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FIGURE 1.
Temporal and spatial expression profile of XH2AX during embryogenesis. A, RT-PCR analysis of temporal mRNA expression of XH2AX. Developmental stages are indicated above each lane. Histone H4 served as a loading control. −RT, control reaction without reverse transcriptase. B–I, whole-mount in situ hybridization showing the spatial expression of XH2AX during early Xenopus development. B and C, blastula stage: animal hemisphere view (B) and vegetal hemisphere view (C); D and E, gastrula stage: animal hemisphere view (D) and vegetal hemisphere view (E); F, neurula stage: anterior view with posterior right; G and H, tail bud stage: lateral view with anterior left (G) and dorsal view with anterior left (H); I, stages 33–34. The black lines represent the angle of sectioning for J–L. J–L, transverse section through a stage 33–34 embryo stained by whole-mount in situ hybridization for XH2AX mRNA. ba, branchial arches; br, brain; ey, eye; hg, hatching gland; mb, midbrain; ov, otic vesicle; so, somite.
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FIGURE 2.
Depletion of XH2AX causes defective anterior neural formation. Control-MO or XH2AX-MO was injected at the one- or two-cell stage in the animal pole region, and embryos were cultured until the tadpole stage. A, the MO sequence targeting XH2AX. B, XH2AX-MO (30 ng) specifically knocked down the translation of the overexpressed C-terminal HA-tagged histone H2AX protein. α-Tubulin served as a specificity control. C, phenotypes of XH2AX-depleted embryos. D, β-gal mRNA (200 pg)-injected embryos were used as a control phenotype. E, injection of Control-MO did not result in severe defects. F–H, hematoxylin-stained transverse section of the head region of tadpole embryos from Type A (F and G) or Control-MO (H). I, quantitative results of relative defects in whole embryos. *, p < 0.05. J, RT-PCR analysis of whole embryos expressing XH2AX-MO. XH2AX-MO caused repression of anterior neural markers (Otx2, Rx1, and Pax6), the pan-neural marker (N-CAM), and the neural differentiation marker (N-tubulin) without changing the mesoderm marker actin and the posterior marker HoxB9. Control-MO did not change the anterior neural markers tested above. K, XH2AX-MO (20 ng) was injected into the DMZ or VMZ of four-cell stage embryos. L, ventrally XH2AX-MO-injected embryos showed a normal phenotype. M, dorsally injected XH2AX-MO caused a weak head defect with small eyes and shortened axis. N, RT-PCR analysis of whole embryos dorsally or ventrally expressing XH2AX-MO. Dorsally (but not ventrally) expressed XH2AX-MO repressed anterior neural markers, including neural markers, but not the actin and globin mesoderm markers, compared with the whole embryo (W.E.) that was not injected. −RT, control reaction without reverse transcriptase.
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FIGURE 3.
XH2AX is required for anterior neural development. A–D, one- or two-cell stage embryos were injected with XH2AX-MO (30 ng) alone or in combination with the indicated dose of FLAG-XH2AX-8aa mRNA for rescue experiments. A, XH2AX-MO (30 ng) specifically knocked down the translation of the overexpressed C-terminal HA-tagged histone H2AX protein, but not the FLAG-H2AX-8aa protein, which lacks the MO target site. α-Tubulin served as a control for specificity. B, rescued phenotypes were classified into three types: Type A, severely defective embryo; Type B, moderately defective embryo; and Type C, fully rescued embryo. The control was an uninjected embryo. C, quantitative results of relative rescue in whole embryos are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, RT-PCR analysis of sibling embryos: FLAG-XH2AX-8aa mRNA (150 pg) rescue of anterior neural markers (Otx2, Rx1, and Pax6) and the pan-neural marker (N-CAM) that were repressed by XH2AX-MO. W.E., whole embryo as a positive control for PCR; −RT, control reaction without reverse transcriptase. E–G, whole-mount in situ hybridizations on embryos injected as described for A with a Pax6 probe, an anterior neural marker. FLAG-XH2AX-8aa mRNA (G) rescued Pax6 expression previously repressed by XH2AX-MO (F and F′). E, control-MO-injected embryo.
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FIGURE 4.
XH2AX is required for the induction of anterior neural markers by activin. A–I, animal caps, explanted from embryos injected with Control-MO or XH2AX-MO (30 ng) either alone or in combination with the indicated dose of FLAG-XH2AX-8aa mRNA, were incubated with or without the activin protein (40 ng/ml) until stage 24, anterior neural markers (Otx2, Rx1, and Pax6), pan-neural markers (N-CAM and Sox3), a mesoderm marker (actin), and EF1α as a loading control. W.E., whole embryo as a positive control for PCR; −RT, control reaction without reverse transcriptase. A, RT-PCR analysis of animal cap explants. XH2AX-MO selectively blocked the activin-induced expression of Sox3, N-CAM, Pax6, and Otx2, but not actin (lane 4). Control-MO did not change activin induction of any of the genes (lane 2). B–G, phenotype of animal caps explanted from embryos injected as described for A–I. XH2AX-MO blocked activin-induced elongation of animal caps, and this phenotype was rescued by XH2AX-8aa mRNA in a dose-dependent manner. H, RT-PCR analysis of samples shown in B–G. XH2AX-8aa mRNA injection rescued activin induction of Sox3, Pax6, N-CAM in XH2AX-MO-injected animal caps. I, Western blot of embryonic extracts probed with anti-FLAG antibody, showing that all injected constructs were translated equally when injected into Xenopus embryos. Actin served as a loading control.
