Kawaguchi A et al. (2012), Comparative expression analysis of the H3K27 de...

XB-ART-45255

Int J Dev Biol 2012 Jan 01;564:295-300. doi: 10.1387/ijdb.113360ak.

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Comparative expression analysis of the H3K27 demethylases, JMJD3 and UTX, with the H3K27 methylase, EZH2, in Xenopus.

Kawaguchi A , Ochi H , Sudou N , Ogino H .

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The regulated removal of the gene-silencing epigenetic mark, trimethylation of lysine 27 of histone H3 (H3K27me3), has been shown to be critical for tissue-specific activation of developmental genes; however, the extent of embryonic expression of its demethylases, JMJD3 and UTX, has remained unclear. In this study, we investigated the expression of jmjd3 and utx genes in Xenopus embryos in parallel with that of the H3K27 methylase gene, ezh2. At the blastula stage, jmjd3, utx and ezh2 showed similar expression patterns in the animal cap and marginal zone that give rise to the ectoderm and mesoderm, respectively. The three genes maintained similar expression patterns in the neural plate, preplacodal ectoderm and axial mesoderm during the gastrula and neurula stages. Later, expression was maintained in the developing brain and cranial sensory tissues, such as the eye and ear, of tailbud embryos. These findings suggest that the H3K27 demethylases and methylase may function continuously for progressive switching of genetic programs during neural development, a model involving the simultaneous action of both of the demethylases for the de-repression of silent genes and the methylase for the silencing of active genes.

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Species referenced: Xenopus
Genes referenced: cntnap1 cul1 cyb5d1 efhc2 ezh2 fundc1 kdm6a kdm6b nppa prl.2 tbx2 tbxt uqcc6 zdhhc3

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	Fig. 3. Developmental expression of xtjmjd3, xtutx and xtezh2. (A)Temporal expression was analyzed in whole embryos by RT-PCR at indicated stages. xtodc was used as a loading control (Sindelka et al., 2006). (B-Z) In situ hybridization analysis of blastula, gastrula and neurula embryos. (B,C,G,H,K,L) Expression at the blastula stage. Lateral views of the whole embryos (B,G,K), and their sections are shown (C,H,L). The animal pole is at the top. A black triangle in C indicates dotted expression of xtjmjd3intheupperpartofthevegetalregion.(D-EI-JM-N Expression at the gastrula stage. Lateral views of the whole embryos (D,I,M), sections (E,J,N), and high magnification views of the sections focusing on the involuting marginal zone (EJN are shown. The animal pole is at the top, and dorsal is to the right.The expression of the X. tropicalis brachyury gene (xtbra) was also examined for comparison with xtjmjd3 (F), since Jmjd3 is involved in the regulation of brachyury in mouse ES cells (see Discussion). (O-Q,S-U,W-Y) Expression at the early neurula stage. (O,S,W) Frontal views. White broken lines indicate the planes of the parasagittal sections shown in P, T and X. (PTX High magnification views of the parasaggital sections in the anterior region. (Q,U,Y) Transverse sections of the dorsal part of the embryos. (R,V,Z) Expression at the late neurula stage. Lateral views of whole embryos are shown. A white triangle indicates posterior expression of xtezh2. ac, animal cap; mz, marginal zone; dbg, dorsal blastoporal groove; anp, anterior neural plate; ppe, preplacodal ectoderm; np, neural plate; pp, precaudal plate; dm, dorsal mesoderm; ae, anterior endoderm; nc, notochord; pm, paraxial mesoderm; ar, archenteron roof; br, brain; mnc, migrating neural crest.
	Fig. 4. Expression of xtjmjd3, xtutx and xtezh2 in tailbud embryos and tadpoles. (A,G,M) Lateral views of tailbud embryos subjected to whole-mount in situ hybridiza- tion. Insets show embryos hybridized with a sense probe for xtjmjd3, xtutx or xtezh2. (B-E,H-K,N-Q) Transverse sections sub- jected to in situ hybridization. The planes of the sections are illustrated in the top white box. (BHN High magnification views of eyes in the transverse sections shown in B, H, and N. Arrows indicate the outer layer of the optic cup. (C,I,O) Transverse sections hybridized with a sense probe for xtjmjd3, xtutx or xtezh2. (DJP High magnification views of otic placodes in the transverse sections shown in D, J and P. (F,L,R) Transverse sections of tadpole eyes subjected to in situ hybridization. br, brain; ov, otic vesicle; ba, branchial arch; mc, mesencephalon; dc, diencephalon; oc, optic cup; le, lens; mnc, migrating neural crest; rc, rhombencephalon; nc, notochord; sc, spinal cord; gcl, ganglion cell layer; inl, inner nuclear layer; prl, photoreceptor layer.
	Fig. 2. Overexpression of XtJMJD3 or XtUTX reduced H3K27me3 levels in cell culture. COS-7 cells were transfected with an empty expression vector, pCS2+ (A-C), an XtJMJD3 expression vector, pCS2+xtjmjd3 (D- F), an XtUTX expression vector, pCMVSPORT6-xtutx (G-I), or a mouse JMJD3 expression vector, pCS2+mJmjd3 (J-L). A GFP expression vector, pCAGGS-GFP, was co-transfected in each case to identify the transfected cells. The blue, red and green are nuclear DAPI staining, anti-H3K27me3 antibody staining and anti-GFP antibody staining, respectively. White ar- rows indicate nuclei of transfected cells.
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in Xenopus tropicalis embryos, NF stage 32, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in bissected Xenopus tropicalis embryo, NF stage 8, as assayed by in situ hybridization, dorsal right.
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in bissected Xenopus tropicalis embryo, NF stage 10.25, as assayed by in situ hybridization, dorsal right.
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up (O) and sagittal/median section, dorsal up (P and P').
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in bissected Xenopus tropicalis embryo, NF stage 8, as assayed by in situ hybridization, dorsal right.
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in bissected Xenopus tropicalis embryo, NF stage 10.25, as assayed by in situ hybridization, dorsal right.
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up (S) and sagittal/median section, dorsal up (T and T').
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in bissected Xenopus tropicalis embryo, NF stage 8, as assayed by in situ hybridization, dorsal right.
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in bissected Xenopus tropicalis embryo, NF stage 10.25, as assayed by in situ hybridization, dorsal right.
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up (W) and sagittal/median section, dorsal up (X and X').
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	kdm6b (lysine (K)-specific demethylase 6B) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 42, retinal section.
	kdm6a (lysine (K)-specific demethylase 6A) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 42, retinal section.
	ezh2 (enhancer of zeste 2 polycomb repressive complex 2 subunit ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 42, retinal section.