Gene Expression Patterns 14 (2014) 105–110
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish Kazuya Takayama a, Nobuyoshi Shimoda b, Shunsuke Takanaga a, Shunya Hozumi a, Yutaka Kikuchi a,⇑ a b
Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima 739-8526, Japan Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522, Japan
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Article history: Received 13 September 2013 Received in revised form 6 January 2014 Accepted 25 January 2014 Available online 6 February 2014 Keywords: Zebrafish Danio rerio dnmt3aa dnmt3ab dnmt4 Pronephric duct Digestive organs Hematopoietic cells Blastema
a b s t r a c t Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells. Ó 2014 Elsevier B.V. All rights reserved.
1. Results and discussion In vertebrates, spatially and temporally regulated gene expression is required for cell proliferation and differentiation during development, and one of the critical factors for the regulation of gene expression is epigenetic modifications including DNA methylation and chromatin modifications (Turek-Plewa and Jagodzin´ski, 2005; Mellor et al., 2008; Bogdanovic´ et al., 2012; Smith and Meissner, 2013). In mammals, cytosines in CpG dinucleotides are predominately methylated at the 5 position by 3 DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) (Goll and Bestor, 2005; Jones, 2012). Genetic and biochemical evidence in mice have revealed that although Dnmt1 mainly functions in the maintenance of pre-existing DNA methylation (Li et al., 1992; Lei et al., 1996), Dnmt3a and Dnmt3b contribute to the establishment of de novo DNA methylation patterns during development (Okano et al., ⇑ Corresponding author. Tel.: +81 82 424 7440; fax: +81 82 424 0734. E-mail address: [email protected] (Y. Kikuchi). http://dx.doi.org/10.1016/j.gep.2014.01.005 1567-133X/Ó 2014 Elsevier B.V. All rights reserved.
1999). Two de novo Dnmt proteins, Dnmt3a and Dnmt3b, showed distinct expression patterns during mouse development; Dnmt3a is ubiquitously expressed after E10.5, whereas Dnmt3b is specifically detected in progenitor cells during hematopoiesis, spermatogenesis, and neurogenesis (Okano et al., 1999; Watanabe et al., 2004; Watanabe et al., 2006). These distinct expression patterns result in different developmental defects; Dnmt3a-deficient mice normally develop to term, but die about 4 weeks after birth (Okano et al., 1999); Dnmt3b-deficient mice show an embryonic lethal phenotype with multiple developmental defects (e.g. rostral neural tube defects, liver hypotrophy, ventricular septal defect, and haemorrhage) (Okano et al., 1999; Ueda et al., 2006); Missense mutations in Dnmt3b cause similar phenotypes leading to patients with immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome (Ueda et al., 2006). These results indicate that these two genes have non-overlapping functions. In addition to the developmental stages, recent reports have revealed that Dnmt3a play roles in somatic stem cells; Dnmt3a regulates the expression of neurogenic genes in postnatal neural stem cells (Wu et al., 2010); Dnmt3a
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is essential for differentiation, but not for self-renewal, of hematopoietic stem cell (HSCs) (Challen et al., 2012). In contrast to mammals, 8 zebrafish Dnmt genes were cloned and phylogenetic analyses revealed that 6 of them [dnmt3 (also known as dnmt3b3), dnmt3aa (also known as dnmt8, dnmt3a2), dnmt3ab (also known as dnmt6, dnmt3a1), dnmt3b (also known as dnmt7, dnmt3b2), dnmt4 (also known as dnmt3b1), and dnmt5 (also known as dnmt3b4)] are orthologues of mammalian Dnmt3a and Dnmt3b (Xie et al., 1999; Shimoda et al., 2005; Campos et al., 2012). We have previously reported that expression of dnmt3b is ubiquitous during zebrafish development (Shimoda et al. 2005). Despite partial gene expression analyses by in situ hybridization and real-time PCR (Smith et al., 2005; Rai et al., 2010; Smith et al., 2011; Campos et al., 2012), detailed spatiotemporal expression patterns of dnmt3, dnmt3aa, dnmt3ab, dnmt4, and dnmt5 have not yet been reported. We examined the expression patterns of these 5 genes, and found that 3 of them (dnmt3aa, dnmt3ab, and dnmt4) show tissue-specific expression patterns during development (data not shown). In addition, we recently found that the DNA methylation levels (5-methyl cytosine, 5mC and 5-hydroxymethyl cytosine, 5hmC) are transiently reduced at the early stages of fin regeneration (36 h post amputation, hpa), and these levels of 5mC and 5hmC are gradually recovered in the course of fin regeneration (Hirose et al., 2013). Based on our results, we hypothesized that some Dnmt genes might be expressed in fin regenerate and
