Loss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos - PubMed (original) (raw)

Loss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos

I Stancheva et al. EMBO J. 2001.

Erratum in

Abstract

DNA methylation is necessary for normal embryogenesis in animals. Here we show that loss of the maintenance methyltransferase, xDnmt1p, triggers an apoptotic response during Xenopus development, which accounts for the loss of specific cell populations in hypomethylated embryos. Hypomethylation-induced apoptosis is accompanied by a stabilization in xp53 protein levels after the mid-blastula transition. Ectopic expression of HPV-E6, which promotes xp53 degradation, prevents cell death, implying that the apoptotic signal is mediated by xp53. In addition, inhibition of caspase activation by overexpression of Bcl-2 results in the development of cellular masses that resemble embryonic blastomas. Embryonic tissue explant experiments suggest that hypomethylation alters the developmental potential of early embryo cells and that apoptosis is triggered by differentiation. Our results imply that loss of DNA methylation in differentiated somatic cells provides a signal via p53 that activates cell death pathways.

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Figures

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Fig. 1. Post-MBT depletion of xDnmt1 results in phenotypic abnormalities and hypomethylation. (A) Wild-type control at stage 35. (B) Antisense xDnmt1 RNA injection at the 2-cell stage depletes the maternal form of xDnmt1 (MD) and results in headless and axis-truncated phenotypes (55%), equivalent to stage 35. (C) Embryos depleted of the zygotic form of xDnmt1 RNA by antisense RNA expressed from a CMV-driven promoter (ZD) that is activated after MBT develop axis truncation (34%) and open neural tubes (30%), equivalent to stage 35. (D) RT–PCR showing the level of antisense xDnmt1 and endogenous histone H4 (Hist H4) RNAs in maternal (left set) and zygotic (right set) depleted embryos. M is a size marker; stages are indicated on top of each lane. (E) Western blot showing that xDnmt1p is absent at different stages in maternal (MD) and zygotic (ZD) _xDnmt1_-depleted embryos. (F) Southern blots of genomic DNA from MD and ZD embryos. The DNA was digested with either _Hpa_II (methylation sensitive) or _Msp_I (methylation insensitive), and probed with a 750 bp satellite I probe. This sequence is 750 bp in length, contains two _Hpa_II sites and is present in ∼2–4 × 104 copies in the genome (Lam and Carroll, 1983). In MD embryos (lanes 3 and 4), satellite I DNA is hypomethylated at blastula and remethylated at gastrula stages (lanes 7 and 8). In the ZD embryos, loss of methylation occurs later during gastrula (lanes 11 and 12) and neurula (lane 13) stages. Embryos that received the antisense RNAs are marked with +, wild type is indicated by –.

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Fig. 2. Whole-mount TUNEL staining of normal and hypomethylated Xenopus embryos. Normal and methylation-deficient embryos were assayed by TUNEL for the appearance of apoptotic cells. Late blastula (A, E and I), gastrula (B, F and J), neurula (C, G and K) and tailbud (D, H and L) were assayed in WT (A–D), MD (E–H) and ZD (I–L) embryos. An MD gastrula embryo with a high number of TUNEL-positive cells, corresponding to +++ in Table I, is shown in (F). A ZD gastrula with a few TUNEL-positive cells, + in Table I, is shown in (J). A ZD neurula with the highest levels of TUNEL-positive cells, ++++, is shown in (K). Embryos in (A), (B), (C), (D) and (I) are TUNEL negative. Note that the positive staining (purple) is subsequent to, or coincident with loss of DNA methylation (Figure 1F) in MD and ZD embryos, respectively. Abbreviations: An, animal pole; bp, blastopore; DM, dorsal mesoderm; VM, ventral mesoderm; PHE, presumptive head ectoderm; NF, neural fold; NP, neural plate; FB, forebrain; NT, neural tube; Ant., anterior; and Post., posterior.

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Fig. 3. Hypomethylated embryos show evidence of nuclear fragmentation and activation of caspases. (A) In vitro translated tPARP protein undergoes cleavage in extracts derived from maternal (MD) and zygotic (ZD) _xDnmt1_-depleted embryos but not in wild-type (WT) extracts. Note that xDnmt1 depletion results in tPARP cleavage during gastrula (Gast.) and neurula (Neu.) but not blastula (Blast.) stages. Note that ZD extracts have a lower caspase activity. Caspase activity can be inhibited by z-DEVD-fmk in MD and ZD neurula extracts (Neu + fmk). (B) Nuclei from wild-type neurula stage embryos stained with 4′-6-diamidine-2-phenylindole (DAPI). (C) Nuclei isolated from MD neurula stage embryos stained with DAPI. Arrow indicates a fragmented nucleus. (D) Nuclei isolated from ZD neurula stage embryos stained with DAPI. Arrow indicates a fragmented nucleus. (E) DNA isolated from MD staged embryos and separated in native agarose gels does not show fragmentation at blastula stages 7 and 10. Low molecular weight fragments appear during and after gastrulation at stages 13 and 19, and are less prominent at 23 and 35. (F) DNA from ZD embryos shows increasing fragmentation beginning at neurula stage 19 and at later stages of development (23 and 35).

