Suzuki M et al. (2016), In vivo tracking of histone H3 lysine 9 acetyla...

XB-ART-51894

Genes Cells 2016 Apr 01;214:358-69. doi: 10.1111/gtc.12349.

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In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

Suzuki M , Takagi C , Miura S , Sakane Y , Suzuki M , Sakuma T , Sakamoto N , Endo T , Kamei Y , Sato Y , Kimura H , Yamamoto T , Ueno N , Suzuki KT .

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Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

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Species referenced: Xenopus laevis
Genes referenced: aopep hpse

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	Figure 1 H3K9ac-Mintbody enhanced green ﬂuorescent protein (EGFP) ﬂuorescence pattern during early embryogenesis. (A, B)EGFP ﬂuorescence pattern of Mintbody mRNA-injected embryos during early embryogenesis. EGFP Fluorescence images at NFstage 32 (A) and 41 (B) were captured using a ﬂuorescence stereomicroscope. S, somite. (C–F) CMV:Mintbody F0 embryosduring early embryogenesis. EGFP ﬂuorescence images at NF stage 31 (C), 41 (D), 45 (E) and 48 (F). S, somite; O, olfactoryepithelium.
	Figure 2 Enhanced green ﬂuorescent protein (EGFP) ﬂuorescence pattern of CMV:H3K9ac-Mintbody F0 embryos during tailregeneration. EGFP ﬂuorescence (A) and bright-ﬁeld (B) images of a representative embryo just before tail amputation at NF stage41. Continuous EGFP ﬂuorescence images at 0 dpa (C), 1 dpa (D), 2 dpa (E), 3 dpa (F) and 5 dpa (G). Dotted lines trace theshapes of tails. Arrowheads indicate amputation sites. Dorsal side is oriented at the top of each panel. (D0) Merged EGFP ﬂuores-cence and bright-ﬁeld images of (D), high magniﬁcation. (H) Localization and tissue distribution of Mintbody in the regeneratingtail. Immunohistochemistry was carried out using anti-EGFP-antibody (green) in the sagittal section of an F0 embryo at 1 dpa.Nuclei were counterstained with DAPI (blue). Dorsal side is oriented at the top of the panel. Note that nuclear accumulation ofMintbody is dominantly seen in regenerating notochord. NC, notochord; NA, neural ampulla; WE, wound epithelium. (I) Aschematic illustration of regeneration bud during Xenopus tail regeneration at 1 dpa in lateral view. Dorsal side is oriented at thetop. At 1–2 dpa, regenerating notochord has a bullet-shaped structure.
	Figure 3 Regenerating notochord cells possessing robust H3K9ac-Mintbody signals migrate and reconstruct notochord. Continu-ous images of tail regeneration were captured from just after amputation to 73 hpa at the indicated time. Straight lines indicate theamputation site. Curly and square brackets show the proximal notochord and regenerating notochord cells, respectively. Left andright panels show Enhanced green ﬂuorescent protein ﬂuorescence and bright-ﬁeld images at each indicated time, respectively.Dorsal side is oriented at the top of each panel.
	Figure 4 Reactive oxygen species (ROS) production and the increase of H3K9ac-Mintbody signal at the onset of tail regenera-tion. To visualize ROS production, wild-type embryos were exposed to H2DCFDA 2 h before observation at each indicatedtime. Mintbody signals in F1 embryos were observed from 0 to 24 hpa at each time point. Dotted lines trace the shapes of tails.NC, notochord. Dorsal side is oriented at the top of each panel.
	Figure 5 Reactive oxygen species (ROS) inhibition impairs the increase of H3K9ac-Mintbody signal in tail regeneration. (A) Ascheme of ROS inhibition in tail regeneration. F1 embryos at NF stage 41 were pre-treated with apocynin (APO), an inhibitor ofROS production, for 2 h before amputation; then, tails were amputated. Mintbody signal was observed at 1 dpa. Xenopus imagesfrom Nieuwkoop & Faber (1994) were modiﬁed. (B, C) Representative images of dimethyl sulfoxide (DMSO)- and APO-treatedembryos. Mintbody signals were seen in the notochord of control DMSO-treated embryos (B; n = 22 positive in notochord/28total, 79%), whereas no or faint signal was seen in the notochord of APO-treated embryos (C; n = 5 positive in notochord/47total, 11%). The data were obtained from two independent experiments using three different F0 parents. Dotted lines trace theshapes of tails. NC, notochord; WE, wound epithelium. Dorsal side is oriented at the top of each panel.
	Figure 6 Schematic model of reactive oxygen species (ROS)production and H3K9ac modiﬁcation at the onset of tailregeneration. After amputation, ROS are immediately pro-duced near the amputation site and its production is sustainedduring tail regeneration. Acetylation of H3K9 is subsequentlyinduced at the amputation site, in particular, in the notochordfrom 16 to 24 hpa.
