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    • ack1 inhibitor Enhancers play a central role

    2018-10-24

    Enhancers play a central role in cell-type-specific and stage-specific regulation of gene expression and are capable of acting over a distance ranging from several to hundreds, and in rare cases even thousands of kilobases from their target genes (Bulger and Groudine, 2011; Ong and Corces, 2011, 2012). In developing mouse embryos, Oct4 gene expression is regulated spatially and temporally by at least two enhancers, DE and PE (Yeom et al., 1996). Here we have elucidated the control of Oct4 expression by its DE and PE elements during the totipotent cycle (Figure 7A). The Oct4 gene is expressed under the control of the DE during the pre-implantation stage, but its expression is controlled by PE in the epiblast subsequent to implantation (Figure 7A; Scholer, 1991). Expression of the Oct4 gene is downregulated in epiblasts and expressed exclusively in the developing germline. In founder PGCs, Oct4 is initially controlled by DE but subsequently by both DE and PE (Figures 1E–1J and 7A). Interestingly, we found that Oct4 was expressed not only in spermatogonia but also in differentiating germ cells; in adult mice, Oct4 expression was regulated by DE in spermatogonia but by PE in differentiating germ cells. It is well known that PGCs first emerge at the proximal region of the epiblast adjacent to the extraembryonic ectoderm and then migrate to form the PGC cluster at 7.25 dpc (Ginsburg et al., 1990; Saitou et al., 2002). However, while our dual reporter system could not distinguish the PGC population at 7.25 dpc, Oct4-ΔPE-GFP PGCs were apparent in 8.5-dpc embryos. Intriguingly, migrating PGCs in 9.5- and 10.5-dpc embryos contained two subpopulations, GFP+RFP− and GFP+RFP+ cells, and the GFP+RFP+ cell population increased during PGC migration into the genital ridge. Since genome-wide epigenetic modification, including imprinting erasure, occurs in migrating PGCs (Sasaki and Matsui, 2008), further experiments are required to examine the differences between these two migrating PGC populations and whether they can be distinguished using genome-wide epigenetic markers. This study showed that ICM ack1 inhibitor initially express only GFP but express both GFP and RFP during the derivation of ESCs (Figure 2B). This result supports the findings of recent studies indicating that the ability of ICM cells to self-renew as ESCs is acquired during epiblast specification (Boroviak et al., 2014; Tang et al., 2010). Boroviak et al. (2014) also suggested that early ICM cells are distinct from ESCs and that ESCs exhibit the greatest degree of identity to the embryonic day 4.5 (E4.5) epiblast. Our results confirm that the switch in enhancer activity occurs at approximately the E4.5 stage (Yeom et al., 1996), and enhancer activity of DE and PE overlapped in early post-implantation epiblast (5.5–6.5 dpc). Self-renewing ESCs cultivated in conventional ESC culture medium (supplemented with LIF and serum) constitute a cell population at various stages of pluripotency (Hayashi et al., 2008; Martinez Arias and Brickman, 2011; Miyanari and Torres-Padilla, 2012). A defined ESC culture system, containing 2i (MEK inhibitor PD0325901 and glycogen synthase kinase 3 inhibitor CHIR99021) (Ying et al., 2008) together with LIF (2i + LIF) has been used to select naive pluripotent cells (Silva et al., 2008). Here we show that ESCs from 2i + LIF medium still contain two cell populations when cultured in serum-containing medium and that the mixed population could be converted to homogeneous naive PSCs by using serum-free N2B27 medium. This result indicates that serum contains a factor(s) that induce differentiation or inhibit maintenance of naive pluripotency. Previous reports have suggested that serum contains ERK phosphorylation-inducing factors (Yamaji et al., 2013). Silva et al. (2008) also showed that the presence of MEK inhibitor in serum-containing medium accelerated the activation of Nanog and Rex1 during reprogramming. This is also supported by the findings that GFP+RFP− ESCs cultured in serum-containing medium were molecularly distinguishable from GFP+RFP− ESCs cultured in serum-free medium. Collectively, the GFP+RFP− state is a necessary but not sufficient condition for naive PSCs, since special medium is also required for maintaining naive PSCs. Several reporter systems suggested that undifferentiated ESCs heterogeneously expressed Nanog (Chambers et al., 2007), Rex1 (Marks et al., 2012; Toyooka et al., 2008), Stella (Hayashi et al., 2008), Esrrb (van den Berg et al., 2008), and Tbx3 (Niwa et al., 2009). The heterogeneity of transcription factors in ESCs regulated self-renewal capacity, expression of developmental genes, and differentiation potential (Torres-Padilla and Chambers, 2014). However, the GFP+RFP− and GFP+RFP+ ESCs in serum-containing medium are similar to each other at the transcriptional, epigenetic, and functional levels. Our dual transgenic system allows monitoring of enhancer activity of DE and PE, but not Oct4 expression levels. Although enhancer activity fluctuates between GFP+RFP− and GFP+RFP+ in serum-containing medium, these cells highly expressed Oct4 gene.