[Frontiers in Bioscience 14, 1068-1087, January 1, 2009]
Specification of the germ cell lineage in mice
Mitinori Saitou1,2
1
TABLE OF CONTENTS
1. ABSTRACT
Specification of the germ cell lineage is fundamental in development and heredity. In mice, and presumably in all mammals, germ cell fate is not an inherited trait from the egg, but is induced in pluripotent epiblast cells by signaling molecules. Recent studies are beginning to uncover the signaling requirements and key transcriptional regulators for the specification of the germ cell lineage in mice, as well as the distinct properties that the specified germ cells acquire uniquely. Accordingly, the evidence suggests that germ cell specification is an integration of the repression of the somatic program, re-acquisition of potential pluripotency, and ensuing genome-wide epigenetic reprogramming. The accumulated knowledge will be critical for the reconstitution of this key lineage in vitro, which may provide a useful foundation for reproductive and regenerative medicine.
2. INTRODUCTION
Specification of germ cell fate is fundamental in development and heredity. There are essentially two key pathways by which the specification is realized. One is through the inheritance of determinants, generally known as germ plasm, that are maternally deposited into specific blastomeres (preformation) and the other through the induction by specific signals from a pluripotent cell population that arises in the middle of embryonic development (epigenesis) (1, 2). Although the preformation mode is seen in many model organisms of modern biology, including C. elegans and D. melanogaster, recent studies have suggested that the epigenesis mode, which is seen in mice and probably all mammals, is more prevalent across and ancestral to the Metazoa (1). Despite the difference in the mode of germ cell specification, recent studies are beginning to show that both modes involve common cellular events, most notably, the repression of the somatic program, although the mechanisms involved are markedly different among the organisms (3-6). Furthermore, it has also become increasingly evident that the specified germ cells utilize a number of common molecular pathways for their further development, reflecting conserved properties shared by this key lineage across essentially all the species (3). In this article, I will discuss the present knowledge on the mechanisms of germ cell specification in mice, which seems to involve an integration of the repression of somatic program, re-acquisition of potential cellular pluripotency, and ensuing genome-wide epigenetic reprogramming. These mechanisms may be related to those responsible for inducing pluripotency in somatic cells (7-12).
3. FROM FERTILIZATION TO THE SPECIFICATION OF THE NEW GERM CELL LINEAGE
The developmental program in the mouse is initiated upon the fusion of a highly specialized female gamete, the oocyte, with a male counterpart, the sperm. One of the first events to occur in the fertilized oocyte is the re-organization of the paternal genome, which includes replacement of the protamines with maternally deposited histones and subsequent apparent genome-wide DNA demethylation (13-16). These events take place prior to the S phase. It is notable that genome-wide DNA demethylation from the paternal genome is a conserved phenomenon across several mammalian species, which suggests the functional significance of this event (17). Apparently, this resetting of the epigenetic modifications is a well-programmed process in the initiation of embryonic development (18).
The resultant totipotent zygote, with key maternal factors, replicates haploid parental genomes and begins to undergo cleavage divisions, with the major zygotic transcription starting at the late 2-cell stage (19, 20). The first sign of cell fate specification seems to occur in the 8- to 16-cell stage, when the cells located outside start to show signs of differentiation towards trophectoderm (TE) cells and the cells located inside remain undifferentiated and maintain pluripotency (21). Although the precise mechanisms regulating this process are unknown, reciprocal inhibition of the key lineage determinants Oct4 and Cdx2 seems to play a role in the segregation of these two lineages (22, 23). More recently, a TEA DNA binding domain-containing transcription factor, Tead4, was shown to be critical for up-regulating Cdx2 specifically in the outside cells at the morulae stage embryos, which in turn is essential for TE differentiation (24, 25). Upon formation of the blastocoel cavity, the developing embryos are referred to as blastocysts. At around embryonic day (E) 3.5, the blastocysts consist of two clearly discernable cell types, the TE and the inner cell mass (ICM) cells. The ICM is the source of all the cells in the adult body, including germ cells, and is a pluripotent cell population (26, 27).
Following substantial genome-wide DNA demethylation in the preimplantation stages (28, 29), embryonic DNA methylation patterns start to be imposed through lineage-specific de novo methylation that begins in the ICM of a blastocyst (30, 31). Genome-wide DNA methylation levels increase rapidly, and this increase is mediated by the de novo DNA methyltransferases Dnmt3a and Dnmt3b (30). Another repressive modification, histone H3 Lysine-9 di-methylation (H3K9me2), increases after the two-cell stage, which thus precedes de novo DNA methylation (32).
