Kumar Anoop, Velloso Cristiana P, Imokawa Yutaka, Brockes Jeremy P
Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom.
PLoS Biol. 2004 Aug;2(8):E218. doi: 10.1371/journal.pbio.0020218. Epub 2004 Aug 17.
The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.
在蝾螈肢体再生过程中，多核的有丝分裂后肌纤维会转变为可分裂的单核后代细胞（细胞化），但这一非凡过程背后的细胞事件和分子调控尚不清楚。同源框基因Msx1已被作为肌肉分化的拮抗剂进行研究，其在培养的小鼠肌管中的表达可诱导约5%的细胞发生细胞化和存活片段化，但其与蝾螈再生内源性程序的相关性尚不清楚。我们从幼体蝾螈的肢体中分离出肌纤维，并将其接种到培养基中培养。大多数纤维因解离而被激活，从而动员其细胞核并发生细胞化或断裂成可存活的多核片段。随后，我们将谱系示踪剂显微注射到单根纤维中，并对标记的后代细胞进行分析，同时进行延时显微镜观察。表现出形态可塑性的纤维选择性地表达Msx1 mRNA和蛋白质。针对Msx1的吗啉代反义寡核苷酸的摄取导致肌细胞核中Msx1蛋白的表达特异性降低，并显著抑制细胞化和片段化。蝾螈的肌纤维通过激活内源性细胞化和片段化程序对解离作出反应。谱系追踪表明，循环的单核后代细胞来源于单个肌纤维。Msx1表达的诱导是激活该程序所必需的。我们对有丝分裂后蝾螈细胞可塑性调控的理解应为促进其他情况下的再生提供策略。