	Figure S1. (A) A scheme for generating Mintbody transgenic Xenopus laevis using the microhomology-mediated end-joining (MMEJ)-dependent knock-in technique. Details of this knock-in technique are described in Nakade et al. (2014). Donor vectors are able to be knocked-in to the tyr loci via MMEJ repair using TALENs. The knock-in phenotype exhibited both albinism and EGFP fluorescence. (B, C) Phenotypes of CMV:Mintbody F0 embryos. Embryos were divided into three groups according to their phenotypes. (B) Representative phenotypes: EGFP-positive in somites, fluorescence signal in the nuclei of somites was entirely observed; Mosaic, highly mosaic fluorescence signal was observed; Non-fluorescent, no fluorescence signal was observed. (C) Percentage of phenotypes from three independent experimental groups: Uninjected, in vitro fertilized only embryos; TALEN-R, only right TALEN mRNA and donor vector were co-injected as a negative control of transgenesis; TALEN-R/L, a pair of TALEN mRNAs and donor vector were co-injected. Total numbers of individuals in this experiment are shown at the top of each graph.
	Figure S2. EGFP fluorescence pattern of EF1a:Mintbody F0 embryo during tail regeneration. EGFP fluorescence (A) and bright-field (B) images of a representative F0 embryo at NF stage 41, just before tail amputation. Continuous images from a representative embryo at just after amputation (C), 1 dpa (D), 2 dpa (E), 3 dpa (F), and 5 dpa (G). Dotted lines trace the shapes of tails. Dorsal side is oriented at the top of each panel. Note that this EF1a:Mintbody embryo was generated by the I-SceI transgenic method. Strong nuclear Mintbody signal was 1" shown in 3 of 4 (75%) transgenic embryos in regenerating notochord at 1 dpa.
	Figure S3. EGFP fluorescence pattern of CMV:Mintbody F1 embryos and tadpoles during tail regeneration. EGFP fluorescence (A) and bright-field (B) images of a representative embryo just after tail amputation at NF stage 41. Continuous EGFP fluorescence images at 0 dpa (C), 1 dpa (D), 2 dpa (E), 3 dpa (F), and 4 dpa (G). Dotted lines trace the shapes of tails. Arrowheads indicate amputation sites. (H) Localization and tissue distribution of Mintbody in the regenerating tail. Immunohistochemistry was performed using an anti-EGFP-antibody (green) in the sagittal section of F1 embryo at 4 dpa. Nuclei were counterstained with Hoechst 33342 (blue). Note that nuclear accumulation of Mintbody is seen in regenerating notochord, neural ampulla and wound epithelium. NC, notochord; NA, neural ampulla; WE, wound epithelium. Continuous EGFP fluorescence images of regenerating tail in a representative F1 tadpole just after tail amputation at NF stage 50(I), 1 dpa (J), 2 dpa (K), 3 dpa (L), and 4 dpa (M). (J´), (K´), and (L´) are high-magnification image of (J), (K), and (L), respectively. Dotted lines trace the shapes of tails. Arrowheads indicate amputation sites. NC, notochord. All transgenic tadpoles amputated at NF stage 49-53 showed strong Mintbody signal in regenerating notochord at 1-2 dpa (n=7). Dorsal side is oriented at the top of each panel.
	Figure S4. Enhancement of Mintbody signal by TSA treatment in F1 embryos. Representative Mintbody embryos at 0 h (A, C) and 45 h after (B, D) treatment. Embryos were treated with 100 nM TSA or 0.1% EtOH (solvent control) for 45 h. Enhancement of Mintbody signal was seen in all 1" TSA treated embryos but not in EtOH treated embryos. (n=3/3, each). The media containing TSA or EtOH were changed every 24 h. Note that REMI F1 embryos are not albino unlike MMEJ-mediated transgenic F0 embryos.
	Figure S5. Time-course observation of CMV:Mintbody F1 embryo during tail regeneration. Continuous images of tail regeneration were captured from just after amputation to 63 hpa. Dotted lines trace the shapes of tails. Straight line indicates amputation site. Note that regenerating notochord cells possessing robust Mintbody signals migrated and reconstructed notochord. Dorsal side is oriented at the top of each panel.
	Figure S6. Regeneration rate following treatment with various concentrations of APO. Embryos at NF stage 41 were treated with each concentration of APO for 2 h prior to amputation. Then embryos were exposed to this inhibitor for 3 days and reared for up to 7 days in normal media. The media containing APO or DMSO were changed every 24 h. Regeneration rate was evaluated at 7 dpa according to previously reported criteria (regeneration index; Tseng et al. 2007).