The first lineage that differentiates from the ICM is the primitive endoderm (PE), which delineates the inner surface of the ICM at E4.5 (33). The undifferentiated cells in the ICM are now called the primitive ectoderm, which maintains pluripotency. Upon implantation, TE cells that are in direct contact with the ICM or the primitive ectoderm (polar TE) proliferate and grow into a thick column of extraembryonic ectoderm (ExE) cells. The primitive ectoderm cells then form, through a process of cavitation (34), a cup-shaped epithelial sheet, which is called the epiblast. It is from these epiblast cells that all the somatic cells as well as the germ cells in the adult body arise. The initial patterning of embryogenesis, including anterior-posterior polarity formation, the gastrulation that forms mesodermal and definitive endodermal cells, and germ cell specification is mediated through signaling molecules from the ExE and PE-derived visceral endoderm (VE) that cover the epiblast (33).
4. EXPERIMENTAL EMBRYOLOGY ON THE ORIGIN OF THE GERM CELL LINEAGE
In mammals, the origin of the germ cell lineage in embryogenesis had long been unknown due to the absence of the characteristic germ plasm in the egg as seen in other organisms such as X. laevis and D. melanogaster. It was in 1954 that Chiquoine found that the primordial germ cells (PGCs), the first population of the germ cell lineage that gives rise to both oocytes and sperm, can be identified as cells bearing high alkaline phosphatase (AP) activity in the endoderm of the yolk sac immediately beneath the primitive streak of a young 8-day embryo (35). These AP-positive PGCs migrate through the developing hindgut endoderm, eventually colonizing in the genital ridges after E9.5, where they begin to differentiate into functional gametes through highly complex developmental pathways. Much later, in 1990, the origin of the PGCs was traced back to a small cluster of AP-positive cells just posterior to the definitive primitive streak in the extraembryonic mesoderm (ExM), separated from the embryo by the amniotic fold, of E7.0-7.25 embryos (36) (Figure 1). It was suggested that PGCs are set aside as early as E7.0, possibly as one of the first "mesodermal" cell types to emerge. Subsequently, based on a clonal analysis of the fate of epiblast cells of E6.0 and E6.5 embryos, it was proposed that precursors of PGCs reside in the proximal epiblast close to the ExE in both stages of embryos and they are not lineage restricted while they are in the epiblast (37). This analysis also predicted that the founder population of PGCs, located in the extraembryonic mesoderm, consists of approximately 45 cells. Transplantation of distal epiblast cells of E6.5 embryos into
the proximal region of the other embryos resulted in the contribution of the donor cells to the germ cell lineage, indicating that epiblast cells as late as E6.5 are pluripotent and can form germ cells when placed in an appropriate microenvironment (38).
5. SIGNALING FOR GERM CELL SPECIFICATION
The uncovering of signaling molecules and their signal transducers necessary for the specification of the germ cell lineage was accomplished by gene knockout studies (39, 40) (Table 1). Most notably, Bmp4, which is initially expressed in the ExE directly contacting the proximal epiblast from around E5.5, was the first to be demonstrated as essential for PGC specification (41). In the Bmp4 mutants on a mixed genetic background, PGCs detectable by AP staining, as well as the allantoic mesoderm, were not formed. In Bmp4 heterozygous mutants, the number of founder PGCs was reduced to less than half. Chimera analysis indicated that it is the Bmp4 expression in the ExE that is essential for the formation of PGCs. This study thus demonstrated for the first time that germ cell specification in mice depends on a secreted signal from the previously segregated, extraembryonic lineage. Subsequently, it was shown that Bmp4 expressed in the ExM is not required for the specification of PGCs but is necessary for the correct localization and survival of PGCs (42).
Bmp8b, which is expressed exclusively in the ExE from as early as E5.5, was also shown to be critical for PGC specification (43). On a predominantly C57BL/6 background, almost no PGCs or a reduced number of PGCs were formed in the homozygous and heterozygous mutants, respectively. Additionally, it was shown that embryos that are double heterozygotes for the Bmp8b and Bmp4 mutations have similar defects in PGC numbers as Bmp4 heterozygotes, indicating that the effects of the two Bmps are not additive.
Bmp2, expressed primarily in the VE of pregastrula and gastrula embryos, also plays a role in the establishment of the germ cell lineage (44). The number of PGCs is significantly reduced in Bmp2 heterozygous and homozygous embryos on a largely C57BL/6 background. Notably, Bmp2 and Bmp4 have an additive effect on PGC generation, whereas Bmp2 and Bmp8b do not. Thus, these findings indicate that at least three Bmps, Bmp4 and Bmp8b from ExE and Bmp2 from VE, are necessary for the proper establishment of PGCs in vivo.