could function in fin regeneration processes possibly through the regulation of gene expression. Thus, in this study, we analyzed the spatiotemporal expression of dnm3aa, dnmt3ab, and dnmt4 genes during embryonic and larval development and during fin regeneration in zebrafish. 1.1. dnmt3aa expression during zebrafish development As previously reported, phylogenetic analysis have revealed that Dnmt3aa and Dnmt3ab are closely related to mammalian Dnmt3a, while Dnmt4 is closely related to mammalian Dnmt3b (Shimoda et al., 2005; Campos et al., 2012). To analyze the expression pattern of these dnmt3-related genes, we first examined the spatiotemporal expression of dnmt3aa during embryonic and larval development of zebrafish by whole-mount in situ hybridization. A previous report showed that the maternal transcripts of dnmt3aa and dnmt3ab can be detected and the expression of these genes is upregulated following the midblastula transition (MBT) (Smith et al., 2011). After the MBT, faint and ubiquitous expression of dnmt3aa was observed in whole embryos (data not shown). At the 10 somite (s) stage, dnmt3aa began to be expressed in the intermediate mesoderm fated to become the pronephric epithelia as a pair of bilateral stripes (Fig. 1A and B). dnmt3aa expression in the pronephric duct (pd), neural tube, and somitic mesoderm was evident at the 18s stage (Fig. 1C and D), and the expression,
Fig. 1. Expression patterns of dnmt3aa in zebrafish embryos and larvae. (A–P) dnmt3aa expression was examined by whole-mount in situ hybridization at the 10s and 18s stages as well as at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, C, E, H, K, and N). Vegetal pole views of the tail-bud region, anterior to the left (B and D). Dorsal views, anterior to the left (F, I, L, and O). The boxed areas in (F), (I), (L), and (O) are shown enlarged in (G), (J), (M), and (P), respectively. (Q–S) Transverse sections showing dnmt3aa expression. The transverse sections were cut at the levels indicated by the dashed black lines in (K). br, brain; im, intermediate mesoderm; in, intestine; li, liver; nt, neural tube; pa, pancreas; pd, pronephric duct; pf, pectoral fin buds; ph, pharyngeal arches; re, retina; sb, swim bladder; sc, spinal cord; sm, somitic mesoderm.
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except in the somitic mesoderm, continued at 24 h post-fertilization (hpf) (Fig. 1E–G). As development proceeded, although the dnmt3aa expression in the spinal cord disappeared at 48 hpf, dnmt3aa was expressed in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, pectoral fin buds (pf), intestine, swim bladder (sb), and pd from 48 hpf to 72 hpf (Fig. 1H–M). Transverse sections clearly indicated that dnmt3aa is expressed in the intestine, liver, pancreas, and a pair of bilateral strips of pd (Fig. 1Q– S). At 96 hpf, dnmt3aa expression in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, sb, and intestine was maintained, whereas the expression in the pf and pd was downregulated (Fig. 1N–P). Restricted expression of dnmt3aa in the intermediate mesoderm during somitogenesis and pd until 72 hpf raises the possibility that Dnmt3aa may play a critical role in the formation of the kidney in zebrafish. 1.2. dnmt3ab expression during zebrafish development dnmt3ab was ubiquitously expressed in whole embryos at the 4s stage, and the expression was restricted to the neural tube at the 18s stages (Fig. 2A–C). At 24 hpf, expression of dnmt3ab was detected in the spinal cord and auditory capsule (Fig. 2D and E).
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As development proceeded, dnmt3ab was clearly expressed in the brain, pharyngeal arches, and intestine, and it was faintly expressed in the pf, spinal cord and retinae (Seritrakul and Gross, 2014) at 48 hpf; these expression patterns were maintained until 72 hpf (Fig. 2F–I). In addition, transverse sections indicated that dnmt3ab is expressed in intestine and sb, but not in liver and pancreas at 72 hpf (Fig. 2L and M). At 96 hpf, expression of dmnt3ab was detected in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, sb, and intestine (Fig. 2J and K). 1.3. dnmt4 expression during zebrafish development dnmt4 was ubiquitously expressed until early somitogenesis, and its expression began to be restricted to the optic vesicle (ov) and optic tectum at the 10s stage (Fig. 3A and B). At the 18s stage, dnmt4 was expressed in the ov, putative midbrain–hindbrain boundary (mhb) region, and a faint expression was detected in the presomatic mesoderm (Fig. 3C and D). At 24 hpf, dnmt4 expression domains in the optic cup and putative mhb were maintained (Fig. 3E and F), and the dnmt4 expression was detected in the pharyngeal endoderm (Fig. 3E). From 48 to 72 hpf, dnmt4 transcripts were detected in the ciliary marginal zone (cmz) (Seritrakul and
Fig. 2. Expression patterns of dnmt3ab in zebrafish embryos and larvae. (A–K) dnmt3ab expression was examined by whole-mount in situ hybridization at the 4s and 18s stages, and at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, B, D, F, H, and J). Dorsal views, anterior to the left (C, E, G, I, and K). (L and M) Transverse sections showing dnmt3ab expression. The transverse sections were cut at the levels indicated by the dashed black lines in (I). ac, auditory capsule; ae, anterior endoderm; br, brain; in, intestine; nt, neural tube; li, liver; pa, pancreas; pf, pectoral fin buds; ph, pharyngeal arches; re, retina; sb, swim bladder; sc, spinal cord.
