Fig. 4. Apoptosis in Xenopus embryos injected with RNA encoding Bix3. (A-F) One group of embryos was left uninjected whereas another (G-I) was injected with 400 pg of Bix3 RNA at the one-cell stage. At stage 8.5, half of the uninjected embryos were transferred to 0.1 mg/ml cycloheximide (D-F), whereas the other half was allowed to develop normally (A-C). Embryos were collected at the indicated stages and used for TUNEL staining. Positive cells are dark blue. Note that loosely adherent cells in Bix3-injected embryos, as seen in Fig. 2, are lost during the TUNEL procedure. (J) tPARP assay shows that Bix1 does not activate caspase activity whereas induction of apoptosis by Bix3 was first detectable by stage 11. Cycloheximide induces caspase activity from early gastrula stage 10. Intact tPARP has a relative molecular mass of 60 kD (upper arrows in both gels), and this is cleaved into fragments of 36 kD and 24 kD (lower arrows). (K) Human Bcl2 delays the onset of Bix3-induced apoptosis. Caspase assays were performed on control embryos at the indicated stages, embryos injected with RNA encoding Bix3, or embryos injected with Bix3 RNA together with RNA encoding human Bcl2 (500 pg). Note that human Bcl2 delays the onset of caspase activity. (L-N) Partial rescue of the cell adhesion and apoptosis defect by human Bcl2. Bix3 RNA alone (50 pg; L), or in combination with 500 pg of human Bcl2 RNA (M), was injected in the animal pole of one-cell stage embryos. Embryos were left to develop and photographed at mid-gastrula stage 11.5-12. Note that human Bcl2 does not prevent the appearance of darkly pigmented cells in the animal hemisphere but does reduce or delay the incidence of cell disaggregation. Human Bcl2 delayed disaggregation in five out of seven experiments of this sort. (N) Quantitation of an experiment of the type illustrated in (L) and (M). Embryos undergoing cell disaggregation were scored at the indicated stages.
Image published in: Trindade M et al. (2003)
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