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Ecdysteroid receptor signaling is required for limb regeneration in the fiddler crab
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.
A novel model uncovers striking similarities during limb regeneration between arthropods and vertebrates
In previous posts I have mentioned the importance of studying how regeneration takes place at both cellular and molecular levels in a large variety of animals. This will help us not only to understand how the process of regeneration itself occurs in different animals but will provide us with basic data for comparative analyses that can unravel common and specific regenerative strategies throughout phylogeny.
The laboratory of Michalis Averof reports now on the regeneration capacities of the crustacean Parhyale hawaiensis and shows striking similarities during limb regeneration in these animals compared to vertebrates (http://www.ncbi.nlm.nih.gov/pubmed/24385602). Parhyale can regenerate all their appendages throughout their lifetime. Here, the authors used morphological, cellular and genetic markers to describe limb regeneration. After amputation, wound closure takes place within a day. Then a blastema consisting of proliferative cells is formed around day 2 and 3. By day 4-6 the distal tip of the regenerated limb is visible by the expression of Distal. Finally, the muscles regenerate within a week from moulting.
An important question that the authors wanted to address here was about the origin of the regenerative cells in Parhyale. In order to distinguish between pluripotent and lineage-restricted progenitor cells they marked different cell lineages (at the 8-blastomere stage) and followed their contribution during limb regeneration in adults. At this stage, 3 blastomeres (El, Er and Ep) are fated to produce the ectoderm, 3 more (ml, mr, Mav) to mesoderm, 1 (en) to endoderm and 1 (g) to the germline. They injected those embryos with a transposon carrying a fluorescence marker driven by a ubiquitous promoter activated after heat-shock. After injecting about 4,000 embryos they got 79 individuals in which specific lineages were labelled. Limbs from these animals were then amputated and allowed to regenerate. Remarkably, descendants of blastomeres El, Er and Ep gave rise exclusively to ectodermal derivatives (epidermis and neurons) whereas descendants of ml, mr and Mav gave rise only to muscle. No contributions from the endoderm or germline lineages were found in the regenerated limb. Moreover none labelled lineage contributed to both ectodermal and mesodermal derivatives suggesting that neither pluripotent progenitors nor trans-differentiation across ectoderm and mesoderm appears to occur in Parhyale. Therefore, and similarly to what happens in the axolotl limb, in Parhyale, new regenerated ectodermal cells appear to originate from the pre-existing ectodermal lineage whereas new mesodermal cells originate from the mesodermal one. Further experiments should determine whether these lineage-restricted progenitor cells for regeneration derive from stem cells or differentiated cells that re-enter the cell cycle.
Because the authors were able to specifically label ectodermal and mesodermal lineages of the left or right sides of the body, they found out that the descendants of blastomeres El and ml, for instance, contribute to the regeneration of the appendages of the left side (descendants of Er and mr contribute only to regeneration in the right side). Thus, regenerative cells have a local origin respect to the amputated limb. Finally, the authors found some cells closely associated to the muscle fibers that reminded the satellite cells in vertebrates. Satellite cells are stem cells for muscle regeneration and are recognized by the expression of Pax3/7. Interestingly, these cells in Parhyale also expressed Pax3/7 and had a mesodermal origin. In order to study the function of these satellite-like cells (SLCs) the authors transplanted individual labelled SLCs from the limbs of transgenic animals into the amputated limbs of control animals and found out that in some cases these GFP-labelled cells gave rise to regenerated muscle fibers. Thus, SLCs might function as muscle progenitor cells.
In summary, this study introduces Parhyale as a regeneration model and points out several similarities between crustacean and vertebrate limb regeneration such as lineage-restricted progenitor cells, local origin of the regenerative cells and the presence of Pax3/7 positive muscle progenitors.
regeneration in nature 