Nishikawa A, Hayashi H
Department of Physiology, Saitama Medical School, Japan.
Differentiation. 1995 Nov;59(4):207-14. doi: 10.1046/j.1432-0436.1995.5940207.x.
No examination to date has been made of apoptosis during vertebrate muscle development. The authors recently reported programmed muscle cell death to be important in tail degeneration as well as in the larval-to-adult conversion of the dorsal body muscles of Xenopus laevis during metamorphosis [30]. In the present study, we examined programmed cell death (PCD) of the dorsal body and tail muscle morphologically and biochemically, with special attention to whether apoptotic processes, such as chromatin fragmentation and apoptotic body-formation actually occur, and whether triiodothyronine (T3) induces such processes. Light microscopic observation indicated muscle fibers break down into short fragments (sarcolytes or muscle apoptotic bodies) during the metamorphic climax, not only in tail but also in larval-type fibers of dorsal body muscles. Apoptotic bodies first appeared near the base of the tail in early climax (stage 59) when the T3 level is quite high, and thereafter expanded in an anterior direction in the dorsal body and posteriorly in the tail. The ratio of apoptotic area to total muscle area became maximum (10%-30% in dorsal body muscles and 50% in the tail) at the climax (stages 63-64). During these stages, genomic DNA fragmented into oligonucleosome-sized units (200 bp, 400 bp, 600 bp ...) in both body and tail muscles. To confirm whether this chromatin fragmentation is associated with apoptotic bodies, in situ DNA nick end labeling (TUNEL) was applied to sections of the dorsal body and tail muscles. Labeled muscle nuclei could be found only in muscle apoptotic bodies but not in intact muscle fibers, indicating DNA fragmentation was associated with cell fragmentation during metamorphosis. It thus follows that morphological (apoptotic body formation) and biochemical (fragmentation of chromatin) processes occurring during PCD of dorsal body and tail muscles are identical. To determine whether T3 regulates programmed muscle cell death, the effects of T3 on DNA ladder formation were examined in tails cultured in vitro. The oligonucleosomal DNA ladder was found to form only in tails incubated with T3, thus showing T3 to induce programmed muscle cell death without interaction with other endocrine organs during metamorphosis.
迄今为止，尚未对脊椎动物肌肉发育过程中的细胞凋亡进行过研究。作者最近报道，程序性肌肉细胞死亡在非洲爪蟾变态发育过程中的尾部退化以及背侧体肌从幼体到成体的转变中起着重要作用[30]。在本研究中，我们从形态学和生物化学角度研究了背侧体肌和尾部肌肉的程序性细胞死亡（PCD），特别关注是否实际发生了诸如染色质片段化和凋亡小体形成等凋亡过程，以及三碘甲状腺原氨酸（T3）是否诱导了这些过程。光学显微镜观察表明，在变态高峰期，肌肉纤维分解为短片段（肌溶产物或肌肉凋亡小体），不仅在尾部，背侧体肌的幼体型纤维中也如此。凋亡小体最早出现在变态早期高潮（第59阶段）时的尾基部附近，此时T3水平相当高，此后在背侧体肌中向前扩展，在尾部向后扩展。在高潮期（第63 - 64阶段），凋亡面积与总肌肉面积的比率达到最大值（背侧体肌为10% - 30%，尾部为50%）。在这些阶段，体肌和尾肌中的基因组DNA均断裂成寡核小体大小的单位（200 bp、400 bp、600 bp……）。为了确认这种染色质片段化是否与凋亡小体相关，我们对背侧体肌和尾部肌肉切片应用了原位DNA缺口末端标记（TUNEL）法。仅在肌肉凋亡小体中发现了标记的肌细胞核，而在完整的肌肉纤维中未发现，这表明在变态过程中DNA片段化与细胞片段化相关。因此可以得出结论，背侧体肌和尾部肌肉PCD过程中发生的形态学（凋亡小体形成）和生物化学（染色质片段化）过程是相同的。为了确定T3是否调节程序性肌肉细胞死亡，我们在体外培养的尾部中检测了T3对DNA梯带形成的影响。发现仅在与T3孵育的尾部中形成了寡核小体DNA梯带，这表明在变态过程中T3可诱导程序性肌肉细胞死亡，且无需与其他内分泌器官相互作用。
