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.