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Fig. 3. Expression patterns of dnmt4 in zebrafish embryos and larvae. (A–R) dnmt4 expression was examined by whole-mount in situ hybridization at the 10s and 18s stages as well as at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, C, E, G, K, and O). Dorsal views, anterior to the bottom (B and D). Dorsal views, anterior to the left (F, H, L, and P). Magnified views of the agm (I, M, and Q) and cht (J, N, and R) in (G), (K), and (O). (S–U) Cross and transverse sections showing dnmt4 expression at 72 hpf. The cross and transverse sections were cut at the level indicated by the dashed black lines in (L). ac, auditory capsule; cht, caudal hematopoietic tissue; cmz, ciliary marginal zone; dm, dorsal midbrain; in, intestine; li, liver; mhb, midbrain-hindbrain boundary; oc, optic cup; ot, optic tectum; ov; optic vesicle; pa, pancreas; pe, pharyngeal endoderm; pf, pectoral fin buds; ph, pharyngeal arches; psm, presomitic mesoderm.
Gross, 2014), pharyngeal arches, auditory capsule, pf, intestine, pancreas, and liver (Fig. 3G, H, K, and L). The sagittal sections showed, according to brain anatomy and by comparing with sonic hedgehog a expression (Barth and Wilson, 1995), that dnmt4 is expressed in a part of the tectum, in the dorsal and ventral thalamus, and in the pallium region (Supplemental Fig. 1). Histological analyses confirmed that dnmt4 expression is restricted to the cmz in the retina by cross section (Fig. 3L and S), and is restricted to the liver, pancreas, and intestine by transverse sections (Fig. 3L, T, and U). Additionally, dnmt4-expressing cells were observed in the aorta–gonad–mesonephros (agm) and caudal hematopoietic tissue (cht) regions (Fig. 3I, J, M, and N). At 96 hpf, dnmt4 expression was markedly reduced in cells of the agm and cht regions, as well as in digestive organs (Fig. 3O–R).
in mice and zebrafish (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010). To investigate the expression of dnmt4 in HSCs/hematopoietic cells in the agm and cht during development, we used cloches5 (clos5) mutant embryos, in which differentiation of hematopoietic and endothelial cells are severely impaired (Stainier et al. 1995). At 72 hpf, dnmt4-expressing cells were observed in the agm and cht of siblings, but not in clos5 mutant embryos (Fig. 4). These results suggest that dnmt4 is expressed in HSCs/hematopoietic cells in the agm and cht. In mice, analyses of conditional knockout mice for Dnmt3a in HSCs have revealed that Dnmt3a is essential for HSC differentiation, but not for self-renewal (Challen et al., 2012). Based on the expression pattern of zebrafish dnmt4, the zebrafish Dnmt4 protein may be involved in the differentiation to hematopoietic cell lineages.
1.4. dnmt4 is expressed in hematopoietic cells
1.5. Expression of dnmt genes during zebrafish fin regeneration
Previous studies have reported that HSCs originate from the ventral dorsal aorta in the agm, and they then migrate to the cht
We have previously reported that the level of both 5mC and 5hmC is transiently reduced at 36 hpa and DNA demethylation-related
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Fig. 4. dnmt4 is expressed in HSCs/hematopietic cells in agm and cht in zebrafish larvae. (A–H) dnmt4 expression in sibling and clos5 mutant larvea was examined by wholemount in situ hybridization at 72 hpf. Lateral views, anterior to the left (A–C, and E–G). Magnified views of the agm (B and F) and cht (C and G) in (A) and (E). (D and H) Transverse sections showing dnmt4 expression. The transverse sections were cut at the levels indicated by the dashed black lines in (C) and (G). clos5, cloches5; sib, sibling.
genes are expressed during fin regeneration (Hirose et al., 2013). The level of both 5mC and 5hmC was gradually recovered in the course of fin regeneration, suggesting that Dnmt proteins are involved in fin regeneration (Hirose et al., 2013). To identify the Dnmt genes involved in fin regeneration, we examined the expression patterns of 7 zebrafish dnmt genes (dnmt1, dnmt3, dnmt3aa, dnmt3ab, dnmt3b, dnmt4, and dnmt5) in fin regenerates. Only 3 dnmt genes (dnmt3aa, dnmt3ab, and dnmt4) were expressed in blastema cells. Although faint expression of dnmt3ab and dnmt4 was observed (Fig. 5C, D, G, and H), dnmt3aa was clearly expressed in the blastema cells at 72 hpa (Fig. 5A, B, E, and F). No expression of these three genes was observed before amputation, immediately after amputation, and at 36 hpa (Supplemental Fig. 2). These results suggest that these 3 Dnmts may function in the recovery of 5mC levels and regulation of spatiotemporal gene expression during fin regeneration. 1.6. Comparison of dnmt3-related genes in zebrafish and mouse Based on our results, expression of zebrafish dnmt3aa, dnmt3ab, and dnmt4 in specific cells, such as hematopoietic cells and neural cells, appears to be related to the phenotypes of Dnmt3 knockout
mice. However, other expression domains, such as the pronephric duct, digestive organs, pectoral fins, and retinae, appear not to be related to the phenotypes of Dnmt3 knockout mice. Therefore, the function of dnmt3-related genes during development could mostly differ between zebrafish and mouse. Future studies of individual zebrafish dnmt mutants would be useful for the elucidation of Dnmt3-related protein function in organogenesis and regeneration.
