Ecdysteroid receptor signaling disruption obstructs blastemal cell proliferation during limb regeneration in the fiddler crab, Uca pugilator.

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.