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Fig. 4. Hypomethylated Xenopus embryos accumulate stable forms of p53. (A) Western blotting of staged extracts from wild-type (WT), maternally depleted xDnmt1 (MD) and post-MBT-depleted (ZD) embryos. The p53 antibody identifies two forms of xp53 at 60 and 46 kDa; note that both forms decrease as normal development proceeds. In the MD and ZD embryos the active 46 kDa form is preferentially stabilized. (B) As a loading control, all western blots were re-incubated with anti-PCNA antibodies; only the wild type is shown (WT). (C) The 46 kDa form of xp53 is stabilized in UV-treated gastrula embryos, which also leads to the induction of p21. In contrast to UV treatment, MD embryos (MD gastrula) do not induce p21 at gastrulation. (D) 14-3-3 proteins accumulate after MBT in extracts from maternally (MD) and zygotically depleted (ZD) xDnmt1 embryos but not in the wild-type (WT) embryos.

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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.

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Fig. 6. Developmental competence of _xDnmt1_-depleted ectodermal explants. The expression of specific transcripts was monitored by RT–PCR analysis of untreated and treated explants derived from wild-type embryos (WT) and embryos co-injected with antisense xDnmt1 RNA and Bcl-2 sense RNA (MD/Bcl-2). Note that the pattern of expression for differentiation-specific markers is disrupted in the _xDnmt1_-depleted explants in the absence (–) and presence (+) of activin. Histone H4 (HistH4) serves as a control as its levels do not change in response to xDnmt1 depletion or activin (+) treatment. Developmental competence was monitored by expression of Xbra, Goosecoid, muscle-specific actin (M.actin), Xnot, NCAM and epidermal keratin (Ep.ker.). A further control shows the expression of these markers in normal (WT) and maternal _xDnmt1_-depleted (MD) gastrula embryos. Note the expression of differentiation-specific markers is also disrupted in the _xDnmt1_-depleted gastrula embryos.

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Fig. 7. Ectopic hypomethylation in cells rescued from apoptosis results in embryonic blastomas. (A) Scheme for single-cell injection into a dorsal blastomere of 32- to 64-cell embryos. (B) _β_-galactosidase and Bcl-2 sense RNAs were co-injected into a single dorsal animal blastomere of a 32- to 64-cell blastula (as indicated on the drawing). The injected embryos were allowed to develop until stage 35 and assayed for β-galactosidase activity. Note the normal appearance of the embryos and that the eye stains blue. (C) Antisense xDnmt1 RNA and _β_-galactosidase sense RNA were co-injected into a single dorsal animal blastomere of a 32- to 64-cell blastula. When assayed for β-galactosidase activity at stage 35 this resulted in staining on the forehead of the embryos amidst patches of dead cells that colocalize with β-galactosidase staining (blue). (D) Antisense xDnmt1 RNA, Bcl-2 and _β_-galactosidase sense RNA were co-injected into a single dorsal animal blastomere of a 32- to 64-cell blastula, which was allowed to develop until stage 35 and assayed for β-galactosidase activity. This resulted in the appearance of a large outgrowth of cells that grew out of the forehead and were β-galactosidase positive (blue). (E) Antisense xDnmt1 RNA and _β_-galactosidase sense RNA were co-injected into a single dorsal animal blastomere of a 32- to 64-cell blastula. When assayed for β-galactosidase activity at stage 35 this resulted in the absence of an eye amidst patches of dead cells (see G) that colocalize with β-galactosidase staining (blue). (F) Antisense xDnmt1 RNA, Bcl-2 and _β_-galactosidase sense RNA were co-injected into a single dorsal animal blastomere of a 32- to 64-cell blastula, which was allowed to develop until stage 35 and assayed for β-galactosidase activity. This resulted in the appearance of a large outgrowth of cells that grew in place of the eye and were β-galactosidase positive (blue). (G) The embryo in (E) was also assayed for apoptosis by TUNEL staining. A region corresponding to the β-galactosidase staining (blue) is shown under higher magnification to show the presence of TUNEL-positive cells (purple) highlighted by arrows. (H) The embryo in (F) was also assayed for apoptosis by TUNEL staining. A region corresponding to the β-galactosidase staining (blue) is shown under higher magnification to show the absence of TUNEL-positive cells (purple).

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