2. Experimental procedures 2.1. Ethics statement All animal experiments were conducted according to relevant national and international guidelines ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan). Ethics was approved from the Hiroshima University Animal Research Committee (Permit Number: F13-1). 2.2. Nomenclature for the zebrafish dnmt genes The nomenclature for the zebrafish dnmt3 gene family in this article follows the ZFIN Zebrafish Nomenclature Guidelines. The ZFIN, previous, and recently proposed (Campos et al., 2012) nomenclatures of the dnmt3 gene family are summarized in Supplemental Table 1. 2.3. Zebrafish husbandry and fin amputation
Fig. 5. Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during zebrafish fin regeneration. (A–H) In situ hybridization analyses with antisense riboprobes (A–D) and sense riboprobes (E–H) of dnmt3aa, dnmt3ab, and dnmt4 in fin regenerates at 72 hpa. Longitudinal sections of wildtype fin regenerates (B and F). Dashed black lines indicate the amputation planes.
Zebrafish embryos were maintained as previously described by Westerfield (1995). Embryos were incubated in 1/3 Ringer’s solution (39 mM NaCl, 0.97 mM KCl, 1.8 mM CaCl2, 1.7 mM HEPES, pH 7.2) at 28.5C, and their developmental stages were determined according to Kimmel et al. (1995). The clos5 mutant used in this study was kindly provided by Dr. Atsushi Kawakami (Tokyo Institute of Technology). Embryos homozygous for the clos5 mutation were collected along with wildtype siblings as controls. Adult wildtype 3- to 6-month-old zebrafish (AB strain) were used for all experiments. For caudal fin amputation, fish were anesthetized using tricaine and approximately two-thirds of the fin was cut with a blade. After fin amputation, these fish were allowed to regenerate in the aquarium until a defined time point at 28.5 °C (Hirose et al., 2013). The blastema starts to form at approximately 24 hpa and the amputated fins have restored at approximately 14 days post amputation.
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2.4. Whole-mount in situ hybridization and histological analysis To avoid cross-hybridization among dnmt3aa, dnmt3ab, and dnmt4, the N-terminus region of dnmt3aa, dnmt3ab, or dnmt4 was cloned into the pCRÒII-TOPOÒ vector (Invitrogen). For synthesis of antisense riboprobes, the plasmid template for dnmt3aa, dnmt3ab, or dnmt4 was linearized with ApaI and transcribed with SP6 RNA polymerases. For sonic hedgehog a (Krauss et al., 1993) antisense probes, the template was linearized with HindIII and transcribed with T7 RNA polymerases. For sense riboprobes, the templates were linearized with HindIII for dnmt3ab and dnmt4 and KpnI for dnmt3aa, and then transcribed with T7 RNA polymerases. Whole-mount in situ hybridization was performed using conventional nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase (Westerfield, 1995; Mizoguchi et al., 2008). For histology, the NBT/BCIP-stained in situ embryos were dehydrated, embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim), and cut at 15 lm. The sectioned in situ samples were imaged using an Olympus FV1000D confocal microscope. Acknowledgements We thank the members of Kikuchi and Atsushi Suzuki laboratories in Hiroshima University for helpful discussion and critical comments. We also thank Dr. A. Kawakami for providing clos5 mutant embryos. This study was supported by Grants from Grant-inAid for Scientific Research from the MEXT (KAKENHI 23616002) to Y.K. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gep.2014.01.005. References Barth, K.A., Wilson, S.W., 1995. Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development 121, 1755–1768. Bertrand, J.Y., Chi, N.C., Santoso, B., Teng, S., Stainier, D.Y., Traver, D., 2010. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464, 108–111. Bogdanovic´, O., van Heeringen, S.J., Veenstra, G.J., 2012. The epigenome in early vertebrate development. Genesis 50, 192–206. Boisset, J.C., van Cappellen, W., Andrieu-Soler, C., Galjart, N., Dzierzak, E., Robin, C., 2010. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120. Campos, C., Valente, L.M., Fernandes, J.M., 2012. Molecular evolution of zebrafish dnmt3 genes and thermal plasticity of their expression during embryonic development. Gene 500, 93–100. Challen, G.A., Sun, D., Jeong, M., Luo, M., Jelinek, J., Berg, J.S., Bock, C., Vasanthakumar, A., Gu, H., Xi, Y., Liang, S., Lu, Y., Darlington, G.J., Meissner, A., Issa, J.P., Godley, L.A., Li, W., Goodell, M.A., 2012. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat. Genet. 44, 23–31.