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FIGURE 5.
Depletion of Chk1 causes defective anterior neural formation. A, Chk1-MO (20 ng) was injected into the DMZ or VMZ of four-cell stage embryos. B, Chk1-MO specifically knocked down the translation of an overexpressed C-terminal HA-tagged Chk1 protein. Actin served as a control for specificity. C and D, dorsally injected Chk1-MO caused severe head and eye defects with a shortened axis (C) compared with the normal embryo that was not injected (D). E, RT-PCR analysis of whole embryos dorsally or ventrally expressing Chk1-MO. Dorsally (but not ventrally) expressed Chk1-MO repressed anterior neural markers (Pax6, Rx1, and Otx2) and the neural marker (N-CAM), but not the actin and globin mesoderm markers, compared with whole embryos (W.E.) not injected. EF1α served as a loading control. −RT, control reaction without reverse transcriptase. F, whole-mount in situ hybridization showing the spatial expression of Chk1 at the tadpole stage. The upper panel shows a magnified anterior region of an embryo.
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FIGURE 6.
XH2AX is phosphorylated by Chk1 at Thr16 of the N terminus. A, in vivo interaction between XH2AX and Chk1. Lysates from embryos injected with mRNAs for HA-tagged Xenopus Chk1 and Myc-tagged XH2AX expression were used to immunoprecipitate (IP) Myc-XH2AX (left panels) or HA-Xenopus Chk1 (right panels). IB, immunoblot. B, schematic representation of the structure of XH2AX. The histone fold domain (HFD) is a globular domain comprising the nucleosome core. αN, α-helix of the N-terminal tail; αC, α-helix of the C-terminal tail. C, Coomassie Brilliant Blue (CBB) staining of a bacterially expressed GST-XH2AX deletion mutant. D–G, Western blot using an anti-phosphothreonine antibody after an in vitro kinase reaction. D, Chk1 phosphorylated a threonine residue of XH2AX. E, Chk1 phosphorylated a threonine residue in the N terminus (but not the C terminus) of XH2AX. F, Chk1 phosphorylated a threonine residue in the N terminus of XH2AX (*), whereas Chk2 had little effect. G, phosphorylation of a threonine residue in the N terminus of XH2AX (*) was absent when Thr16 was mutated to alanine. H–J, Western blot using anti-phospho-Thr16 XH2AX antibody after an in vitro kinase reaction. H, anti-phospho-Thr16 XH2AX antibody was used for detecting phosphorylation of XH2AX. The upper arrow indicates full-length GST-XH2AX, and the lower arrow indicates the N terminus of GST-XH2AX. Asterisks indicate phosphorylation of full-length XH2AX and its N terminus. I, phosphorylation of XH2AX at Thr16 by Chk1 is shown by an in vitro kinase assay using immunoprecipitated FLAG-tagged Chk1. Extracts from embryos injected or uninjected (control) with FLAG-Chk1 mRNA were immunoprecipitated using an anti-FLAG antibody, and the kinase reaction was performed with the indicated GST-recombinant protein as a substrate. Phosphorylation of the threonine residue was analyzed by Western blotting using anti-phospho-Thr16 XH2AX antibody. Western blotting with anti-FLAG antibody showed that equal amounts of Chk1 immunoprecipitates were loaded. The upper arrow indicates the N terminus of GST-XH2AX, and the lower arrow indicates the IgG light chain. The asterisk indicates phosphorylated XH2AX. J, immunoprecipitated Chk1 from DMZ-expressed FLAG-Chk1 mRNA phosphorylated Thr16 of XH2AX. Extracts from embryos dorsally or ventrally injected with FLAG-Chk1 mRNA were immunoprecipitated using an anti-FLAG antibody and subjected to a kinase reaction with the indicated GST-recombinant protein as a substrate. Western blotting using anti-FLAG antibody showed that equal amounts of Chk1 were immunoprecipitated. The upper arrow indicates the N terminus of GST-XH2AX, and the lower arrow indicates the IgG light chain. The asterisk indicates phosphorylated XH2AX.
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FIGURE 7.
Thr16 of XH2AX has a critical role in neurogenesis. A and B, one or two-cell stage embryos injected with XH2AX-MO (30 ng) alone or in combination with 150 pg of FLAG-XH2AX-8aa or FLAG-XH2AX-T16A mRNA and cultured until the tadpole stage. A, severely defective embryo. B, fully rescued embryo. C, quantitative results of relative rescue in whole embryos shown in A and B. **, p < 0.01; ***, p < 0.001. D–I, animal caps explanted from embryos injected with XH2AX-MO (30 ng) alone or in combination with FLAG-XH2AX-8aa or FLAG-XH2AX-T16A mRNA were incubated with or without the activin protein (40 ng/ml) until stage 24. D–H, phenotype of animal cap explans. I, RT-PCR analysis of samples shown in E–H. The anterior neural marker Pax6 and the pan-neural markers N-CAM and Sox3 were used; EF1α served as a loading control. W.E., whole embryo as a positive control of PCR; −RT, control reaction without reverse transcriptase.
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h2ax (H2A.X variant histone) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
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h2ax (H2A.X variant histone) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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