Currently, it is unknown through which receptors these Bmps signal for PGC specification. A Bmp ligand initiates signaling by binding to and bringing together type I and type II receptor serine/threonine kinases on the cell surface. This allows receptor II to phosphorylate the receptor I kinase domain, which then propagates the signal through phosphorylation of the conserved C-terminal residues of the Smad1, 5, or 8 proteins, which, by forming heterodimers with Smad4, translocate to the nucleus and function as transcriptional regulators (45, 46). Bmps use three different type II receptors, Bmp type II receptor (Bmpr-II) and activin type II receptors (Actr-IIA and Actr-IIB), and three type I receptors, Activin receptor-like kinase (Alk) 3/Bmpr-IA, Alk6/Bmpr-IB, and Alk2. BmprII, which encodes a type II receptor for Bmp2 and Bmp4, is expressed uniformly in the embryonic and extraembryonic tissues at E6.5. Inactivation of BmprII results in early embryonic lethality, due to impaired growth of the epiblast and a failure to form mesoderm or gastrulate (47). Alk3, which encodes a type I receptor for most, if not all, Bmps, is expressed ubiquitously throughout development, including the epiblast at around E6.0. Embryos homozygous for Alk3 show a reduced size of the epiblast as early as E6.5 and form no mesoderm and fail to gastrulate (48). The early and severe phenotypes of these mutants preclude the possibility of analyzing if it is these receptors that are involved in PGC specification. Alk2, which encodes a type I receptor for Bmp7 and possibly for Bmp2 and 4, is expressed specifically in the VE before gastrulation and later both in embryonic and extraembryonic cells during gastrulation (49, 50). Alk2-deficient embryos are morphologically normal before gastrulation but show severe gastrulation defects and fail to form proper mesoderm. This gastrulation defect can be rescued in chimeric embryos generated by injection of Alk2 mutant ES cells into wild-type blastocysts, indicating that Alk2 function in the VE is essential for the proper gastrulation (49, 50). Interestingly, there is a report showing that AP-positive PGCs are absent in Alk2 mutants (51), although the mechanism through which Alk2 in the VE contributes to PGC specification remains unclear.
Consistent with the requirement of Bmps for PGC specification, several Smad proteins transducing Bmp signals are known to be required for PGC specification. Smad1 and Smad5, which are expressed widely in the epiblast during gastrulation, are both necessary for PGC specification (52-54). Both Smad1-deficient embryos and Smad5-deficient embryos have severely reduced numbers of PGCs. Smad1 and Smad5 double heterozygous embryos show much smaller numbers of PGCs compared to that in either single heterozygote, indicating that Smad1 and Smad5 have an additive effect on PGC formation (55). Whether Smad1 and Smad5 have redundant functions or distinct roles in PGC specification remains to be clarified. Smad4, which functions by forming a heterodimer either with Smad1 or Smad5 for Bmp signal transduction, is also shown to be essential for PGC formation (56). Collectively, all the evidence to date demonstrates that both the Bmp signals emanating from ExE and those emanating from VE are essential for PGC specification. However, the molecular mechanisms by which these Bmp signals induce only a subset of proximal epiblast cells to commit to the PGC fate are still largely unexplored.
Interestingly, recent studies have shown that key signals for embryonic patterning converge, at least in some part, on Smad proteins (57-59). The Smad proteins consist of two conserved globular domains (MH1 and MH2 domains) connected by a linker region. The MH1 domain binds DNA, whereas the MH2 domain binds membrane receptors for activation by phosphorylation (as described above), nucleoporins for nuclear translocation, and other Smads and nuclear factors to form transcriptional complexes. It has been shown that MAPKs and GSK3 successively phosphorylate the conserved residues in the linker region, which are then recognized by Smurf ubiquitin ligase for degradation (58, 59). Therefore, signals through MAPKs and GSK3 negatively regulate and hence determine the duration of the Bmp signals. Notably, Aubin et al. generated two point mutants of Smad1: Smad1C that mutates the conserved receptor phosphorylation sites at the C-terminus and Smad1L that mutates the MAPK consensus sites in the linker region (57). The Smad1C/C mutants showed similar phenotypes to the Smad1 null mutants and exhibited severe defects in PGC specification. In contrast, the Smad1L/L mutants were viable and survived to adulthood with specific phenotypes in stomach homeostasis. However, interestingly, the Smad1L/L mutants also showed defects in PGC specification, which were rescued by the functionally inactive Smad1C allele: the Smad1L/C mutants showed relatively normal PGC specification. These findings suggest that the tight balance of Bmp and MAPK signalings through Smad1 is critical for PGC specification.
Notably, in the mutants of Smad2, which transduces the signals of Tgf and Nodal, there appear many ectopic clusters of PGCs (52). This likely reflects the fact that all the epiblast cells of Smad2 mutants seem to acquire a proximal posterior property (60, 61) at least in part due to the failure to form anterior visceral endoderm, a key signaling center secreting inhibitors of posteriorizing signals and conferring an anterior property to the epiblast (33, 62).