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish Kazuya Takayama a, Nobuyoshi Shimoda b, Shunsuke Takanaga a, Shunya Hozumi a, Yutaka Kikuchi a,⇑ a b
Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima 739-8526, Japan Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522, Japan
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Article history: Received 13 September 2013 Received in revised form 6 January 2014 Accepted 25 January 2014 Available online 6 February 2014 Keywords: Zebrafish Danio rerio dnmt3aa dnmt3ab dnmt4 Pronephric duct Digestive organs Hematopoietic cells Blastema
a b s t r a c t Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells. Ó 2014 Elsevier B.V. All rights reserved.
1. Results and discussion In vertebrates, spatially and temporally regulated gene expression is required for cell proliferation and differentiation during development, and one of the critical factors for the regulation of gene expression is epigenetic modifications including DNA methylation and chromatin modifications (Turek-Plewa and Jagodzin´ski, 2005; Mellor et al., 2008; Bogdanovic´ et al., 2012; Smith and Meissner, 2013). In mammals, cytosines in CpG dinucleotides are predominately methylated at the 5 position by 3 DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) (Goll and Bestor, 2005; Jones, 2012). Genetic and biochemical evidence in mice have revealed that although Dnmt1 mainly functions in the maintenance of pre-existing DNA methylation (Li et al., 1992; Lei et al., 1996), Dnmt3a and Dnmt3b contribute to the establishment of de novo DNA methylation patterns during development (Okano et al., ⇑ Corresponding author. Tel.: +81 82 424 7440; fax: +81 82 424 0734. E-mail address: [email protected] (Y. Kikuchi). http://dx.doi.org/10.1016/j.gep.2014.01.005 1567-133X/Ó 2014 Elsevier B.V. All rights reserved.
1999). Two de novo Dnmt proteins, Dnmt3a and Dnmt3b, showed distinct expression patterns during mouse development; Dnmt3a is ubiquitously expressed after E10.5, whereas Dnmt3b is specifically detected in progenitor cells during hematopoiesis, spermatogenesis, and neurogenesis (Okano et al., 1999; Watanabe et al., 2004; Watanabe et al., 2006). These distinct expression patterns result in different developmental defects; Dnmt3a-deficient mice normally develop to term, but die about 4 weeks after birth (Okano et al., 1999); Dnmt3b-deficient mice show an embryonic lethal phenotype with multiple developmental defects (e.g. rostral neural tube defects, liver hypotrophy, ventricular septal defect, and haemorrhage) (Okano et al., 1999; Ueda et al., 2006); Missense mutations in Dnmt3b cause similar phenotypes leading to patients with immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome (Ueda et al., 2006). These results indicate that these two genes have non-overlapping functions. In addition to the developmental stages, recent reports have revealed that Dnmt3a play roles in somatic stem cells; Dnmt3a regulates the expression of neurogenic genes in postnatal neural stem cells (Wu et al., 2010); Dnmt3a
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is essential for differentiation, but not for self-renewal, of hematopoietic stem cell (HSCs) (Challen et al., 2012). In contrast to mammals, 8 zebrafish Dnmt genes were cloned and phylogenetic analyses revealed that 6 of them [dnmt3 (also known as dnmt3b3), dnmt3aa (also known as dnmt8, dnmt3a2), dnmt3ab (also known as dnmt6, dnmt3a1), dnmt3b (also known as dnmt7, dnmt3b2), dnmt4 (also known as dnmt3b1), and dnmt5 (also known as dnmt3b4)] are orthologues of mammalian Dnmt3a and Dnmt3b (Xie et al., 1999; Shimoda et al., 2005; Campos et al., 2012). We have previously reported that expression of dnmt3b is ubiquitous during zebrafish development (Shimoda et al. 2005). Despite partial gene expression analyses by in situ hybridization and real-time PCR (Smith et al., 2005; Rai et al., 2010; Smith et al., 2011; Campos et al., 2012), detailed spatiotemporal expression patterns of dnmt3, dnmt3aa, dnmt3ab, dnmt4, and dnmt5 have not yet been reported. We examined the expression patterns of these 5 genes, and found that 3 of them (dnmt3aa, dnmt3ab, and dnmt4) show tissue-specific expression patterns during development (data not shown). In addition, we recently found that the DNA methylation levels (5-methyl cytosine, 5mC and 5-hydroxymethyl cytosine, 5hmC) are transiently reduced at the early stages of fin regeneration (36 h post amputation, hpa), and these levels of 5mC and 5hmC are gradually recovered in the course of fin regeneration (Hirose et al., 2013). Based on our results, we hypothesized that some Dnmt genes might be expressed in fin regenerate and
