Iwano H et al. (2004), Xenopus MBD3 plays a crucial role in an early s...

XB-ART-3745

Dev Biol 2004 Apr 15;2682:416-28. doi: 10.1016/j.ydbio.2003.12.032.

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Xenopus MBD3 plays a crucial role in an early stage of development.

Iwano H , Nakamura M , Tajima S .

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DNA methylation plays a crucial role in gene silencing via recruitment of the proteins that specifically recognize methyl-CpG. In the present study, we have shown that two splicing isoforms of MBD3, xMBD3 and xMBD3LF, are the major methyl-CpG binding proteins in Xenopus eggs and early stage embryos. They were highly expressed in the eyes and central nerve system of tadpoles. Inhibition of the expression of xMBD3 by antisense oligonucleotides severely affected embryogenesis. Low-dose injection of antisense oligonucleotides specifically affected eye formation. An identical phenotype was observed on the forced expression of xMBD3 mutated in the methyl-CpG binding domain (MBD) and xMBD3LF, those of which lack methylated DNA binding activity. On the other hand, the eye-defective phenotype was not induced on the injection of truncated forms of mutant xMBD3 or xMBD3LF that contained MBD. We propose that MBD3, distinct from the case in mouse, plays a crucial role in the recognition of methylated genes as an intrinsic component of the complex to guide the corepressor complex during an early stage of Xenopus embryogenesis.