6. KEY MOLECULES AND EVENTS ASSOCIATED WITH GERM CELL SPECIFICATION
6.1. The founder PGCs repress the Hox genes
Despite these advances on the embryological origin of and signaling requirements for the PGCs, very little was known regarding the genes that determine the PGC fate and the intrinsic properties of PGCs until relatively recently. This was in part due to the difficulty of analyzing the founder population of PGCs, which is small in number and embedded deeply in somatic neighbors. However, single-cell gene expression analysis of the founder PGCs and their somatic neighbors revealed that the PGCs indeed show distinct properties (63). Most notably, PGCs that showed high levels of an interferon-inducible transmembrane protein fragilis/mil-1/ifitm3 (63-65) and exclusive expression of a small nuclear-cytoplasmic shuttling protein stella/Pgc7/Dppa3 (63, 66, 67) were found to specifically repress the Hox genes, including Hoxb1 and Hoxa1, which are highly up-regulated in somatic neighbors (63). It has been proposed that the repression of the Hox genes reveals one of the mechanisms by which the PGCs escape from a somatic fate and retain their pluripotency. At the same time, it was identified that founder PGCs, similarly to their somatic neighbors, express the typical mesodermal markers T (Brachyury) and Fgf8, suggesting that the PGC fate is induced in a population of cells originally destined for a somatic mesodermal fate (63).
Since expression of stella and repression of the Hox genes are highly correlated in PGCs, it was postulated that stella may repress the expression of the Hox genes in PGCs. However, a gene knockout study showed that stella is dispensable in PGC specification (68). However, interestingly, it was found that stella-deficient oocytes lose their developmental potential (68), which was due to aberrant genome-wide DNA demethylation in a single-cell zygote (69). As discussed above, upon fertilization, the paternal haploid genome has been known to specifically remove its genome-wide DNA methylation (13, 14). Interestingly, in the absence of Pgc7/stella, genome-wide DNA demethylation occurs not only from the paternal genome but also from the maternal genome. Moreover, some of the specific methylations on the imprinted genes of the paternal genome, which are protected from genome-wide DNA demethylation in normal embryos, are also demethylated in Pgc7/stella (-/-) embryos. These findings indicate that Pgc7/stella functions to prevent DNA demethylation from the maternal allele and from imprinted genes on the paternal allele in normal development (69).
6.2. Blimp1 is a critical regulator for germ cell specification
Further single-cell analysis has identified Blimp1/Prdm1 as a gene specifically expressed in founder PGCs (70). Blimp1 encodes a potent transcriptional repressor with a N-terminal PR/SET domain, a proline-rich region, five C2H2 zinc fingers, and a C-terminal acidic domain, and was originally cloned as a gene specifically induced upon terminal differentiation of B-cells into plasma cells (B lymphocyte-induced maturation protein-1) (71). Blimp1 was later shown to be both necessary and sufficient for the terminal differentiation of B-cells (72, 73), leading to a notion that Blimp1 is a "master regulator" of plasma cell differentiation (74). Careful expression analysis has shown that Blimp1 is first expressed in a few of the most proximal posterior epiblast cells at around E6.25 prior to the onset of gastrulation. Blimp1-positive cells increase in number and form a cluster of approximately 20 cells at E6.75 (mid-streak (MS) stage) and 40 cells with strong AP activity at E7.25 (early-bud (EB) stage) (Figure 2). Genetic lineage tracing showed that all the Blimp1-positive cells at early stages contribute almost invariantly to stella-positive PGCs. It was therefore concluded that Blimp1-positive cells appearing in the most proximal epiblast at E6.25 are lineage-restricted PGC precursors (70, 75).
These observations were unexpected in light of the initial studies by clonal analysis of the fate of epiblast cells, which indicated that germ cell restriction is not evident in the early epiblast cells (37). It is possible that the clonal analysis may have failed to label one of the very few early Blimp1-positive epiblast cells. The epiblast clones contributing to PGCs in the clonal analysis might be the ones that were initially negative for Blimp1, but following division of these cells, some of them may have become Blimp1-positive and given rise to PGCs while the others gave rise to somatic descendants. It will be necessary to precisely clarify the mechanism that is responsible for the initial increase in the numbers of Blimp1-positive cells to resolve this issue fully. It is currently unknown how long the Blimp1-positive germ cell precursors continue to be recruited from the epiblast cells, and how the cell cycle of the Blimp1-positive cells is subsequently regulated, which together might be the key to understanding the properties of these PGC precursors.
Blimp1 function is essential for PGC specification (70, 76, 77). In Blimp1 mutants, PGC specification seems to be halted at a very early stage, with only about 20 AP-positive PGC-like cells formed (70). These PGC-like cells do not subsequently increase in number and fail to show migration. Gene expression analysis of Blimp1 mutant PGC-like cells indicates that these cells fail to consistently repress the Hox genes, with inadequate acquisition of some of the genes specific for PGCs, including stella (70). Although further analysis will
be required for a more precise understanding of the function of Blimp1 in PGC specification, the findings described above indicate that Blimp1 plays a central role in PGC specification.
Studies in other contexts have shown that Blimp1 functions as a transcriptional repressor by recruiting a repressor complex of Groucho family proteins (78) and HDAC2 (79) through its proline-rich region and Zinc fingers, and the histone methyltransferase G9a for histone H3 lysine9 di-methylation (80), for which the Blimp1 zinc fingers are essential to generate the appropriate complex (81). Blimp1 can also activate the expression of some genes via its carboxyl terminal acidic domain (82). Therefore, the Blimp1 protein has a modular structure that can potentially perform diverse functions in a context-dependent manner.
