That some crustaceans are able to regenerate their limbs is something known since the 18th century. Limb regeneration has been also observed in other arthropods like some insects. In all these animals, regeneration is closely associated to the molting process. Unfortunately, for many of these species we do not have the proper tools to analyze regeneration at the molecular and/or gene expression level. Therefore, it is important to develop those tools in these species so they can become good models to study the regeneration process. In a paper from the laboratory of David S. Durica the authors applied for the first time RNAi to study regeneration in a brachyuran crab, the fiddler crab Uca pugilator (http://www.ncbi.nlm.nih.gov/pubmed/23142248).
Upon predation or injury these animals can cast off their legs at a predetermined breakage plane by a process called autotomy. After autotomy, the wound is sealed and a scab is formed. Two days following autotomy the epidermal cells underlying the scab divide to originate a blastema. The exact origin of the cells that form the blastema is not completely clear yet although it seems that migrating epidermal cells dedifferentiate and proliferate to give rise to it (however, it is not known the degree of dedifferentiation that those epidermal cells suffer). Seven to nine days after autotomy a blastema emerges; this protuberance is called papilla. This papilla (limb bud) will grow and differentiate into limb segments. When the animal gets ready to molt, this limb goes through a second growth phase called proecdysial growth marked by hypertrophy.
Ecdysteroids are steroid hormones that play pivotal roles during growth, development and reproduction in arthropods. Previous studies had shown how the level of ecdysteroids varies along the different phases of limb growth and development before and during the molt and, therefore, the authors wanted to analyze the function of this signaling during limb regeneration. Ecdysteroids bind to nuclear receptors that are transcription factors that bind to conserved Hormone Response Element (HRE) sequences. Here, the authors used RNAi to silence EcR and RXR the genes encoding the ecdysteroid receptor heterodimer. These genes had been shown to be expressed in the blastema and limb buds at the proecdysial growth phase during regeneration, which further supported the hypothesis of a role of this signaling pathway during blastema formation.
The authors checked the efficiency of their RNAi experiments and used different injection protocols to deliver the dsRNA of these two genes at different time points after autotomy. In general, what they observed was that the emergence of the blastema in form of a papilla was significant reduced compared to controls. Moreover, these defects were more penetrant when dsRNA was delivered earlier after autotomy. Somehow this suggests that the ecdysteroid receptor signaling may be especially important during the first days after autotomy. After autotomy, epidermal cells migrate underneath the scab, proliferate and secrete also a very thin cuticle. The first segment of the limb is formed by the invagination of this cuticle (around 7 days following autotomy). In the dsEcR/dsRXR-injected animals no cuticular invaginations were seen and, compared to controls, a much thicker cuticle was secreted from the epidermal cells that migrate underneath the scab.
In order to check whether the lack of papilla formation and blastema arrest could be due to defects in cell proliferation the authors performed some BrdU labellings. After EcR and RXR RNAi a significant decrease of proliferating cells was observed. In contrast, in controls they found division of epidermal cells underneath the scab as well as in cells along the nerve. However, the silencing of these receptors did not seem to affect the normal migration of epidermal cells towards the wound. Overall, these experiment suggest that the silencing of EcR and RXR resulted in the failure of epidermal cells to proliferate and give rise to a normal blastema. Finally, those animals failed to molt and died which suggested that the RNAi of EcR and RXR might result in the inability to correctly respond to hormonal signaling at the end of the molt cycle.
In summary the ecdysteroid receptor signaling appears to be required for the proliferation and differentiation of the blastema cells during fiddler crab limb regeneration.