1在小分裂球中的功能，我们构建了由动物帽中分裂球和注射了融合蛋白mRNA的胚胎的小分裂球四重体组成的嵌合胚胎。这些嵌合体发育成没有植物性结构的滞育囊胚，其中小分裂球后代构成囊胚壁。我们还分析了由表达micro1的动物帽和中分裂球组成的嵌合体的表型。这些嵌合体发育成羽腕幼虫，其中中分裂球后代作为初级间充质细胞内陷并形成一整套骨骼杆。后肠和部分中肠也由宿主中分裂球产生。然而，幼虫中未形成前肠和非骨骼发生中胚层。从这些观察结果中，我们得出结论，micro1对于小分裂球分化和中/后肠诱导活性都是必需且充分的，并且我们还表明micro1可能无法履行小分裂球的所有功能。