Accordingly, Blimp1 has been shown to be expressed in many cell types during development and subsequently in the adults in several vertebrate species, and in at least some of these species it appears to play a critical role during the establishment of the identity of diverse cells. For example, in the zebrafish, Blimp1 is required for the specification of at least three cell types, i.e., slow-twitch muscle fibers (83) and the common progenitors of the neural crest and sensory neurons (84, 85), the latter two of which, interestingly enough, also require Bmp signaling. In the Xenopus and zebrafish, Blimp1 is expressed in the anterior mesendoderm and prechordal plate, where it is required for regulating various functions of these tissues (86, 87). Notably, germ cell specification in Xenopus and zebrafish is predetermined by the inheritance of germ plasm, and consequently, no defects in germ cell formation have been reported in these organisms in the absence of Blimp1. In mice, Blimp1 is expressed in the analogous anterior visceral endoderm (AVE) and anterior definitive endoderm (ADE)/prechordal plate, and then widely in specific cell types derived from all three germ layers (76, 77, 88). Blimp1-deficient embryos die at around E10.5, but the early axis formation, anterior patterning and neural crest formation proceed normally in these animals. Blimp1 deficiency instead disrupts morphogenesis of the caudal branchial arch, which leads to widespread blood leakage, tissue apoptosis and failure to correctly elaborate the labyrinthine layer of the placenta (76). Furthermore, Blimp1 was shown to play critical roles in multipotent progenitor cell populations in the posterior forelimb, caudal pharyngeal arches, secondary heart field and sensory vibrissae and maintains key signaling centers at these diverse tissue sites (77). In the adult, Blimp1 was shown to define a progenitor population that governs cellular input to the sebaceous gland (89) and to be a critical regulator of keratinocyte transition from the granular to the cornified layer (90). Blimp1 also plays a critical role in controlling T-lymphocyte homeostasis (91, 92).
Interestingly, a recent study has provided evidence that Blimp1 forms a novel complex with an arginine-specific methyltransferase, Prmt5 (93). Prmt5 belongs to the protein arginine methyltransferase family and functions by conferring -NG, N'G-symmetric di-methylation to arginine residues in diverse target proteins, which include histone H4, H2A, and H3, and spliceosomal proteins SmD1, SmD3, and SmB/B' (94). In cultured 293T and P19 embryonal carcinoma cells, transiently overexpressed Blimp1 co-immunoprecipitates Prmt5. Blimp1 and Prmt5 show apparent co-localization in the nuclei of PGCs, at least from E8.5 to E10.5, and the levels of symmetrical di-methylation of histone H4 arginine3 (H4R3me2) in the PGC nuclei seem to be elevated in this period. Notably, at around E11.5, both Blimp1 and Prmt5 translocate from the nucleus to the cytoplasm in PGCs, which coincides with the down-regulation of H4R3me2 in the PGC nuclei. Thus, the Blimp1/Prmt5 complex may play an essential role in maintaining the germ cell lineage during its migration period (93). Whether the Blimp1/Prmt5 complex may play a role in PGC specification remains to be determined.
Remarkably, studies in Drosophila show that the homologue of Prmt5, Dart5/Capsuleen, is essential for the maturation of spermatocytes in males and, notably, for germ cell specification in females (95, 96). As described above, the germ cell fate in Drosophila is determined by "preformation" but not by "epigenesis" as in mammals. Accordingly, embryonic blastomeres that acquire posteriorly localized pole plasm, which contains maternally deposited germ cell determinants, take on the germ cell fate (3, 6). In Dart5/Capsuleen mutants, assembly of the pole plasm, especially the localization of an essential pole plasm component Tudor, is critically impaired (95, 96). The arginine methyltransferase activity of Dart5/Capsuleen seems essential for this process. The spliceosomal Sm proteins are identified as in vivo substrates of symmetric arginine di-methylation by Dart5/Capsuleen. However, the localization of Tudor in the pole plasm and the methylation of the Sm proteins appear to be separable processes (96). Further studies are needed to clarify the mechanism by which Dart5/Capsuleen functions in germ cell specification in Drosophila.
6.3. A molecular program for germ cell specification
The identification of Blimp1 as a gene that marks the lineage-restricted PGC precursors as early as E6.25 and the development of a method for a more quantitative amplification of mRNAs expressed in single cells enabled the measurement of expression dynamics of key genes associated with PGC specification (97). Importantly, it was shown that Blimp1-positive PGC precursors at E6.75 show an expression profile similar to that in their Blimp1-negative somatic neighbors. At E7.25, although the expression of Oct4, a key regulator of pluripotency (7, 98-101), is similar between Blimp1-positive PGCs and their somatic neighbors, Blimp1-positive PGCs specifically express stella and regain expression of Sox2, another essential gene associated with pluripotency (7, 102, 103). This specific re-acquisition of Sox2 would enable the exclusive formation of Oct4-Sox2 complex in PGCs and may herald the regaining of the potential pluripotency in PGCs, which is manifested by their exclusive potential to derive pluripotent embryonic germ (EG) cells in culture (104, 105) (see section 7.3). The Hox genes were found to be repressed as early as E7.25 and the genes such as T (Brachyury) and Fgf8 are repressed later than E7.75 in Blimp1-positive PGCs. Therefore, the transitions from Blimp1-positive PGC precursors to stella-positive PGCs and to more advanced migrating PGCs seem to involve a highly dynamic, stage-dependent transcriptional orchestration that begins with the regaining of the pluripotency-associated gene network, followed by stepwise activation of PGC-specific genes, differential repression of the somatic mesodermal program, as well as potential modulation of signal transduction capacities and unique control of epigenetic regulators (see section 7.4). Future studies involving genome-wide analysis of gene expression dynamics during germ cell specification will provide a more comprehensive view of the events associated with this process.