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Species referenced: Xenopus
Genes referenced: mbd2 mbd3 mmut myc pax6
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	Fig. 1. Expression of xMBD2, xMeCP2, and xMBD3 during early Xenopus embryogenesis. (A) Schematic illustration of the cDNAs and the primers used for RT-PCR of xMBD3 and xMBD2. Boxes indicate the coding regions. The gray boxes denote the MBD. For xMBD3LF, the extra sequence inserted into the MBD of xMBD3 is indicated by a black box. The primer sets that amplify xMBD3 + xMBD3LF (a), xMBD3LF (b), the 3V region of xMBD2 (c), and the MBD of xMBD2 (d) are shown. (B) Expression of xMBD2, xMeCP2, and xMBD3 was detected by RT-PCR. xMBD2-5Vshows the amplification by the primer set (d) that amplifies the MBD of xMBD2. Ornithine decarboxylase (ODC) and histone H4 were used as controls. (C) xMBD3 and xMBD3LF proteins were detected during early development by Western blotting with anti-xMBD3 C-terminal antibodies. Four embryos were used for the detection in each case.
	Fig. 2. Spatial expression patterns of xMBD3 transcripts in Xenopus embryos on whole-mount in situ hybridization. (A) Animal pole (left), vegetal pole (middle), and lateral (right) views at stage 8. Dorsal (B) and anterior (C) views of neural groove embryos at stage 19. Dorsal (D) and lateral (E) views of tailbud embryos at stage 23. (F) Lateral view of a tadpole stage embryo.
	Fig. 3. The antisense MO for xMBD3 inhibited the translation of xMBD3 and xMBD3LF. The antisense MO or control MO was injected into two blastomeres in the indicated total amounts. xMBD3 was detected by Western blotting with anti-xMBD3 C-terminal antibodies at the indicated stages. Five embryos were analyzed by SDS-PAGE, and xMBD3 proteins were determined by Western blot analysis of the same membrane in each case.
	Fig. 4. xMBD3 MO delayed early neural development in Xenopus. Embryos were injected at the two-cell stage, into two blastomeres, with 60 ng per blastomere of the control MO (A and E), 10 (B and F), 30 (C and G), and 60 ng (D and H) per blastomere of the antisense MO. The images were recorded when normal embryos had reached stages 20 and 32. The embryos injected with the control MO and total 20 ng of the antisense MO developed to apparently similar stages, stages 20 (A and B) and 32 (E and F), to those of normal embryos. On the other hand, the development of the embryos injected with total 60 ng of the antisense MO apparently reached stages 17–18 (C) and 22– 25 (G), and those with total 120 ng reached stages 11– 12 (D) and were then arrested at these stages (H), while normal embryos had reached stages 20 and 32, respectively.
	Fig. 5. Injection of the antisense MO induced the eye-defective phenotype. Embryos at the two-cell stage were injected with 10 ng per blastomere of the antisense MO (A and B), 60 ng per blastomere of the control MO (C and L), or 10 ng of the antisense MO and 50 pg of modified xMBD3LF mRNA (J and K) mRNA per blastomere into two blastomeres. In panels D–I, embryos at the four-cell stage were injected with 10 ng per blastomere of the antisense MO (D, E, G, and H) or 60 ng per blastomere of the control MO (F and I) into two blastomeres, either dorsally (D–F) or ventrally (G–I). The images were recorded when the embryos had reached stages 36–38. Arrows in panels A, B, D, and E indicate the prospective eye regions.
	Fig. 6. Schematic illustrations of xMBD3 (xMBD3LF), the mutants, and the truncated forms. The constructs used for the injection experiments are shown. The number in each illustration is the amino acid number. All the constructs had a Myc-tag sequence added at their 5V end. The truncated constructs were in a vector with a nuclear localization signal before the Myc-tag sequence. Methyl-CpG binding domains (MBD), coiled-coil sequences, and Glu- and Asp-rich sequences (E, D rich) are indicated by boxes. Black boxes indicate the 19 amino acid sequence inserted into the MBD of xMBD3LF through alternative splicing. At the right of the illustrations, the results of the injection experiments on eye-defective and apoptotic phenotypes, and the ability of methyl-CpG binding are shown.
	Fig. 7. Injected mRNA was effectively translated into proteins in Xenopus embryos. Whole-cell extracts prepared from 18 embryos each at the 33– 34 stage embryos injected at the four-cell stage with 0.5 ng mRNA per blastomere of xMBD3LF (1), xMBD3 (2), mut-xMBD3 (3), xMBD3LFMBD (4), xMBD3-MBD (5), mut-xMBD3-MBD (6), xMBD3LFDCC (7), xMBD3DCC (8), xMBD3DMBD (9), and h-galactosidase (10) into two dorsal (D) or ventral (V) blastomeres were immunoprecipitated with anti- Myc monoclonal antibody (9E10), then analyzed by Western blotting with alkaline phosphatase-coupled secondary antibodies.
	Fig. 8. Effect of overexpression of various xMBD3 constructs on the eye phenotype. Embryos were injected with 0.5 ng mRNA per blastomere of xMBD3 (A), xMBD3LF (B), mut-xMBD3 (C), xMBD3LF-MBD (D), mut-xMBD3-MBD (E), xMBD3LFDCC (F), xMBD3DMBD (G), xMBD3-MBD (H), xMBD3DCC (I), and h-galactosidase (h-gal) (J) into two dorsal blastomeres at the four-cell stage. An uninjected embryo is also shown (K). The images were recorded when the embryos had reached stages 36–38. Arrows indicate the prospective eye regions in the embryos that showed a defect in eye formation.
	Fig. 9. Injection of xMBD3 mRNA induced apoptotic cell clusters. Embryos were injected with 0.5 ng mRNA per blastomere of xMBD3 (A– C), xMBD3LF (D– F), or h-galactosidase (h-gal) (G and H) into two dorsal blastomeres at the four-cell stage. The images were recorded at around the gastrula or neurula stage (A, B, D, E, and G) and at the stage of hatching (C, F, and H). Panels B and E show TUNNEL staining. Abnormal cell clusters appearing in the embryos injected with xMBD3 are indicated by circles.
	Fig. 10. Effect of the antisense MO on expression of the Pax6 gene. Embryos were injected at the two-cell stage with 15 ng per blastomere of the control MO (A) or the antisense MO (B and C) into two blasotmeres, and at the four-cell stage with 5 ng per blastomere of the control MO (D) or the antisense MO (E– G), into two dorsal blastomeres. The expression of the Pax6 gene at the neurula (A–C) and tadpole (D–G) stages was detected by in situ hybridization. The expression of Pax6, especially at around prospective eye positions, was disturbed by the antisense MO. Each lane in panel H indicates the amplified DNA by RT-PCR performed with total RNA prepared from one embryo injected with the antisense MO (MO) or uninjected embryo (None) at stage 18 and stages 32– 34 for Pax6 and histone H4. PCR without reverse transcriptase reaction ( ) is indicated as control.
	mbd3 (methyl-CpG binding domain protein 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, lateral view, anterior left, dorsal up.
	mbd3 (methyl-CpG binding domain protein 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	mbd3 (methyl-CpG binding domain protein 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.