Fig. 5. The apoptotic phenotype of xDnmt1-deficient explants is dependent upon differentiation and can be rescued by co-injection of HPV-E6 and Bcl-2 mRNAs. (A) Animal cap explants from either antisense MD or MD/Bcl-2 co-injected blastulae are TUNEL negative in the absence of activin. An explant derived from an MD embryo is shown. (B) Animal cap explants from MD blastulae treated with a low dose of activin (2 U/ml TGF) exhibit a low level of TUNEL staining (blue). (C) High dose of activin (10 U/ml) causes death of >50% of MD animal cap explant cells as detected by the intensity of TUNEL staining (blue). (D) p53 levels are elevated compared with wild type (WT) in explants derived from antisense xDnmt1 RNA injected embryos (MD) non-induced (–) or induced (MD ac 10 U/ml TGF) with activin. A blot was reprobed for PCNA as a loading control. (E) Co-injection of a defective HPVE6 (MD/HPV-6E6) is unable to prevent hypomethylation-induced apoptosis as shown by TUNEL-positive staining (dark brown) in activin (10 U/ml TGF)-treated MD animal cap explants. (F) Co-injection of a wild-type HPVE6 (MD/HPV-18E6) is able to prevent hypomethylation-induced apoptosis in activin (10 U/ml TGF)-treated MD animal cap explants. (G) Co-injection of a wild-type HPV-E6 (MD/18E6) mRNA promotes p53 degradation in gastrula embryos whilst an N-terminal deletion of HPV-E6 (MD/6E6) does not. (H) Injection of a human Bcl-2 (hBcl-2) mRNA by itself does not lead to apoptosis in activin-treated (10 U/ml TGF) WT animal cap explants. (I) Co-injection of a human Bcl-2 mRNA (MD/hBcl-2) is able to prevent hypomethylation-induced apoptosis in activin (10 U/ml TGF)-treated MD animal caps. (J) Co-injection of a Bcl-2 mRNA does not interfere with p53 stability in wild-type (WT/Bcl-2) or maternal MD gastrula embryos. (K) 14-3-3 proteins accumulate in maternally depleted (MD), but not in the wild-type (WT) cap extracts upon activin induction. Co-injection of functional HPV-18E6 (MD/18E6 ac 10 U/ml TGF) prevents the accumulation of 14-3-3 in activin-treated MD animal cap explants. All the explants were collected 6 h after treatment and TUNEL stained for apoptosis or used for preparation of extracts.