Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA.
Evol Dev. 2013 Jul-Aug;15(4):228-42. doi: 10.1111/ede.12029. Epub 2013 May 14.
The discovery of genetic mechanisms that can transform a morphological structure from a plesiomorphic (=primitive) state to an apomorphic (=derived) one is a cardinal objective of evolutionary developmental biology. However, this objective is often impeded for many lineages of interest by limitations in taxonomic sampling, genomic resources, or functional genetic methods. In order to investigate the evolution of appendage morphology within Chelicerata, the putative sister group of the remaining arthropods, we developed an RNA interference (RNAi) protocol for the harvestman Phalangium opilio. We silenced the leg gap genes Distal-less (Dll) and dachshund (dac) in the harvestman via zygotic injections of double-stranded RNA (dsRNA), and used in situ hybridization to confirm RNAi efficacy. Consistent with the conserved roles of these genes in patterning the proximo-distal axis of arthropod appendages, we observed that embryos injected with Dll dsRNA lacked distal parts of appendages and appendage-like structures, such as the labrum, the chelicerae, the pedipalps, and the walking legs, whereas embryos injected with dac dsRNA lacked the medial podomeres femur and patella in the pedipalps and walking legs. In addition, we detected a role for these genes in patterning structures that do not occur in well-established chelicerate models (spiders and mites). Dll RNAi additionally results in loss of the preoral chamber, which is formed from pedipalpal and leg coxapophyses, and the ocularium, a dorsal outgrowth bearing the eyes. In one case, we observed that an embryo injected with dac dsRNA lacked the proximal segment of the chelicera, a plesiomorphic podomere that expresses dac in wild-type embryos. This may support the hypothesis that loss of the cheliceral dac domain underlies the transition to the two-segmented chelicera of derived arachnids.
发现能够将形态结构从原始状态转变为衍生状态的遗传机制，是进化发育生物学的主要目标。然而，对于许多感兴趣的谱系来说，由于分类采样、基因组资源或功能遗传方法的限制，这一目标常常受到阻碍。为了研究节肢动物的姐妹群螯肢动物门的附肢形态的进化，我们开发了一种 RNA 干扰（RNAi）协议，用于 harvestman Phalangium opilio。我们通过对合子注射双链 RNA（dsRNA）来沉默 harvestman 中的腿部间隙基因 Distal-less（Dll）和 dachshund（dac），并使用原位杂交来确认 RNAi 的效果。这些基因在模式化节肢动物附肢的近-远轴中具有保守作用，因此我们观察到，注射 Dll dsRNA 的胚胎缺乏附肢和类似附肢的结构的远端部分，如唇、螯肢、步足和步行足，而注射 dac dsRNA 的胚胎则缺乏步足和步行足的中节肢骨股骨和髌骨。此外，我们发现这些基因在模式化未在既定螯肢模型（蜘蛛和螨虫）中出现的结构中发挥作用。Dll RNAi 还导致前口腔室的缺失，该室由步足和腿的 coxapophyses 形成，以及眼的背部长出的眼盖。在一种情况下，我们观察到，注射 dac dsRNA 的胚胎缺乏螯肢的近端节段，这是一个原始的节肢骨，在野生型胚胎中表达 dac。这可能支持这样一种假说，即衍生蛛形纲动物的两段式螯肢的形成是由于 dac 缺失所致。