could function in fin regeneration processes possibly through the regulation of gene expression. Thus, in this study, we analyzed the spatiotemporal expression of dnm3aa, dnmt3ab, and dnmt4 genes during embryonic and larval development and during fin regeneration in zebrafish. 1.1. dnmt3aa expression during zebrafish development As previously reported, phylogenetic analysis have revealed that Dnmt3aa and Dnmt3ab are closely related to mammalian Dnmt3a, while Dnmt4 is closely related to mammalian Dnmt3b (Shimoda et al., 2005; Campos et al., 2012). To analyze the expression pattern of these dnmt3-related genes, we first examined the spatiotemporal expression of dnmt3aa during embryonic and larval development of zebrafish by whole-mount in situ hybridization. A previous report showed that the maternal transcripts of dnmt3aa and dnmt3ab can be detected and the expression of these genes is upregulated following the midblastula transition (MBT) (Smith et al., 2011). After the MBT, faint and ubiquitous expression of dnmt3aa was observed in whole embryos (data not shown). At the 10 somite (s) stage, dnmt3aa began to be expressed in the intermediate mesoderm fated to become the pronephric epithelia as a pair of bilateral stripes (Fig. 1A and B). dnmt3aa expression in the pronephric duct (pd), neural tube, and somitic mesoderm was evident at the 18s stage (Fig. 1C and D), and the expression,
Fig. 1. Expression patterns of dnmt3aa in zebrafish embryos and larvae. (A–P) dnmt3aa expression was examined by whole-mount in situ hybridization at the 10s and 18s stages as well as at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, C, E, H, K, and N). Vegetal pole views of the tail-bud region, anterior to the left (B and D). Dorsal views, anterior to the left (F, I, L, and O). The boxed areas in (F), (I), (L), and (O) are shown enlarged in (G), (J), (M), and (P), respectively. (Q–S) Transverse sections showing dnmt3aa expression. The transverse sections were cut at the levels indicated by the dashed black lines in (K). br, brain; im, intermediate mesoderm; in, intestine; li, liver; nt, neural tube; pa, pancreas; pd, pronephric duct; pf, pectoral fin buds; ph, pharyngeal arches; re, retina; sb, swim bladder; sc, spinal cord; sm, somitic mesoderm.
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except in the somitic mesoderm, continued at 24 h post-fertilization (hpf) (Fig. 1E–G). As development proceeded, although the dnmt3aa expression in the spinal cord disappeared at 48 hpf, dnmt3aa was expressed in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, pectoral fin buds (pf), intestine, swim bladder (sb), and pd from 48 hpf to 72 hpf (Fig. 1H–M). Transverse sections clearly indicated that dnmt3aa is expressed in the intestine, liver, pancreas, and a pair of bilateral strips of pd (Fig. 1Q– S). At 96 hpf, dnmt3aa expression in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, sb, and intestine was maintained, whereas the expression in the pf and pd was downregulated (Fig. 1N–P). Restricted expression of dnmt3aa in the intermediate mesoderm during somitogenesis and pd until 72 hpf raises the possibility that Dnmt3aa may play a critical role in the formation of the kidney in zebrafish. 1.2. dnmt3ab expression during zebrafish development dnmt3ab was ubiquitously expressed in whole embryos at the 4s stage, and the expression was restricted to the neural tube at the 18s stages (Fig. 2A–C). At 24 hpf, expression of dnmt3ab was detected in the spinal cord and auditory capsule (Fig. 2D and E).
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As development proceeded, dnmt3ab was clearly expressed in the brain, pharyngeal arches, and intestine, and it was faintly expressed in the pf, spinal cord and retinae (Seritrakul and Gross, 2014) at 48 hpf; these expression patterns were maintained until 72 hpf (Fig. 2F–I). In addition, transverse sections indicated that dnmt3ab is expressed in intestine and sb, but not in liver and pancreas at 72 hpf (Fig. 2L and M). At 96 hpf, expression of dmnt3ab was detected in the brain, retinae (Seritrakul and Gross, 2014), pharyngeal arches, sb, and intestine (Fig. 2J and K). 1.3. dnmt4 expression during zebrafish development dnmt4 was ubiquitously expressed until early somitogenesis, and its expression began to be restricted to the optic vesicle (ov) and optic tectum at the 10s stage (Fig. 3A and B). At the 18s stage, dnmt4 was expressed in the ov, putative midbrain–hindbrain boundary (mhb) region, and a faint expression was detected in the presomatic mesoderm (Fig. 3C and D). At 24 hpf, dnmt4 expression domains in the optic cup and putative mhb were maintained (Fig. 3E and F), and the dnmt4 expression was detected in the pharyngeal endoderm (Fig. 3E). From 48 to 72 hpf, dnmt4 transcripts were detected in the ciliary marginal zone (cmz) (Seritrakul and
Fig. 2. Expression patterns of dnmt3ab in zebrafish embryos and larvae. (A–K) dnmt3ab expression was examined by whole-mount in situ hybridization at the 4s and 18s stages, and at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, B, D, F, H, and J). Dorsal views, anterior to the left (C, E, G, I, and K). (L and M) Transverse sections showing dnmt3ab expression. The transverse sections were cut at the levels indicated by the dashed black lines in (I). ac, auditory capsule; ae, anterior endoderm; br, brain; in, intestine; nt, neural tube; li, liver; pa, pancreas; pf, pectoral fin buds; ph, pharyngeal arches; re, retina; sb, swim bladder; sc, spinal cord.