7. KEY MOLECULES AND EVENTS IN PGCS AFTER THEIR SPECIFICATION
7.1. Migration and proliferation of PGCs
The specified PGCs that form a tight cluster at the base of allantois initiate their migration individually toward future genital ridges at around E7.5 (106, 107). They actively migrate out of the initial cluster into the endoderm epithelium and are incorporated into the forming hindgut diverticulum and embedded in the hindgut epithelium by E8.5. They are highly motile in the hindgut and apparently very rapidly exit it at around after E9.0 to reach the mesentery (108, 109). During the E10.0-E10.5 period, they migrate directionally from the dorsal body wall into the genital ridges (109). A number of mutations have been identified that affect the viability or behavior of the PGCs during their migration period (see section 7.2). However, the precise molecular mechanisms governing the complex PGC migration process remain to be determined.
Classical studies have suggested that, once specified, PGCs proliferate constantly with a doubling time of 16 hours during their migration and colonization of the genital ridges (37, 106). However, a recent study involving a more accurate counting of the PGC number by PGC reporter mice and fluorescent activated cell sorting analysis of the cell cycle of the PGCs showed that from at around E8.0 to E9.0 when PGCs are in the hindgut epithelium, a majority of them (~60%) are arrested at the G2-phase of the cell cycle and proliferate relatively slowly (110) (see also section 7.4.1). Therefore, it appears that Blimp1- and stella-positive PGCs increase their number relatively constantly up to approximately 100 by E8.0, after which most of them enter the G2 arrest of the cell cycle. When PGCs exit from the hindgut epithelium after E9.0, they are released from the G2 arrest and resume rapid proliferation.
- Molecules essential for early PGC development
The mutations of the signaling molecules and Blimp1 directly affect the germ cell specification process itself. There are a number of genes that are known to play critical roles in the early phase of PGC development, presumably after the PGC fate is specified (Table 1).
Classical genetic studies have shown that the Steel and W loci are critical for the migration, proliferation and/or survival of PGCs (111, 112). The Steel locus encodes a secreted ligand, the stem cell factor/Kit ligand (SCF/Kitl), and the W locus encodes its cell surface tyrosine kinase receptor, Kit (113, 114). Detailed studies have shown that in Kit mutants, PGC specification seems apparently normal but the numbers of PGCs do not increase after E8.5 in their migration period (111, 115). The molecular mechanisms through which the SCF-Kit signaling pathway controls early PGC development are still obscure, although in vitro culture experiments and in vivo studies have shown that SCF prevents apoptosis of PGCs (116-119) through the AKT/mTOR/Bax pathway (119-121). Since Kit is expressed in PGCs as early as E7.25 (97, 122), the SCF-Kit signaling may play a role in PGC specification.
The POU domain transcription factor Oct4, the first and the most representative gene shown to be associated with pluripotency (7, 98-101, 123), has been demonstrated to be essential for the survival of PGCs: The germ-cell-specific deletion of Oct4 results in the apoptosis of PGCs (124). This is in contrast to the Oct4 deficiency in pre-implantation embryos, which leads to the loss of the pluripotency in the ICM cells: The Oct4-deficient ICM cells are restricted to differentiation towards TE cells (100). Therefore, the loss of Oct4 function at different developmental stages and in different cell types leads to different biological effects. How precisely Oct4 deficiency leads to apoptosis in PGCs remains to be determined. It is also critical to explore the function of Sox2 in PGC development.
Another classical mutant that is known to affect early PGC development is the ter mutation (125-127). The ter/ter mutation causes germ cell deficiency and a high incidence of congenital testicular teratomas on a 129/SV background (125-127). However, on C57BL/6 and LTXBJ backgrounds, the ter/ter mutation causes only germ cell loss but not germ cell tumors, indicating that the ter gene causes germ cell deficiency singly in both sexes and elevates the incidence of congenital testicular teratomas on a 129/Sv-ter male background (128). Recently, the ter mutation was identified as a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end gene (129, 130). Dnd1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex (131), and shows expression in PGCs as early as E6.75 (97). In ter/ter mutants, the PGC number decreases as early as E8.0, indicating an essential role of Dnd1 in early PGC development. It is of note that a conserved protein is essential for PGC development both in the zebrafish and the mouse, whose germ cell specification depends on preformation and epigenesis modes, respectively. The precise role of this gene product in PGC development remains to be investigated.
