Lee SY, Lau AT, Jeong CH, Shim JH, Kim HG, Kim J, Bode AM, Dong Z.

CA077646 NCI NIH HHS , CA111536 NCI NIH HHS , CA120388 NCI NIH HHS , ES016548 NIEHS NIH HHS , R37 CA081064 NCI NIH HHS , R01 ES016548-04 NIEHS NIH HHS , R37 CA081064-13 NCI NIH HHS , R01 ES016548 NIEHS NIH HHS , R01 CA077646 NCI NIH HHS , R01 CA111536 NCI NIH HHS , R01 CA120388 NCI NIH HHS

	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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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