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Fig. 3. Expression patterns of dnmt4 in zebrafish embryos and larvae. (A–R) dnmt4 expression was examined by whole-mount in situ hybridization at the 10s and 18s stages as well as at 24, 48, 72, and 96 hpf. Lateral views, anterior to the left (A, C, E, G, K, and O). Dorsal views, anterior to the bottom (B and D). Dorsal views, anterior to the left (F, H, L, and P). Magnified views of the agm (I, M, and Q) and cht (J, N, and R) in (G), (K), and (O). (S–U) Cross and transverse sections showing dnmt4 expression at 72 hpf. The cross and transverse sections were cut at the level indicated by the dashed black lines in (L). ac, auditory capsule; cht, caudal hematopoietic tissue; cmz, ciliary marginal zone; dm, dorsal midbrain; in, intestine; li, liver; mhb, midbrain-hindbrain boundary; oc, optic cup; ot, optic tectum; ov; optic vesicle; pa, pancreas; pe, pharyngeal endoderm; pf, pectoral fin buds; ph, pharyngeal arches; psm, presomitic mesoderm.
Gross, 2014), pharyngeal arches, auditory capsule, pf, intestine, pancreas, and liver (Fig. 3G, H, K, and L). The sagittal sections showed, according to brain anatomy and by comparing with sonic hedgehog a expression (Barth and Wilson, 1995), that dnmt4 is expressed in a part of the tectum, in the dorsal and ventral thalamus, and in the pallium region (Supplemental Fig. 1). Histological analyses confirmed that dnmt4 expression is restricted to the cmz in the retina by cross section (Fig. 3L and S), and is restricted to the liver, pancreas, and intestine by transverse sections (Fig. 3L, T, and U). Additionally, dnmt4-expressing cells were observed in the aorta–gonad–mesonephros (agm) and caudal hematopoietic tissue (cht) regions (Fig. 3I, J, M, and N). At 96 hpf, dnmt4 expression was markedly reduced in cells of the agm and cht regions, as well as in digestive organs (Fig. 3O–R).
in mice and zebrafish (Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010). To investigate the expression of dnmt4 in HSCs/hematopoietic cells in the agm and cht during development, we used cloches5 (clos5) mutant embryos, in which differentiation of hematopoietic and endothelial cells are severely impaired (Stainier et al. 1995). At 72 hpf, dnmt4-expressing cells were observed in the agm and cht of siblings, but not in clos5 mutant embryos (Fig. 4). These results suggest that dnmt4 is expressed in HSCs/hematopoietic cells in the agm and cht. In mice, analyses of conditional knockout mice for Dnmt3a in HSCs have revealed that Dnmt3a is essential for HSC differentiation, but not for self-renewal (Challen et al., 2012). Based on the expression pattern of zebrafish dnmt4, the zebrafish Dnmt4 protein may be involved in the differentiation to hematopoietic cell lineages.
1.4. dnmt4 is expressed in hematopoietic cells
1.5. Expression of dnmt genes during zebrafish fin regeneration
Previous studies have reported that HSCs originate from the ventral dorsal aorta in the agm, and they then migrate to the cht
We have previously reported that the level of both 5mC and 5hmC is transiently reduced at 36 hpa and DNA demethylation-related
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Fig. 4. dnmt4 is expressed in HSCs/hematopietic cells in agm and cht in zebrafish larvae. (A–H) dnmt4 expression in sibling and clos5 mutant larvea was examined by wholemount in situ hybridization at 72 hpf. Lateral views, anterior to the left (A–C, and E–G). Magnified views of the agm (B and F) and cht (C and G) in (A) and (E). (D and H) Transverse sections showing dnmt4 expression. The transverse sections were cut at the levels indicated by the dashed black lines in (C) and (G). clos5, cloches5; sib, sibling.
genes are expressed during fin regeneration (Hirose et al., 2013). The level of both 5mC and 5hmC was gradually recovered in the course of fin regeneration, suggesting that Dnmt proteins are involved in fin regeneration (Hirose et al., 2013). To identify the Dnmt genes involved in fin regeneration, we examined the expression patterns of 7 zebrafish dnmt genes (dnmt1, dnmt3, dnmt3aa, dnmt3ab, dnmt3b, dnmt4, and dnmt5) in fin regenerates. Only 3 dnmt genes (dnmt3aa, dnmt3ab, and dnmt4) were expressed in blastema cells. Although faint expression of dnmt3ab and dnmt4 was observed (Fig. 5C, D, G, and H), dnmt3aa was clearly expressed in the blastema cells at 72 hpa (Fig. 5A, B, E, and F). No expression of these three genes was observed before amputation, immediately after amputation, and at 36 hpa (Supplemental Fig. 2). These results suggest that these 3 Dnmts may function in the recovery of 5mC levels and regulation of spatiotemporal gene expression during fin regeneration. 1.6. Comparison of dnmt3-related genes in zebrafish and mouse Based on our results, expression of zebrafish dnmt3aa, dnmt3ab, and dnmt4 in specific cells, such as hematopoietic cells and neural cells, appears to be related to the phenotypes of Dnmt3 knockout
mice. However, other expression domains, such as the pronephric duct, digestive organs, pectoral fins, and retinae, appear not to be related to the phenotypes of Dnmt3 knockout mice. Therefore, the function of dnmt3-related genes during development could mostly differ between zebrafish and mouse. Future studies of individual zebrafish dnmt mutants would be useful for the elucidation of Dnmt3-related protein function in organogenesis and regeneration.