nanos3 is a mouse homologue of an evolutionarily conserved Nanos gene, which encodes an RNA-binding protein and plays a critical role in germ cell development and abdominal patterning in Drosophila (132-134). In the absence of maternal Nanos, PGCs fail to migrate into the gonad and do not become functional germ cells (135, 136). In mice, specific expression of nanos3 in PGCs begins as early as E7.25 (97). In the absence of nanos3, PGC specification seems to occur normally but the number of PGCs is reduced after E8.0, and eventually all the PGCs are lost (134). The mechanism of nanos3 function also remains to be elucidated.
The T-cell-restricted intracellular antigen1 (TIA-1)-related protein, TIAR, has also been shown to be critical for the survival of migrating PGCs (137). TIA-1 and TIAR belong to the RNA recognition motif (RRM)/ribonucleoprotein family of RNA-binding proteins (138-140). Under conditions of cellular stress, TIA-1 and TIAR are postulated to function as translational repressors by associating with eIF1, eIF3, and the 40S ribosomal subunit and forming a translationally inactive noncanonical preinitiation complex (140, 141). TIA-1 and TIAR have self-aggregating properties and facilitate the accumulation of the transcriptionally inactive preinitiation complexes into discrete cytoplasmic foci called stress granules (140, 141). However, why and how Tiar mutation specifically leads to the loss of PGCs remains to be clarified.
A recent interesting report has shown that Hypoxia-inducible factor-2 (Hif-2) plays a critical role for PGC specification and/or early PGC development, presumably by regulating Oct4 expression in PGCs (142). Hifs are the primary transcriptional regulators of both cellular and systemic hypoxic adaptation in mammals (143, 144). Hifs are heterodimers consisting of a regulated subunit (Hif) and a constitutive subunit (Hif, also known as Arnt (aryl hydrocarbon nuclear translocator)) and regulate the expression of at least 180 genes involved in metabolism, cell survival, erythropoiesis, and vascular remodeling (143). In Hif-2 mutant mice, the AP-positive PGC number seems severely reduced as early as E8.0 (142). In the future, it will be important to explore the precise role of Hif-2 in PGC specification.
7.3. PGCs can de-differentiate into pluripotent EG cells in vitro
Classically, it has been shown that PGCs are the origin of teratocarcinomas, which contain a range of differentiated cell types derived from three germ layers and a population of undifferentiated embryonic cells, known as embryonal carcinoma (EC) cells (145). It is also important to note that, in the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and basic fibroblast growth factor (bFGF), the specified PGCs (from E8.0 to E13.5) can 'de-differentiate' into ES cell-like pluripotent embryonic germ (EG) cells in culture (104, 105). EG cells can differentiate into multiple cell types in vitro and in vivo, and contribute both to the somatic and the germ cell lineage when they are introduced into blastocysts. These findings have led to the notion that PGCs are potentially pluripotent, although PGCs themselves do not contribute to any tissues when they are transferred into blastocysts (105, 146). It is of interest to explore the mechanism by which PGCs de-differentiate into a pluripotent state in vitro and under disease conditions (see also section 8), and conversely, the mechanism by which they normally prevent themselves from reverting to an overtly pluripotent state. In this regard, recent studies have shown that constitutive activation of the PI3 kinase/Akt signaling pathway in PGCs results in teratoma formation in vivo and enhances EG cell derivation in vitro (147, 148). p53 has been shown to be one of the crucial downstream targets of this signaling pathway in PGCs (148).
7.4. Epigenetic reprogramming in migrating PGCs
7.4.1. Genome-wide epigenetic reprogramming in migrating PGCs
In development, a single zygote generates a myriad of diverse cell types with different gene expression programs. These programs are usually fixed in a stable cellular function through epigenetic mechanisms, including DNA methylation (149), histone tail modifications (150-152) and specific nuclear architecture (153). Thus, each somatic cell type acquires a specific and stable epigenetic signature, referred to as "cellular memory," which is often mitotically heritable. In contrast, the genome of the germ cell lineage, which is the sole pathway to the next generation, must be maintained in an epigenetically reprogrammable state for the new generation to continue to be created.
However, as discussed so far, germ cell fate in mice is induced by "epigenesis" in proximal epiblast cells that are otherwise destined toward somatic mesodermal fates. This implies that cells recruited for the germline may have to undergo "epigenetic reprogramming" from a somatic to a potentially totipotent germline phenotype. Indeed, it has long been known that PGCs undergo profound epigenetic reprogramming, which includes genome-wide DNA demethylation (28, 154), erasure of parental imprints (155, 156), and re-activation of the inactive X-chromosome (157), after they colonize the genital ridges. Recent studies, however, are beginning to reveal that epigenetic reprogramming is an integral part of germ cell specification (110, 158, 159) (Figure 3).
