Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA.
Mol Cell Endocrinol. 2013 Jan 30;365(2):249-59. doi: 10.1016/j.mce.2012.10.026. Epub 2012 Nov 8.
To study ecdysteroid signaling during limb regeneration, we have applied RNAi (dsRNA) mediated silencing to EcR/RXR, the genes encoding the ecdysteroid receptor heterodimer, in the fiddler crab Uca pugilator. We injected RNAi into the blastemal chamber during early limb regeneration. Silencing was evaluated by knockdown in receptor transcript abundance, and disruption was evaluated by changes in growth rate and morphology of limb regenerates. q-PCR results indicated a 50% drop in transcript abundance 48h post injection in both RNAi (dsEcR/dsRXR) injected ipsilateral and uninjected contralateral blastemas in experimental animals relative to controls. EcR/RXR transcript levels further decreased over time. Several phenotypes were associated with knockdown. The experimental blastema failed to develop; microscopic examination of the arrested blastema revealed an absence of the cuticular ingrowths characteristic of the beginnings of limb segmentation and cell proliferation assays revealed that the arrested blastema had few dividing cells. Ecdysteroid levels were also lowered in experimental animals; given the bilateral effects of RNAi on limb buds in experimental animals, these results suggest RNAi had a systemic effect. Although hormone titers in experimental animals rose to comparable control levels during the late proecdysial phase of limb regeneration, most experimental crabs failed to molt and died. The overall failure to molt indicates that RNAi receptor knockdown has long-term effects. The combined effects of receptor knockdown indicate that, although circulating ecdysteroid titers are normally low during basal limb bud growth, signaling via the ecdysteroid receptor pathway is necessary for establishment of blastemal cell proliferation and development in the regenerating limbs of U. pugilator.
为了研究蜕皮甾类信号在肢体再生过程中的作用，我们采用 RNAi（dsRNA）介导的沉默技术，对编码蜕皮甾类受体异二聚体的基因 EcR/RXR 在招潮蟹 Uca pugilator 中进行了沉默。我们在肢体再生的早期将 RNAi 注射到芽腔中。通过受体转录物丰度的敲低来评估沉默效果，通过肢体再生的生长速度和形态的变化来评估干扰效果。q-PCR 结果表明，在注射后 48 小时，实验组和未注射组的 RNAi（dsEcR/dsRXR）注射侧和未注射侧芽腔中，受体转录物丰度分别下降了 50%。EcR/RXR 转录物水平随时间进一步下降。几种表型与敲低有关。实验性芽腔未能发育；对阻滞芽腔的显微镜检查显示，缺乏肢段发生和细胞增殖测定开始时特有的表皮内生长，并且表明阻滞芽腔中只有少数分裂细胞。实验动物中的蜕皮甾类水平也降低了；鉴于 RNAi 对实验动物肢体芽的双侧影响，这些结果表明 RNAi 具有全身性效应。尽管实验动物的激素水平在肢体再生的晚期前蜕皮阶段上升到与对照相当的水平，但大多数实验蟹未能蜕皮而死亡。整体蜕皮失败表明 RNAi 受体敲低具有长期影响。受体敲低的综合影响表明，尽管在基础肢芽生长过程中循环蜕皮甾类水平通常较低，但通过蜕皮甾类受体途径进行信号传递对于建立再生肢体的芽腔细胞增殖和发育是必要的。