2. Experimental procedures 2.1. Ethics statement All animal experiments were conducted according to relevant national and international guidelines ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan). Ethics was approved from the Hiroshima University Animal Research Committee (Permit Number: F13-1). 2.2. Nomenclature for the zebrafish dnmt genes The nomenclature for the zebrafish dnmt3 gene family in this article follows the ZFIN Zebrafish Nomenclature Guidelines. The ZFIN, previous, and recently proposed (Campos et al., 2012) nomenclatures of the dnmt3 gene family are summarized in Supplemental Table 1. 2.3. Zebrafish husbandry and fin amputation
Fig. 5. Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during zebrafish fin regeneration. (A–H) In situ hybridization analyses with antisense riboprobes (A–D) and sense riboprobes (E–H) of dnmt3aa, dnmt3ab, and dnmt4 in fin regenerates at 72 hpa. Longitudinal sections of wildtype fin regenerates (B and F). Dashed black lines indicate the amputation planes.
Zebrafish embryos were maintained as previously described by Westerfield (1995). Embryos were incubated in 1/3 Ringer’s solution (39 mM NaCl, 0.97 mM KCl, 1.8 mM CaCl2, 1.7 mM HEPES, pH 7.2) at 28.5C, and their developmental stages were determined according to Kimmel et al. (1995). The clos5 mutant used in this study was kindly provided by Dr. Atsushi Kawakami (Tokyo Institute of Technology). Embryos homozygous for the clos5 mutation were collected along with wildtype siblings as controls. Adult wildtype 3- to 6-month-old zebrafish (AB strain) were used for all experiments. For caudal fin amputation, fish were anesthetized using tricaine and approximately two-thirds of the fin was cut with a blade. After fin amputation, these fish were allowed to regenerate in the aquarium until a defined time point at 28.5 °C (Hirose et al., 2013). The blastema starts to form at approximately 24 hpa and the amputated fins have restored at approximately 14 days post amputation.
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2.4. Whole-mount in situ hybridization and histological analysis To avoid cross-hybridization among dnmt3aa, dnmt3ab, and dnmt4, the N-terminus region of dnmt3aa, dnmt3ab, or dnmt4 was cloned into the pCRÒII-TOPOÒ vector (Invitrogen). For synthesis of antisense riboprobes, the plasmid template for dnmt3aa, dnmt3ab, or dnmt4 was linearized with ApaI and transcribed with SP6 RNA polymerases. For sonic hedgehog a (Krauss et al., 1993) antisense probes, the template was linearized with HindIII and transcribed with T7 RNA polymerases. For sense riboprobes, the templates were linearized with HindIII for dnmt3ab and dnmt4 and KpnI for dnmt3aa, and then transcribed with T7 RNA polymerases. Whole-mount in situ hybridization was performed using conventional nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase (Westerfield, 1995; Mizoguchi et al., 2008). For histology, the NBT/BCIP-stained in situ embryos were dehydrated, embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim), and cut at 15 lm. The sectioned in situ samples were imaged using an Olympus FV1000D confocal microscope. Acknowledgements We thank the members of Kikuchi and Atsushi Suzuki laboratories in Hiroshima University for helpful discussion and critical comments. We also thank Dr. A. Kawakami for providing clos5 mutant embryos. This study was supported by Grants from Grant-inAid for Scientific Research from the MEXT (KAKENHI 23616002) to Y.K. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.gep.2014.01.005. References Barth, K.A., Wilson, S.W., 1995. Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development 121, 1755–1768. Bertrand, J.Y., Chi, N.C., Santoso, B., Teng, S., Stainier, D.Y., Traver, D., 2010. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464, 108–111. Bogdanovic´, O., van Heeringen, S.J., Veenstra, G.J., 2012. The epigenome in early vertebrate development. Genesis 50, 192–206. Boisset, J.C., van Cappellen, W., Andrieu-Soler, C., Galjart, N., Dzierzak, E., Robin, C., 2010. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120. Campos, C., Valente, L.M., Fernandes, J.M., 2012. Molecular evolution of zebrafish dnmt3 genes and thermal plasticity of their expression during embryonic development. Gene 500, 93–100. Challen, G.A., Sun, D., Jeong, M., Luo, M., Jelinek, J., Berg, J.S., Bock, C., Vasanthakumar, A., Gu, H., Xi, Y., Liang, S., Lu, Y., Darlington, G.J., Meissner, A., Issa, J.P., Godley, L.A., Li, W., Goodell, M.A., 2012. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat. Genet. 44, 23–31.
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