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Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateralskin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.
Fig. 1.
Inhibition of Xenopus TOR activity by rapamycin treatment. (A) RT-PCR analyses for TOR and raptor mRNA expressions from unfertilized egg to larva stage. Both TORC1 components are consecutively expressed in Xenopus embryo. (B) Generally conserved model of TORC1 inhibition by rapamycin. Structure of the FKBP12-rapamycin complex interacts with TORC1 in which TOR and raptor are major components. S6 kinase (S6K) is the major output signal activated by TORC1. (C) Western blot analysis of phospho-S6 kinase (p-S6K) in rapamycin-treated embryos. Embryos were treated at 2-cell stage with 80 μM of rapamycin, and extract was collected at stage 42.
Fig. 2.
The effects of TOR signal by rapamycin treatment and dn-Rheb mRNA on developmental speed. (A) Simple comparison of control and rapamycin-treated embryos at same time-course. In embryos treated with rapamycin (20 μM) from 2-cell stage, developmental speed was clearly delayed (100%, n = 15). (B) Time-course of developmental delay following rapamycin treatment. In embryos exposed to each test solution (10, 20, 40, and 80 μM), developmental delays were dose-dependently increased (100%, n = 15, 100%, n = 15, 100%, n = 15, and 100%, n = 15). Same effect was observed in dn-Rheb mRNA (50 pg) injected embryo (100%, n = 15). Refer to the scores before stage 42, the estimated delayed times for stage 45 (95.3 hpf in control embryo) were calculated because structural differences induced by rapamycin made collect staging difficult at this stage. Each stage was determined as follow. Stage 10 was determined when blastopore pigmentation is first detected at the dorsal side. Stage 13 when the blastopore was completely closed. Stage 22 when the neural fold was perfectly closed. Stage 26 when dorsal axis was straighten, and primary melanocyte and optic vesicle bump were observed. Stage 28 when fin structure was first observed. Stage 36 when eyes and cement gland were observed clearly and darkly. Stage 38 when fin region was expanded to 50% of trunk-tail. Stage 40 when boundary between posterior yolk and fin was vertically located. Stage 42 when a part of ventral fin was clearly appeared anterior to anus regions. Stage 45 when coiled-coil structure was observed in gut region. (C) Table for B. Rapamycin treatments were performed after 2-cell stage before control embryos reached scheduled stage. dn-Rheb mRNA was microinjected at 2-cell stage. Every 15 min we counted number of embryos that already reached scheduled stage. Using these scores, each averaged arrival time for scheduled stages was calculated.
Fig. 3.
Pigmentation defects induced by rapamycin treatment. Major pigmentation located at lateral and dorsal side of embryos at stage 42 was obviously detected in control embryos, but weakly in rapamycin-treated (20 μM) embryos (100%, n = 15). PCNT, PNT, PY, and PS indicate pigmentation underlain by cephalic neural tube, neural tube, yolk, and somites. Slightly bigger yolk was frequently observed in treated. Scale bars represent 1 mm.
Fig. 4.
Gastrointestinal malformation induced by rapamycin treatment and dn-Rheb mRNA injection. (A) Control embryo at stage 45 (95.3 hpf) for B and C. (B) Rapamycin-treated embryo at stage 45. Embryos were treated with 40 μM of rapamycin and cultured. Pigmentation defects were stronger than Stage 42. Both size and shape of gut were affected by rapamycin treatment (100%, n = 15). (C) dn-Rheb mRNA (200 pg) injected embryo at stage 45. Both size and shape of gut were also affected by dn-Rheb mRNA injection (100%, n = 15), but pigmentations were observed like control. Dotted lines mean sectioned sites for histological analyses shown in G–I. (D–F) Surgically-resected guts from embryos shown in A–C. Upper and under panels respectively indicate ventral and dorsal views. Secondary coiled structure of gut was not detected in rapamycin-treated and dn-Rheb mRNA injected embryos. 1°, primary coil; 2°, secondary coil. The diagram of gut structure was shown on the bottom-left corner. (G–I) Histological sections created from embryos shown in A–C. In both rapamycin-treated and dn-Rheb mRNA injected embryos, gut formations were disordered and thicker than the case of control embryo. bd, bile duct; li, large intestine; lu, lungs; pa, pancreas; si, small intestine; st, stomach. (J) Long cultured embryos in 20 μM of rapamycin (176 hpf). All embryos were swollen like balloon but survived until 200 hpf maximum. The scale bars of A, B, and C and of D, E, and F represent 2 mm and 0.5 mm, respectively.
