br Results br Discussion Here we provide

Results
Discussion Here, we provide evidence showing the expression of PRDM1 in human fetal germline tissues. Although it has been reported that Prdm1 is absent in zebrafish PGCs (Ng et al., 2006), the expression of PRDM1 in the germ cell lineage appears to be evolutionarily conserved between mouse and human. In the mouse germline, Prdm1 expression was found in several developmental stages: the ICM, proximal epiblast PCG precursors, and migrating PGCs along the hindgut and primitive gonadal ridge (Chang et al., 2002; Chu et al., 2011; Ohinata et al., 2005). Although in this study we were unable to demonstrate whether PRDM1 is expressed in human PGC precursors, as their formation occurs between 5 and 8 weeks of gestation (Clark, 2007; Freeman, 2003), we showed that PRDM1 is expressed in the second trimester of fetal gonads. Given that PRDM1 is coexpressed with OCT4, but not with the migratory germ cell marker VASA, in the developing human fetal testis or ovary of second-trimester abortus, it is plausible that the PRDM1-expressing AZD1152 in human fetal gonads are newly immigrating PGCs that can further differentiate into late germline progenitors/germ cells. Supporting this finding, it has been reported that OCT4 is expressed in a subpopulation of germ cells where the PGCs reside in testis cords in the second trimester of human testis development (Gaskell et al., 2004). Consistent with the in vivo observation, our in vitro differentiation studies demonstrated that PRDM1 is coexpressed with other germ cell markers in differentiating hESCs in early differentiation stages (days 5 and 10), but is not coexpressed with VASA in the late stage (day 20), although VASA has been detected in the fetal germline population as early as 7 weeks of gestation (Castrillon et al., 2000). This could be due to the heterogeneous nature of early germline development, and cells with different expression patterns of germline markers may represent a different subpopulation of germline progenitors that give rise to a larger population of VASA-expressing cells at later developmental stages. The functional role of BMP/Smad and Wnt/β-catenin signaling pathways in early germ cell formation has been studied extensively in mice (Ohinata et al., 2009). It was demonstrated that only a subset of WNT3A-expressing epiblast cells of E5.5–E6.25 embryos respond to BMPs secreted from their surrounding extraembryonic cells, thereby acquiring the competence of early germ cells (Ohinata et al., 2009). Similar to these findings in mice, we previously demonstrated that BMP4 and WNT3A treatment significantly enhances the potential of germline differentiation by hESCs (Chuang et al., 2012). These observations led us to investigate the importance of BMP and/or Wnt signaling for PRDM1 induction in germline differentiation of hESCs. Indeed, we showed that BMP4 and WNT3A significantly increased the expression of germline-related marker genes as well as PRDM1, whereas blocking the activation of either BMP or Wnt signaling with their antagonists significantly reduced the expression of PRDM1. Furthermore, the germline potential was evidently impaired in PRDM1-KD hESCs after BMP4 and WNT3A treatment, suggesting that PRDM1 is most probably a crucial downstream effector of germline inducers such as BMP and Wnt signaling molecules. Thus, these results suggest an evolutionarily conserved BMP4/PRDM1 regulatory axis between human and mouse in controlling germ cell specification. Although we demonstrated BMP4-dependent PRDM1 induction, the mechanism by which BMP signaling activates PRDM1 is still elusive. It was previously suggested that, in mouse, BMP signals may upregulate Prdm1 via BMP-dependent activation of Smad1 and Smad5 (Ohinata et al., 2009). Further studies are necessary to determine whether SMAD proteins directly regulate PRDM1 during human germline differentiation. During mouse PGC specification, Prdm1 served as a key transcriptional regulator responsible for repression of the somatic program and establishment of germline characteristics, including upregulation of pluripotency-associated genes such as Oct3/4, Nanog, and Sox2, in the PGC precursors (Kurimoto et al., 2008). We showed that ectopic expression of PRDM1 in hESCs downregulated SOX2 expression, but had no effect on the expression of OCT4 and NANOG. Further, combined ChIP and reporter assay analyses showed that PRDM1 directly targets the promoter of SOX2 and represses its transcription. However, these results also reveal a discrepancy between human and mouse in terms of the PRDM1-mediated germline specification. Sox2 is expressed in mouse germ cells (Saitou, 2009), whereas it is absent from human PGCs and gonocytes during the early stage of human development (Perrett et al., 2008). The functional significance of the absence of SOX2 from early human germ cells remains unclear. We show here that overexpression of SOX2 in hESCs under the condition favoring germline differentiation reduced the production of VASA+ cells, suggesting that SOX2 may perturb the expression of germline-associated genes. In hESCs, the SOX family proteins SOX2 and SOX3 are redundant and repress mesendoderm differentiation (Wang et al., 2012). It is thus plausible that in human, other SOX proteins may provide substitutive functions that similarly mimic the action of SOX2 in mouse for modulating germline formation. Supporting this notion, SOX17 was found to be coexpressed with OCT4 in early human germ cells (de Jong et al., 2008). This observation further suggests that SOX17 may play a role in conjunction with other factors, such as OCT4, in the generation of human germ cells.