Identification, Expression Pattern And Functional Analysis Of MMP3/10a, MMP3/10b And MMP9 In Digit Regeneration Of Axolotl

Reports of regeneration are more and more to date. As a mode] organism, salmander is well known for their supper abilities of regeneration. From an evolutionary perspective, their digits are homologous to human fingers, and the research on digit regeneration is more promising applications than that in limb since human has a limited a capacity of finger regeneration. But so far, the knowledge about digit regeneration is scarce. This paper studied axolotl(Ambystoma mexicanum) digit regeneration process for the first time, including analysis of spatial and temporal expression patterns of matrix metallop rote inases (MMPs) and their functions during regeneration, as well as morphological and histological analysis.First of all, morphologically, it shows that axolotl digit regeneration cycle is about 5-6 weeks. Histo logically, the digit regeneration process can be divided into three stages:The wound healing stage, in which epidermal cells around the wound migrate and cover the wound surface as a thin cell sheet, termed wound epidermis(WE), in a few hours after amputation; blastema formation stage(3-10 days post amutation), subsequently, a group of meschymal cells emerged, acumulated and further formed an cone-like structure under the thickend WE(also named apical epidermal cap, AEC); differentiation and morphogenesis stage, differentiation is clearly seen internally with the appearance of cartilage cells in the central part of the blastema by 14 day, and followed by a complex mophoregensis process the digit regeneration is accomplished with formation of mature tissues such as connective tissue and bood vessels.Secondly, the spatial and temporal expression patterns of MMPs were investigated by in situ hybridization. It was showed the 3 genes mainly express at wound healing and early blastema formation stages in a specific manner. For MMP3/10a, its expression is mainly concentrated in the circumference of the wound at 1day, further spread to whole WE at 3 day, and then in deep cell layers of AEC at 7, 14 and 21 day respectively. Similar expression patterns were also demonstrated in MMP3/10b and MMP9 although with small distinctions between them. In addition, the relative gene expression levels of the 3 genes were assayed by Realtime quantative PCR method at diffent days post amputation. It was generally shown that the three MMPs genes were dramatically upregulated although with fluctuations throughout the regenation time course, compared to their expression in intact digits. Take MMP9 for example, it shows a highest expression level at lday post amputation(wound healing stage), followed by down-and-up changes and then reached a second peak(20 folds) and third peak (16 folds) at 7 day and 14 day after amputation respectively. Then it decreased to a level of 2 folds at 21 days after amputation.To detect the MMPs activities in digit regeneration, gelatin zymography were performed on the digit regenerates at different time after post amputation. It was revealed that at 6h post amputation, very weak two closed bands with molecular weight of 90 and 80 kDa were detected, indication of latent and active MMP9 respectively. At 1 day and 3 day post amputation, the activity of MMP9 were greatly increased, followed by a weakened activity at 5 day, and then notably elevated at 7day; whereas no obvious bands for MMP9 were detected in intact digits which was used as control. Furthermore, there were no bands corresponding to the other 2 MMPs being demonstrated in this zymography assay.In summary, morphological and histologically, the digit regeneraration in axlotl is similar to their limb regeneration, belonging to epimorphosis. The MMPs expression patterns suggest that they play roles in wound healing and early blastema formation during the digit regeneration process. Definitely, this study provided an reference for the study of digit regeneration.