Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan.
Dev Genes Evol. 2013 Jul;223(4):261-7. doi: 10.1007/s00427-013-0438-8. Epub 2013 Mar 15.
The appendicularian Oikopleura dioica is a chordate that has a remarkably simple adult body with small cell number. Its transparency, stereotyped cell lineages, short life cycle, and small genome make it a promising new experimental model of chordate developmental biology. However, the functions of its various genes are still poorly understood due to lack of a tool for suppression of gene expression. Here, we applied a double-stranded RNA (dsRNA)-based-RNA interference (RNAi) method in O. dioica. For introducing dsRNA into eggs and embryos, we injected dsRNAs into the ovary. dsRNA, which is specific to EGFP or mCherry mRNA, decreased the exogenous mRNA-derived fluorescence in both eggs and embryos. dsRNA specific to the Brachyury gene of O. dioica, which is a homologous gene of a key notochord transcriptional factor in ascidians, triggered degradation of endogenous Brachyury mRNA and induced malformation or loss of the notochord in the tail. This effect was Brachyury sequence specific, as three dsRNAs covering different sequences produced the same phenotype. The result is in accordance with its expression site and also with the key regulatory function of Brachyury in notochord formation in other chordates. RNAi in O. dioica would be a useful tool for gaining insight into the oogenesis and early developmental processes in chordates.
尾索动物海鞘的幼体 Oikopleura dioica 拥有一个极为简单的成年体，其细胞数量很少。其透明性、定型的细胞谱系、短生命周期和小基因组使其成为一个有前途的新型脊索动物发育生物学实验模型。然而，由于缺乏基因表达抑制的工具，其各种基因的功能仍然知之甚少。在这里，我们在 O. dioica 中应用了基于双链 RNA（dsRNA）的 RNA 干扰（RNAi）方法。为了将 dsRNA 导入卵子和胚胎，我们将 dsRNA 注射到卵巢中。针对 EGFP 或 mCherry mRNA 的特异性 dsRNA 降低了卵子和胚胎中外源 mRNA 衍生的荧光。针对 O. dioica 的 Brachyury 基因的 dsRNA，与尾索动物中关键的脊索转录因子的同源基因，触发了内源性 Brachyury mRNA 的降解，并导致尾部的脊索畸形或缺失。这种效应是 Brachyury 序列特异性的，因为覆盖不同序列的三个 dsRNA 产生了相同的表型。该结果与其表达部位以及 Brachyury 在其他脊索动物中形成脊索的关键调节功能一致。O. dioica 的 RNAi 将成为深入了解脊索动物卵子发生和早期发育过程的有用工具。
