| Background: Salamander is an ideal animal model for regeneration research because it has excellent regeneration ability,and can regenerate entire limbs perfectly after amputation.In addition,ectopic limb regeneration can also be induced by peripheral nerve transposition.Therefore,nerve innervation is critical for the success of limb regeneration in newt.Anterior gradient protein 2(nAG)is an important signal molecule in regulating cell proliferation.Studies have shown that nAG was highly expressed at the broken stump of the nerve after limb amputation in Notophtalmus viridescens.The denervated limb has failed to regenerate after amputation,whereas the local expression of nAG through electroporation can rescue denervated limb regeneration.Although previous experiments demonstrated that nAG plays a key role in limb regeneration,it is still unclear about the biological effect of nAG on the cells near the broken stump and the cell types required to induce limb regeneration.Methods and Results: In this study,firstly we generated purified nAG protein by recombinant technique.The nAG polyclonal antibody was prepared and purified.Then the biological effects of nAG recombinant protein on fibroblasts were examined in vitro.The results showed that nAG had significant chemotactic effect on fibroblasts.Secondly,we analyzed the nAG expression and deposition through quantitative real-time PCR(q RTPCR)and tissue immunofluorescence staining.q RT-PCR results showed that nAG was similarly expressed in both amputated limb and spinal cord after the amputation,and the m RNA expression level peaked at 14 days post amputation(dpa).The results of immunofluorescence indicated that nAG protein expression was mainly localized in the wound epidermis and the blastema cells,in particular,highly expressed in Schwann cells at7 dpa and 14 dpa.These results suggest nAG could be secreted by neurons located in the spinal cord and transported to the end of amputated limb through axons to act on Schwann cells.Finally,we constructed the accessory limb model(ALM)in newt.The ectopic blastema was prominent around 10 days post operation(dpo),and the ectopic limb regeneration completed around 50 dpo.We then observed the induction of accessory limb formation by implantation of recombinant nAG(gelatin sponge-loaded)and Schwann cells.The results showed that the synergistic application of nAG and Schwann cells were able to induce the formation of tissue of blastema characteristics,which had distinct thickened epidermis with the presence of a large number of blastema like cells and hemocytes underneath.The expression of early regenerating blastema marker genes including Prrx2,Twist2 and MLP were upregulated in neotissue,along with a significant increase in cell proliferation through Brd U labeling.Additionally,we found implantation of Schwann cells in the axolotl limb wound and electroporation of the am AGR2 plasmid can also induce blastema-like neotissue similar to the ALM.Conclusion: In this study,we successfully expressed nAG recombinant protein,which was found to significantly increase the chemotaxis of fibroblasts.Furthermore,we confirmed that nAG expression changes significantly during the blastema formation in the process of newt limb regeneration.The initiation of limb regeneration can be induced by the synergistic application of anterior gradient protein and Schwann cells to form blastema-like neotissue.Our results provide a preliminary basis for understanding the effect of nAG and Schwann cells during newt limb regeneration initiation. |
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