Preliminary Research Of Regenerative Mechanism Of Arm Regeneration In Starfish Asterias Rollestoni Bell

Asterias rollestoni Bell, which subject to Echinodermata, own remarkable ability to regenerate. Because they are easy to collect and feed in the laboratory, own typical deuterostomia-like developmental characteristics, they can provide a valuable experimental model for studying all aspects of regenerative processes and mechanisms of deuterostomes from molecular to macroscopic level.To clarify the regenerative mechanism of arm regeneration in A. rollestoni, regeneration was induced by arm-tip cutting and was examined in this study by staining of histochemistry, TEM and immunofluorescent researches. The results of histological and cellular researches show that there is no adult stem cell in intact controls. About 4 days post amputation(PA), cells in some areas in the out epidermis and inner parietal peritoneum near the end of the stump are induced by injury to dedifferentiate into stem cells and then proliferate forming stem-cell masses, which then invaginate into the interior dermis. The dermal cells are then induced by those invaginating stem-cell masses to dedifferentiate. Finally, the invaginated dedifferentiated epidermal cells, epithelial cells and dermal cells formed many stem-cell blastemas, potential cell pools for the subsequent arm regeneration. About 2-4 d PA, musculature near the wound re-arranges causing the wound close and a new pre-epidermis which consists of several layers of stem cells is formed covering the wound.The results of cell proliferating activity researches with BrdU incorporation reveal that from 4 d PA to 20 d PA, extensive cell proliferation activities are detected in the whole peritoneum, the distal end pre-epidermis and the dermis below, epidermal blastemas and dermal blastemas. There are most proliferating cells 14 d PA in the peritoneum, the distal end pre-epidermis and the dermis below. Massive proliferating cells are also detected in the epithelium and musculature of the ambulacral plate. The place and time of cell proliferation are just in accordance with those of the appearances of stem cells and stem-cell blastemas, which means that cells in those areas mentioned above dedifferentiate forming stem cells which proliferate too.Moreover, immunofluorescent researches were applied to study the temporal and spatial expressions of desmin, the marker of activated myoblasts (muscle precursor cells) and myogenin, the marker of differentiation of myoblasts. It is suggested that there is no expression of desmin and myogenin in the control. After amputation, the expression of desmin appears first in the distal end epidermis and the peritoneum 4 d PA. The expression in the peritoneum lasts to the 14 d PA when expression of desmin appears in the musculature, while that in the distal end epidermis only lasts to the 8 d PA when the connective tissue below begins to express desmin. Moreover, from the 4 d PA, musculature and epidermis of the tube foot begin to express desmin and the expressions last to the 20 d PA. From 8-14 d PA, musculature of the ambulacral plate expresses myogenin and the peritoneum also expresses myogenin from 14-20 d PA. All these results indicate that precursor cells for muscle regeneration in adult starfish A.rollestoni are mainly from the distal end epidermis and the parietal peritoneum. Cells of the peritoneum in the arm stump dedifferentiate forming stem cells which proliferate and some of them transform into myoblasts, while epidermal cells near the wound dedifferentiate and proliferate too and migrate to the wound plane where some stem cells in the pre-epidermis transform into myoblasts too.Based on all results in this study, mechanism of arm regeneration in starfish A. rollestoni can be concluded as below. Regeneration of arm includes wound healing, formation and re-growth of new regenerating bud at the distal end and the elongation of the arm stump. The wound healing consists of wound closure, reformation of the distal end epidermis and the parietal peritoneum. During the 4 days PA, musculature in the arm stump near the wound migrates to the wound and re-arranges padding the wound. Epidermal cells near the wound dedifferentiate and proliferate forming blastema and/or migrate to the wound plane forming a pre-epidermis consisting of several layers of stem cells covering the wound plane, which then re-differentiates further forming fully developed epidermis. Cells in the parietal peritoneum are also induced by injury to dedifferentiate into stem cells which then proliferate and migrate along the parietal peritoneum to the wound place forming a pre-peritoneum which re-differentiates into fully developed parietal peritoneum. Based on the wound healing, a new regenerating bud is formed at the distal end of the arm stump 6 d PA, and the mechanism of its formation is as below. After proliferation, stem cells in the epidermal blastema migrate below the epidermis to the arm stump end where they re-differentiate forming the dermis of the new bud. Some of these migrating stem cells transform into myoblasts which then re-differentiate together with those form the distal end epidermis into new musculature of the new bud. Meanwhile, part of epidermal cells near the wound undergo proliferate and migrate to the epidermis of the new bud causing the gradual growth of the new epidermis. Moreover, stem cells from the parietal peritoneum migrate along it to the wound and re-differentiate forming the parietal peritoneum of the new bud. The elongation of the arm stump is mainly carried out by repetitious formation and re-differentiation of a lot of body wall-across blastemas in the arm stump. The pattern and mechanism of muscle regeneration in the arm stump is as below. Some stem cells from the peritoneum transform into myoblasts and migrate to the place where muscle develops and re-differentiate forming new muscle tissue, while the other myoblasts from the peritoneum re-differentiate into muscle tissue in the inner part of the peritoneum, which then breaks away from the peritoneum by delamination and then combinates with the musculature above. Moreover, the results also reveal that two regenerative mechanisms, morphallaxis and epimorphosis are both utilized in the arm regeneration process of A. rollestoni.