Developmental Biology 399 (2015) 164–176 Contents lists available at ScienceDirect Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology The requirement of histone modification by PRDM12 and Kdm4a for the development of pre-placodal ectoderm and neural crest in Xenopus Shinya Matsukawa a, Kyoko Miwata b, Makoto Asashima b, Tatsuo Michiue a,n a Department of Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan b Research Center for Stem Cell Engineering National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba City, Ibaraki, Japan article info abstract Article history: In vertebrates, pre-placodal ectoderm and neural crest development requires morphogen gradients and Received 6 September 2014 several transcriptional factors, while the involvement of histone modification remains unclear. Here, we Received in revised form report that histone-modifying factors play crucial roles in the development of pre-placodal ectoderm 21 November 2014 and neural crest in Xenopus. During the early neurula stage, PRDM12 was expressed in the lateral pre- Accepted 23 December 2014 placodal ectoderm and repressed the expression of neural crest specifier genes via methylation of Available online 6 January 2015 histone H3K9. ChIP-qPCR analyses indicated that PRDM12 promoted the occupancy of the trimethylated Keywords: histone H3K9 (H3K9me3) on the Foxd3, Slug, and Sox8 promoters. Injection of the PRDM12 MO inhibited fi Histone modi cation the expression of presumptive trigeminal placode markers and decreased the occupancy of H3K9me3 on PRDM12 the Foxd3 promoter. Histone demethylase Kdm4a also inhibited the expression of presumptive Kdm4a trigeminal placode markers in a similar manner to PRDM12 MO and could compensate for the effects Pre-placodal ectoderm Neural crest of PRDM12. ChIP-qPCR analyses revealed that promotion of the occupancy of H3K9me3 on the Foxd3, Xenopus Slug, and Sox8 promoters was inhibited by Kdm4a overexpression. Taken together, these data indicate that histone modification was essential for pre-placodal ectoderm and neural crest development. & 2015 Elsevier Inc. All rights reserved. Introduction the competence to form pre-placodal ectoderm (Pieper et al., 2012). Indeed, Dlx3 overexpression reduced the expressions of In the vertebrate embryo, the neural plate border is thought to neural plate genes, neural plate border genes, and neural crest be formed by the intermediate activation of bone morphogenetic genes, whereas the injection of Dlx3 morpholino oligomer (MO) protein (BMP) signaling to induce several neural plate border genes expanded their expression (Pieper et al., 2012). (Dincer et al., 2013; Reichert et al., 2013; Tribulo et al., 2003). The On the other hand, a previous report showed that a neural plate border region then further separates into the pre-placodal ectoderm border gene, Msx1, was important for neural crest specification and neural crest, with the former segregating into individual cranial (Monsoro-Burq et al., 2005). A gain-of-function experiment using placodes, such as the adenohypophyseal, olfactory, lens, trigeminal, the Msx1-GR construct showed that Msx1 could induce the lateral line, otic, and epibranchial placodes (reviewed in Baker and expression of several neural crest specifier genes (Tribulo et al., Bronner-Fraser, 2001; Saint-Jeannet and Moody, 2014; Schlosser, 2003). Another study suggested that some neural crest cells could 2010). In contrast, the neural crest differentiates into the peripheral be induced by co-expression of Msx1 and Pax3 in Xenopus animal nervous system, adrenal medulla, melanocytes, facial cartilage, and cap cells in the presence of Wnt and fibroblast growth factor (FGF) tooth dentine (reviewed in Grenier et al., 2009; Minoux and Rijli, signaling (Monsoro-Burq et al., 2005). In the animal cap cells 2010; Steventon et al., 2014; Theveneau and Mayor, 2012). injected with Noggin, Msx1 overexpression could also activate the Pre-placodal ectoderm development requires the expression of expression of Pax3 and Zic1, which play important roles in the a neural plate border gene, Dlx3 (Hans et al., 2004; Kaji and induction and formation of functional neural crest cells (Milet Artinger, 2004; Woda et al., 2003). Dlx3 is regulated by BMP et al., 2013; Monsoro-Burq et al., 2005; Sato et al., 2005). signaling and promotes the expression of pre-placodal ectoderm A boundary formation between the pre-placodal ectoderm and specifier genes such as Six1 and Eya1, known as pan-placodal neural crest may rely on the expression levels of Dlx and Msx, markers (Woda et al., 2003). A recent study also indicated that whose proteins can bind to each other and form an inhibitory Dlx3 expression in nonneural ectoderm was required for acquiring complex (Zhang et al., 1997). Pre-placodal ectoderm genes could be induced by the high transcriptional activity of Dlx (Pieper et al., 2012), whereas neural crest genes were induced by that of Msx n Corresponding author. (Monsoro-Burq et al., 2005). These mutual inhibitions may con- E-mail address: [email protected] (T. Michiue). tribute