Embryonic RNAi analysis in the firebrat, Thermobia domestica : Distal-less is required to form caudal filament
Embryonic RNAi analysis in the firebrat, Thermobia domestica : Distal-less is required to form caudal filament
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DOI：
10.11416/jibs.78.2_99
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发表时间：
2009-06
影响因子：
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通讯作者：
Takahiro Ohde;M. Masumoto;T. Yaginuma;T. Niimi
中科院分区：
文献类型：
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作者：
Takahiro Ohde;M. Masumoto;T. Yaginuma;T. Niimi
不变态昆虫直接发育到成虫阶段，几乎没有形态变化。这些昆虫对于理解昆虫发育中的几个最重要的方面是不可或缺的，例如翅膀的进化和变态。然而，以前的研究集中在全变态或半变态昆虫的基础上，完善的遗传分析工具的可用性。由于无变态昆虫的遗传分析可能是了解昆虫进化分子机制的关键，因此需要无变态昆虫的基因功能分析工具。在此，我们将RNA干扰（RNAi）作为基因功能分析工具应用于无变态昆虫，火萤，Thermobia acutica（图1A）。RNAi是一种转录后基因沉默过程，其允许快速分析未知基因功能（Agrawal等人，2003年）。在这个过程中，双链RNA（dsRNA）被降解为约20至30个核苷酸的小RNA，并进一步解旋为单链小RNA。将靶单链RNA（引导链）加载到称为RISC（RNA诱导沉默复合物）的核酸酶复合物上，然后RISC根据引导链的序列切割特定mRNA（Siomi和Siomi，2009）。自从在秀丽隐杆线虫中首次发现RNAi以来（Fire等人，1998），该技术已经在各种真核生物中得到发展，并且不仅在模型系统中，而且在非模型系统中作为强大的反向遗传分析工具得到应用（Agrawal等人，2003年）。我们使用基于来自Thermobia的无远端（Dll）同源物的序列的dsRNA测试RNAi的效用。Dll编码包括同源结构域作为DNA结合结构域的转录因子（Cohen等人，1989）并且在节肢动物中具有令人惊讶的良好保守的序列（Panganiban等人，1995; Prpic和Tautz，2003;新见等人，2005年）。在果蝇中，Dll蛋白是形成腹侧附属物、腿、触角、口器、生殖器和肛门所必需的（Cohen和Jürgens，1989; Gorfinkiel等人，1997，1999;坎贝尔和汤姆林森，1998;莫雷诺和莫拉塔，1999）。在其他昆虫和非昆虫节肢动物中也报道了这种附器形成的功能，例如叶螨二斑叶螨（Khila和Grbić，2007）、蜘蛛Cupiennius saler（Schoppmeier和Damen，2001）、乳草虫Oncopeltus fasciatus（Angelini和考夫曼，2004）、瓢虫Harmonia axyridis（新见等人，2005），以及红面粉甲虫赤拟谷盗（Tribolium castaneum）（Beermann等人，2001;布赫等人，2002; Suzuki等人，2009年）。由于Dll功能丧失表型表现出明显的附件末端截短，因此它是检验RNAi方法实用性的良好标记。与其功能相对应，已经在广泛的生物体中观察到Dll在肢体远端的表达（Panganiban等人，1995，1997; Grenier等人，1997年）。值得注意的是，Rogers等人（2002）报道了Dll在Thermobia尾丝中的表达。在Thermobia的尾部区域，有两种类型的产物：尾须和尾丝。虽然这些副产物具有相似的形态（图1B），但发育起源不同。有茎变态昆虫的系统发育地位对了解昆虫的进化机制具有重要意义。因此，利用RNA干扰（RNAi）技术进行有效的基因功能分析是无变态昆虫研究的重要一步。我们测试了RNAi的效用，在firebrat，Thermobia astritica（Zygentoma，Lepismatidae）通过专注于同源框基因，Distal-less（Dll），基于其保守的序列和明显的功能丧失表型。在胚胎早期阶段注射Dll双链RNA的Thermobia patients显示截短的附属物，因此，我们得出结论，RNAi方法可用于分析Thermobia中的基因功能。值得注意的是，Dll RNAi诱导尾附属物（尾须）和尾非附属物生长（尾丝）的截短。据了解，虽然这两个尾部结构看起来相似，但它们有不同的起源。我们的数据表明，这两种类型的副产物可能是由类似的发育程序形成的，至少对于Dll来说是这样，尽管它们的起源不同，而且Dll甚至在非附属结构中发挥作用。
Ametabolous insects show direct development to the adult stage with almost no morphological changes. These insects are indispensable for understanding several of the most important aspects in development in insects, such as evolution of wing and metamorphosis. However, previous studies have concentrated on holometabolous or hemimetabolous insects based on the availability of well-established genetic analysis tools. Since the genetic analysis in ametabolous insects may be key to understanding molecular mechanism underlying insect evolution, gene functional analysis tools in ametabolous insects are needed. Here, we applied RNA interference (RNAi) as a gene functional analysis tool in the ametabolous insect, the firebrat, Thermobia domestica (Fig. 1A). RNAi is a posttranscriptional gene silencing process that allows rapid analysis of unknown gene function (Agrawal et al., 2003). In this process, double-stranded RNA (dsRNA) is degraded into small RNAs of ~20 to 30 nucleotides and is further unwound into single-stranded small RNAs. The target single-stranded RNA (guide strand) is loaded onto the