Kumar A, Velloso C P, Imokawa Y, Brockes J P
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom.
Dev Biol. 2000 Feb 15;218(2):125-36. doi: 10.1006/dbio.1999.9569.
Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.
成肌分化的两个重要指标是多核肌管的形成和细胞周期的有丝分裂后停滞，这两个过程都发生在单核细胞融合之后。我们在此表明，在有尾两栖类肢体再生芽基的环境中，这些指标会发生逆转。为了将一种整合（基因）标记引入蝾螈肌管，我们用表达人胎盘碱性磷酸酶（AP）的假型逆转录病毒感染培养中的单核细胞。融合后，肌管表达AP，可通过筛选和显微操作进行纯化，以去除所有单核细胞。当将这种纯化的逆转录病毒标记的肌管植入肢体芽基时，它们能高效产生单核后代。用亲脂性荧光细胞追踪染料标记的纯化肌管也能产生单核细胞；用追踪染料和核染色剂双重标记的肌管能产生双重标记的单核后代。在用BrdU注射植入蝾螈后，广泛的标记表明逆转录病毒标记的肌管内的细胞核进入了S期。可塑性的这两个方面之间的关系是一个关键的进一步问题。