to the boundary formation between early pre-placodal http://dx.doi.org/10.1016/j.ydbio.2014.12.028 0012-1606/& 2015 Elsevier Inc. All rights reserved. S. Matsukawa et al. / Developmental Biology 399 (2015) 164–176 165 ectoderm and neural crest. However, a recent study showed that 53%, n¼49; compare Fig. 2B with C). As melanocytes are derived neural crest region did not expand to the pre-placodal ectoderm from neural crest cells, whether the injection of PRDM12 also region in the embryo injected with Dlx3 MO (Pieper et al., 2012), inhibited early neural crest differentiation was tested. At the early and thus another mechanism likely determines the boundary. neurula stage, PRDM12 overexpression significantly reduced the A recent study implicated the epigenetic factor Kdm4a, which expression of several neural crest markers such as Foxd3, Slug, and acts as a histone demethylase, was expressed in the neural crest Sox9 (repression in 73%, n¼26, 75%, n¼36, 67%, n¼24, respec- region of chick embryos (Strobl-Mazzulla et al., 2010). We hypothe- tively Fig. 2D–I), whereas expression patterns of the pre-placodal sized that histone modification for transcriptional regulation was markers Islet1 and Six1, and neural marker Sox2, were not affected required for pre-placodal ectoderm and neural crest patterning, and (no repression in 86%, n¼22, 94%, n¼33, 95%, n¼21, respectively focused on histone-modifying enzymes expressed in pre-placodal Fig. 2J–O). A recent study revealed that neural crest cells were ectoderm. To investigate which histone methyltransferases could be induced by both antagonizing BMP and promoting late Wnt involved with pre-placodal ectoderm development, we performed a signaling (Sato et al., 2005). Thus, neural crest cell induction can β-catenin-dependent microarray analysis (Tanibe et al., 2008)and be mimicked by co-injection of Chd and Wnt8 mRNAs in animal identified PRDM12, a member of the PRDM family of histone cap cells (Sato et al., 2005). RT-PCR indicated that PRDM12 over- methyltransferase factors. PRDM12 was expressed in the parietal expression in Chd- and Wnt8-injected animal cap cells inhibited region of the central nervous system and cranial placode in the expression of several neural crest markers in a dose- zebrafish and mouse embryos (Kinameri et al., 2008; Sun et al., dependent manner (Fig. 2P, lanes 4–6). PRDM12 contains a PR/ 2008), and regulated cell proliferation in P19 cells (Yang and SET domain that is probably related to histone methylation and Shinkai, 2013). However, the importance of PRDM12 in embryonic multiple zinc-finger domains (Fig. 2A). Generally, histone methyl- development remains largely unknown. In this study, we demon- transferase acts either as a positive regulator of transcription by strate the role of PRDM12 during early Xenopus development and methylating histone H3 at lysine 4 (H3K4) or as a negative the requirement of histone modification by PRDM12 and Kdm4a for regulator via methylation of histone H3 at lysine 9 (H3K9) or pre-placodal ectoderm and neural crest development. lysine 27 (H3K27) (reviewed in Zentner and Henikoff, 2013). To test whether PRDM12 acts as a positive or negative regulator, Western blotting analysis using each anti-trimethylated lysine Results antibody was performed, which indicated that PRDM12 induced the trimethylation of H3K9 (H3K9me3) in a dose-dependent PRDM12 is expressed in the lateral pre-placodal ectoderm after the manner, but not H3K4 and H3K27 (Fig. 2Q). These results demon- gastrula stage and is regulated by BMP and late Wnt signaling strated that PRDM12 played an inhibitory role in neural crest gene expression and promoted the H3K9me3. Following a previous First, the expression of Xenopus PRDM12 was investigated by study (de Souza et al., 1999), two fusion constructs, VP16-PRDM12 reverse transcription-polymerase chain reaction (RT-PCR) and and EnR-PRDM12, were made (Supplementary Fig. S2A). Micro- whole-mount in situ hybridization (WISH) analyses. PRDM12 was injection with VP16-PRDM12 increased the number of melanocytes zygotically expressed after the late gastrula stage (Fig. 1A) and was and induced several neural crest markers in animal cap cells detected at the lateral pre-placodal ectoderm in the early neurula (Supplementary Fig. S2B, C and F). In contrast, EnR-PRDM12 embryo (Fig. 1B). In the mid-neurula stage, PRDM12 expression decreased pigmentation and repressed several neural crest mar- was also observed in the two stripes of neural plate (Fig. 1C) and kers in Chd- and Wnt-injected animal cap cells, as observed in the was partially co-localized with the lateral expression regions of embryos or animal cap cells injected with PRDM12-WT (Supple- Six1, Pax3, Islet1, and Pax6 (Fig. 1D and E; Supplementary Fig. S1A– mentary Fig. S2D, E and G). Because the effects of EnR-PRDM12 C), but not Foxd3 and Six3 (Fig. 1F and G; Supplementary Fig. S1D). were similar to those of PRDM12-WT, PRDM12 could act as a Wnt and BMP signaling alter the expression of pre-placodal negative regulator for the target gene transcription.