Miller J B, Stockdale F E
J Cell Biol. 1986 Dec;103(6 Pt 1):2197-208. doi: 10.1083/jcb.103.6.2197.
The developmental regulation of myoblasts committed to fast, mixed fast/slow, and slow myogenic cell lineages was determined by analyzing myotube formation in high density and clonal cultures of myoblasts isolated from chicken and quail embryos of different ages. To identify cells of different myogenic lineages, myotubes were analyzed for content of fast and slow classes of myosin heavy chain (MHC) isoforms by immunocytochemistry and immunoblotting using specific monoclonal antibodies. Myoblasts from the hindlimb bud, forelimb bud, trunk, and pectoral regions of the early chicken embryo and hindlimb bud of the early quail embryo (days 3-6 in ovo) were committed to three distinct lineages with 60-90% of the myoblasts in the fast lineage, 10-40% in the mixed fast/slow lineage, and 0-3% in the slow lineage depending on the age and species of the myoblast donor. In contrast, 99-100% of the myoblasts in the later embryos (days 9-12 in ovo) were in the fast lineage. Serial subculturing from a single myoblast demonstrated that commitment to a particular lineage was stably inherited for over 30 cell doublings. When myoblasts from embryos of the same age were cultured, the percentage of muscle colonies of the fast, fast/slow, and slow types that formed in clonal cultures was the same as the percentage of myotubes of each of these types that formed in high density cultures, indicating that intercellular contact between myoblasts of different lineages did not affect the type of myotube formed. An analysis in vivo showed that three types of primary myotubes--fast, fast/slow, and slow--were also found in the chicken thigh at day 7 in ovo and that synthesis of both the fast and slow classes of MHC isoforms was concomitant with the formation of primary myotubes. On the basis of these results, we propose that in the avian embryo, there is an early phase of muscle fiber formation in which primary myotubes with differing MHC contents are formed from myoblasts committed to three intrinsically different primary myogenic lineages independent of innervation and a later phase in which secondary myotubes are formed from myoblasts in a single, secondary myogenic lineage with maturation and maintenance of fiber diversity dependent on innervation.
通过分析从不同年龄的鸡和鹌鹑胚胎分离的成肌细胞在高密度和克隆培养中的肌管形成，确定了向快速、快速/慢速混合和慢速肌源性细胞谱系分化的成肌细胞的发育调控。为了鉴定不同肌源性谱系的细胞，通过免疫细胞化学和使用特异性单克隆抗体的免疫印迹分析肌管中快速和慢速肌球蛋白重链（MHC）同工型的含量。早期鸡胚胎的后肢芽、前肢芽、躯干和胸段以及早期鹌鹑胚胎（孵化3 - 6天）的后肢芽中的成肌细胞分化为三种不同的谱系，根据成肌细胞供体的年龄和物种，快速谱系中的成肌细胞占60 - 90%，快速/慢速混合谱系中的占10 - 40%，慢速谱系中的占0 - 3%。相比之下，后期胚胎（孵化9 - 12天）中99 - 100%的成肌细胞属于快速谱系。从单个成肌细胞进行连续传代培养表明，向特定谱系的分化在超过30次细胞倍增中稳定遗传。当培养来自相同年龄胚胎的成肌细胞时，克隆培养中形成的快速、快速/慢速和慢速类型的肌肉集落百分比与高密度培养中形成的每种类型肌管的百分比相同，这表明不同谱系的成肌细胞之间的细胞间接触不影响形成的肌管类型。体内分析表明，在孵化第7天的鸡大腿中也发现了三种类型的初级肌管——快速、快速/慢速和慢速——并且快速和慢速类MHC同工型的合成与初级肌管的形成同时发生。基于这些结果，我们提出在鸟类胚胎中，存在肌肉纤维形成的早期阶段，其中具有不同MHC含量的初级肌管由分化为三种本质上不同的初级肌源性谱系的成肌细胞形成，独立于神经支配；以及后期阶段，其中次级肌管由单一的次级肌源性谱系中的成肌细胞形成，纤维多样性的成熟和维持依赖于神经支配。
