Role of neuregulin in urodele limb regeneration.

摘要

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3 and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt ( Notophthalmus viridescens) spinal cord. We also obtained partial sequence of two other isoforms. Results of in situ hybridization showed that the newt CRD NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into intraperitonial cavity; The denervated blastemas were rescued into a regeneration response. Several recombinant proteins of newt NRG were expressed in bacteria by utilizing either a maltose binding system or a histamine tagged system. The obtained recombinant proteins were used to generate anti-NRG monoclonal antibodies. We also demonstrated, by RT-PCR, that newt NRG and its receptor erbB3 are expressed in blastemas but not in aneurogenic limbs, a result consistent with a Schwann cell inhibitor model developed in this lab.