SPATIAL, TEMPORAL AND HORMONAL-REGULATION OF PROGRAMMED MUSCLE-CELL DEATH DURING METAMORPHOSIS OF THE FROG XENOPUS-LAEVIS

No examination to date has been made of apoptosis during vertebrate mu scle development. The authors recently reported programmed muscle cell death to be important in tail degeneration as well as in the larval-t o-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 bi ochemically, with special attention to whether apoptotic processes, su ch as chromatin fragmentation and apoptotic body-formation actually oc cur, and whether triiodothyronine (T-3) induces such processes. Light microscopic observation indicated muscle fibers break down into short fragments (sarcolytes or muscle apoptotic bodies) during the metamorph ic climax, not only in tail but also in larval-type fibers of dorsal b ody muscles. Apoptotic bodies first appeared near the base of the tail in early climax (stage 59) when the T-3 level is quite high, and ther eafter expanded in an anterior direction in the dorsal body and poster iorly in the tail. The ratio of apoptotic area to total muscle area be came maximum (10%-30% in dorsal body muscles and 50% in the tail) at l ate 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 fragmentatio n is associated with apoptotic bodies, in situ DNA nick end labeling ( TUNEL) was applied to sections of the dorsal body and tail muscles. La beled muscle nuclei could be found only in muscle apoptotic bodies but not in intact muscle fibers, indicating DNA fragmentation was associa ted with cell fragmentation during metamorphosis. It thus follows that morphological (apoptotic body formation) and biochemical (fragmentati on of chromatin) processes occurring during PCD of dorsal body and tai l muscles are identical. To determine whether T-3 regulates programmed muscle cell death, the effects of T-3 on DNA ladder formation were ex amined in tails cultured in vitro. The oligonucleosomal DNA ladder was found to form only in tails incubated with T-3, thus showing T-3 to i nduce programmed muscle cell death without interaction with other endo crine organs during metamorphosis.