STEM CELLS AND REGENERATION RESEARCH ARTICLE 3565 Development 140, 3565-3576 (2013) doi:10.1242/dev.094045 © 2013. Published by The Company of Biologists Ltd INTRODUCTION Stem cells in adults are distinct from progenitor cells in that they can self-renew (Brinster, 2002; Spangrude et al., 1988). Shortly after birth, murine male germ cells acquire stem cell identity; and the mitotic cells that maintain homeostasis through self-renewal during spermatogenesis are known as spermatogonia (Phillips et al., 2010). Spermatogonia are classified as either type A, intermediate or type B. Type A spermatogonia can be subclassified as single (A s ), paired (A pr ), aligned (A al-4 , A al-8 , A al-16 or A al-32 ) or differentiating (A 1 , A 2 , A 3 or A 4 ; supplementary material Fig. S1) (Russell et al., 1999). The A s theory holds that only A s cells retain stem cell capacities (de Rooij and Russell, 2000), whereas A pr and A al cells (known as proliferating spermatogonia) are thought to be progenitor cells that can no longer self-renew (de Rooij and Russell, 2000; Huckins, 1971; Oakberg, 1971). Nevertheless, the capacity to self-renew has never been tested in A pr -A al spermatogonia because, until now, the definitive assay was thought to require transplantation of purified A pr -A al cells, and no appropriate purification method is known. An alternative spermatogonia classification method relies on the differential expression of the marker gene Kit: only Kit-negative (Kit neg ) spermatogonia show stem cell activity in both neonates and adults (Ohbo et al., 2003; Shinohara et al., 2000). Kit expression commences at the A 1 stage, whereas most A s , A pr and A al spermatogonia are Kit neg (supplementary material Fig. S1) (de Rooij and Russell, 2000; Yoshinaga et al., 1991). In this article, we distinguish between A s -A al and A 1-4 -B spermatogonia by Kit expression. Other markers are expressed in A s cells, including GFRα1, Oct4, Plzf, Ngn3 and Nanos2; and their expression profiles assist in understanding stem cell-specific gene networks (supplementary material Fig. S1) (Buageaw et al., 2005; Buaas et al., 2004; Costoya et al., 2004; Ohbo et al., 2003; Sada et al., 2009; Yoshida et al., 2004). All of these markers were detected in subpopulations of A s , A pr and A al cells, raising questions about the heterogeneity of A s spermatogonia. In mice, for example, only a small fraction of A s spermatogonia co-expresses GFRα1 and EGFP in Ngn3-EGFP, whereas the remainder express only one of these genes (Nakagawa et al., 2010; Suzuki et al., 2009). One marker changes its expression pattern during development. Ngn3 is specifically expressed in Kit- negative fraction of neonate spermatogonia, and its expression broadens in adult, overlapping both Kit-negative and Kit-positive spermatogonia (Yoshida et al., 2004; Yoshida et al., 2006; Suzuki et al., 2009). These variable expression patterns in A s to A al cells could reflect oscillatory expression of lineage-associated genes, differences in cell-cycle status or differential responses to micro- environmental signals. This heterogeneous expression pattern was suggested to reflect a metastable state, in which cells maintain a ‘memory of stemness’ until they reach a point of no return, and lose stem cell potential (May and Enver, 2001; Graf and Stadtfeld, 2008). Nuclear architectures have been reported to fix the gene expression program associated with a particular cell fate (Bártová et 1 Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan. 2 Developmental Genetics group, RIKEN Research Center for Integrative Medical Sciences, Yokohama 230-0045, Japan. 3 Department of Molecular Biology, Yokohama City University School of Medicine,, Yokohama 236-0004, Japan. 4 Division of Germ Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. 5 Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Suita 565-0871, Japan. 6 Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo 123-8585, Japan. *Author for correspondence (kohbo@yokohama-cu.ac.jp) Accepted 27 June 2013 SUMMARY Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized; by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of murine spermatogonia toward meiosis. We have exploited a property of incomplete cytokinesis, which causes male germ cells to form aligned chains of characteristic lengths, as they divide and differentiate. These chains revealed the stage of spermatogenesis, so the epigenetic differences of various stages could be characterized. Single, paired and medium chain-length spermatogonia not expressing Kit (a marker of differentiating spermatogonia) showed no expression of Dnmt3a2 and Dnmt3b (two de novo DNA methyltransferases); they also lacked the transcriptionally repressive histone modification H3K9me2. By contrast, spermatogonia consisting of ~8-16 chained cells with Kit expression dramatically upregulated Dnmt3a2/3b expression and also displayed increased H3K9me2 modification. To explore the function of these epigenetic changes in spermatogonia in vivo, the DNA methylation machinery was destabilized by ectopic Dnmt3b expression or Np95 ablation. Forced Dnmt3b expression induced expression of Kit; whereas ablation of Np95, which is essential for maintaining DNA methylation, interfered with differentiation and viability only after spermatogonia become Kit positive. These data suggest that the epigenetic status of spermatogonia shifts dramatically during the Kit-negative to Kit-positive transition. This shift might serve as a switch that determines whether spermatogonia self-renew or differentiate. KEY WORDS: Stem cell differentiation, Epigenetics, Germ cells, Kit-negative identity, Kit-positive identity An epigenetic switch is crucial for spermatogonia to exit the undifferentiated state toward a Kit-positive identity Takayuki Shirakawa 1 , Ruken Yaman-Deveci 2 , Shin-ichi Tomizawa 1 , Yoshito Kamizato 1 , Kuniko Nakajima 1 , Hidetoshi Sone 1 , Yasuyuki Sato 1 , Jafar Sharif 2 , Akio Yamashita 3 , Yuki Takada-Horisawa 2 , Shosei Yoshida 4 , Kiyoe Ura 5 , Masahiro Muto 2 , Haruhiko Koseki 2 , Toshio Suda 6 and Kazuyuki Ohbo 1, * DEVELOPMENT