nuclease complex, designated RISC (RNA-induced silencing complex), and then the RISC cleaves specific mRNA depending on the sequence of the guide strand (Siomi and Siomi, 2009). Since the first discovery of RNAi in Caenorhabditis elegans (Fire et al., 1998), this technique has been developed in various eukaryotic organisms and has been applied as a powerful reverse-genetic analysis tool not only in model system, but also in non-model systems (Agrawal et al., 2003). We tested the utility of RNAi using dsRNA based on the sequence of Distal-less (Dll) homolog from Thermobia. Dll encodes a transcription factor that includes a homeodomain as a DNA-binding domain (Cohen et al., 1989) and has a surprisingly well-conserved sequence among arthropods (Panganiban et al., 1995; Prpic and Tautz, 2003; Niimi et al., 2005). In Drosophila, Dll protein is required for the formation of ventral appendages, legs, antennae, mouthparts, genitalia, and analia (Cohen and Jürgens, 1989; Gorfinkiel et al., 1997, 1999; Campbell and Tomlinson, 1998; Moreno and Morata, 1999). This function of appendage formation has also been reported in other insects and non-insect arthropods, such as the spider mite, Tetranychus urticae (Khila and Grbić, 2007), the spider, Cupiennius saler (Schoppmeier and Damen, 2001), the milkweed bug, Oncopeltus fasciatus (Angelini and Kaufman, 2004), the ladybird beetle, Harmonia axyridis (Niimi et al., 2005), and the red flour beetle, Tribolium castaneum (Beermann et al., 2001; Bucher et al., 2002; Suzuki et al., 2009). Since Dll loss-of-function phenotype appears obvious truncations of the distal regions of appendages, it is a good marker to examine the utility of RNAi method. Corresponding to its function, Dll expression in the distal end of limbs has been observed in a wide range of organisms (Panganiban et al., 1995, 1997; Grenier et al., 1997). Remarkably, Dll expression in the caudal filament of Thermobia was reported by Rogers et al. (2002). In the caudal region of Thermobia, there are two types of outgrowth: cercus and caudal filament. Although these outgrowths have similar morphology (Fig. 1B), the developmental origin is different. The cauAmetabolous insects are important for understanding the mechanism of insect evolution based on their phylogenetic position. Thus, the development and application of an effective gene functional analysis using the RNA interference (RNAi) method is an important step in research on ametabolous insects. We tested RNAi utility in the firebrat, Thermobia domestica (Zygentoma, Lepismatidae) by focusing on the homeobox gene, Distal-less (Dll), based on its conserved sequence and obvious loss-of-function phenotype. Thermobia nymphs that were injected with Dll double-stranded RNA at an early embryonic stage displayed truncated appendages, and thus, we concluded that the RNAi method is useful for analyzing gene function in Thermobia. Remarkably, Dll RNAi induced truncation of the caudal appendage, cerci and the caudal non-appendage outgrowth, caudal filament. It is known that although these two caudal structures look similar, they have different origins. Our data suggests that these two types of outgrowths may be formed by similar developmental program, at least with respect to Dll, despite their different origins and that Dll even plays a role in a non-appendage structure.