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Apoptosis is fundamental to normal vertebrate development. A dramatic example of postembryonic development involving apoptosis is tail regression during amphibian metamorphosis. Earlier studies led us to propose a functional role for the pro-apoptotic protein Bax in tadpoletail regression. However, its physiological relevance has never been analyzed. We have now cloned a cDNA encoding Xenopus laevis bax (xlbax) and used in vivo gene transfer in tailmuscle to analyze the effects of xlbax overexpression. Furthermore, by using an antisense strategy in a similar experimental paradigm, xlbax antisense mRNA was shown to block the apoptotic effects of xlbax and protect against apoptosis in metamorphosing tadpoles. Our results suggest that xlbax is a regulator of muscle fiber death in the regressing tail during metamorphosis.
Figure 2. xlbax expression during natural and induced metamorphosis. A: Total RNA was extracted from isolated tail at different metamorphic stages and assayed by polymerase chain reaction (PCR) for the expression of xlbax, xcaspase3, and xR11 mRNAs. The internal control was Rpl8. After 30 cycles of PCR, 10 μl of the reaction were electrophoresed on 2% agarose gels and stained with ethidium bromide. In the Ct- lane, the reverse transcription product was replaced by water to check for PCR contamination. B: A more quantitative PCR procedure using 32P-labeled dCTP was carried out to compare stage NF54 and NF62. After 20 cycles of PCR, 10 μl of the reaction were electrophoresed on acrylamide gels and the results were quantified by PhosphorImager scanning. The average values SEM of three experiments are expressed as fold induction where 1 equals means for stage NF54. Moreover for each sample, densitometry readings were normalized against the value for rpl8 RNA. Statistical signification as compared with the untreated animals is expressed as **P < 0.01. C: Stage NF55 tadpoles were treated for 1 to 5 days with T3 (10 nM). Total RNA was extracted from isolated tails and assayed by PCR for the expression of xlbax mRNAs as described in B. The figure presents one of two independent experiments.
Figure 3. xlbax mRNA spatiotemporal expression during natural metamorphosis. B,D: Endogenous bax mRNA expression was followed in tailmuscle at stage (St) NF55 (B) and NF62 (D) by in situ hybridization, using an antisense digoxigenin-labeled probe on longitudinal Vibratome sections. A,C: Some sections were hybridized with a sense probe to test for specificity (A, NF55 and C, NF62). Black arrows indicate dead muscle fibers. The in situ hybridization was done twice on independent groups of tadpoles and gave identical results.
Figure 4. xlbax transfection reduces expression of cotransfected green fluorescent protein (GFP) in muscle cells in vivo. Tadpoles were cotransfected in the tailmuscle with 1 μg peGFP-C1 and either 2 μg of a control plasmid vector (pcDNA3) or a plasmid-expressing xlbax (pcDNA3-xlbax). Three days later, animals were photographed directly in toto under a dissecting scope equipped with fluorescent light. A: In vivo detection of GFP expression in control-injected tadpole. B: GFP detection in tadpolemuscle injected with xlbax. Note decreased GFP expression compared with A. C: Numbers of GFP-positive myofiber clusters in control (Ct, A) and xlbax (B) -injected tadpoles. Means SEM are given; n ≥ 10 in all cases; **P ≤ 0.01. D,E: Longitudinal Vibratome sections of tail observed under a microscope equipped with fluorescent light. D: Section of control-injected tadpoletailmuscle. The blue arrow indicates GFP-positive myofibers. E: Section of tadpoletailmuscle injected with xlbax. No GFP-positive myofibers were observed. The red arrow indicates sites of myofibers loss. Each section shown on the figure is from one of two independent experiments providing identical results.
Figure 6. xlbax antisense (AS) transfection delays cotransfected green fluorescent protein (GFP) disappearance during natural metamorphosis in vivo. Stage NF55 tadpoles were cotransfected in the tailmuscle with 1 μg of peGFP-C1 and either 1 μg of a control pcDNA3 vector in the left side of the tail or a plasmid-expressing full-length xlbax AS RNA in the right side of the tail. A: Animals were photographed directly under a dissecting scope equipped with fluorescent light at different stages of metamorphosis NF61 (A), NF63 (B), and NF64 (C). Note that, as metamorphosis progresses (over 15 days), the GFP signal disappears almost totally in the control side (Ct, bottom) but is maintained in the xlbax mRNA AS-injected side. The results represent one of two independent experiments.
Figure 7. xlbax antisense (AS) transfection delays cotransfected green fluorescent protein (GFP) disappearance during T3-induced metamorphosis in tail culture. Stage NF55 tadpoles were cotransfected in the tailmuscle with 1 μg of peGFP-C1, 0.1 μg of pcDNA3-fLuciferase, and either 1 μg of a control pcDNA3 vector or pcDNA3-xlbax AS72. GFP expression was observed directly 3 days after transfection. Both tadpole groups were killed, and the tails were maintained in culture in absence or presence of T3 (10 nM) for 8 days. A,E: GFP expression in the control (Ct) group before tail culture. C,G: GFP expression in antisense (AS) group before tail culture. B: GFP expression in the Ct group after tail culture without T3 treatment. D: GFP expression in the AS group after tail culture without T3 treatment. F: GFP expression in the Ct group after tail culture with T3 treatment. H: GFP expression in the AS group after tail culture with T3 treatment. Each figure represents one of at two independent experiments with identical results.