It has been shown that Blimp1-positive lineage restricted PGC precursors at around E6.75 bear genome-wide epigenetic modifications indistinguishable from their somatic mesodermal neighbors, some of which should share common precursors with the germ cell lineage (110). These modifications include both active and repressive modifications of histone H3-i.e., di- and tri-methylation of H3 lysine4 (H3K4me2 and me3) and acetylation of H3 lysine9 (H3K9ac) (active modifications), mono-, di-, and tri-methylation of H3 lysine9 (H3K9me1, me2, and me3) (repressive modifications), and di- and tri-methylation of H3 lysine 27 (H3K27me2 and me3) (repressive modifications) (110). Therefore, PGCs may not possess specific epigenetic signatures at their outset, although there could be yet-to-be-discovered histone and chromatin modifications that are unique to the PGC precursors.
Subsequently, however, from around E8.0 onwards, PGCs that have started their migration begin to show the genome-wide reduction of the two major repressive modifications, DNA methylation and H3K9me2 (110, 158). The global reduction of H3K9me2 in migrating PGCs seems to occur in a progressive, cell-by-cell manner and by E8.75, nearly all the PGCs show low H3K9me2 levels. Cell cycle analysis of the migrating PGCs indicated that a majority of them (~60%) are in the G2 phase from around E8.0 to around E9.0 (110). Interestingly, concomitant with this period, PGCs seem to transiently pause their global transcription by RNA polymerase II (RNAPII) (110). These observations suggest that the genome-wide reduction of the repressive modifications progresses when the PGCs are arrested in the G2 phase, during which they simultaneously shut off their RNAPII-dependent transcription. Therefore, genome-wide reductions of DNA methylation and H3K9me2 in migrating PGCs are considered to involve active processes.
Gene and protein expression analysis has shown that PGCs repress critical de novo DNA methyltransferases, Dnmt3a and 3b, as early as E7.25 (97, 158), but continue to express a maintenance methyltransferase, Dnmt1, at least at the mRNA level. They also repress, at around E7.75, the histone lysine methyltransferase Glp, which is essential for conferring genome-wide H3K9me1 and me2 in embryonic development (110, 160). Single-cell gene expression analysis of the JmjC domain-protein family, which consists of ~30 proteins across the genome and can demethylate all the methylation states (mono-, di-, and tri-) of H3K4, H3K9, H3K27, and H3K36 (161, 162), has shown that Jhdm2a (Jmjd1a), which encodes H3K9me1 and 2 demethylase, is indeed expressed in migrating PGCs (110). However, its expression is similarly seen in somatic neighbors. These findings suggest that the specific repression of Glp in PGCs may lead to the loss of H3K9 methyltransferase activity in PGCs, which triggers the genome-wide reduction of H3K9me2, either through a turnover of methyl groups or a replacement of the entire H3 molecule with an unmodified H3 molecule. Consistently, a report involving a mass spectrometry analysis of H3K9 methyl groups differentially pulse-labeled by a heavy isotope demonstrated that H3K9me2 turns over without replication (163). Furthermore, it was also reported that chromatin-associated histones and non-chromatin-associated histones are continually exchanged in Xenopus oocytes, and that the maintenance of H3K9me2 at a specific site requires the continual presence of an H3K9 histone methyltransferase (164). Thus, repression of an essential enzyme may shift the equilibrium between methylation and demethylation toward demethylation, leading to the erasure of H3K9me2 in migrating PGCs.
Recently, a loss-of-function experiment for Jhdm2a has been reported (165). Jhdm2a is shown to be essential for spermatogenesis. Jhdm2a functions to remove H3K9me1/2 on the promoters of the genes for transition nuclear protein 1 and protamine 1, which is essential for their proper expression and in turn for packaging and condensation of sperm chromatin. Whether or not Jhdm2a plays a role in the genome-wide reduction of H3K9me2 in PGCs remains to be investigated.
Following the genome-wide loss of DNA methylation and H3K9me2, genome-wide H3K27me3, another repressive modification mediated by the polycomb repressive complex 2 (PRC2), becomes up-regulated in migrating PGCs at around E8.25 onwards (110, 158). Both the percentage of PGCs with high levels of H3K27me3 and the degree of H3K27me3 up-regulation in individual PGCs increase as the development proceeds, indicating that, as in the case for the demethylation of H3K9me2, H3K27me3 up-regulation proceeds progressively, in a cell-by-cell manner.
PRC2 consists of three core components, Ezh2, Eed and Suz12 (166, 167). Single-cell gene expression analysis has shown that genes for these three core components are expressed at similar levels both in the PGCs and their somatic neighbors by at least E7.75 (97). Considering the fact that PGCs globally shut off RNAP II-dependent transcription after around E8.0, the up-regulation of genome-wide H3K27me3 in PGCs after E8.25 may not depend on the de novo transcription of some specific factors. It was reported that in Suv39h-deficient cells, which essentially lack H3K9me3 at the pericentromeric heterochromatin, H3K27me3 is ectopically up-regulated in this location, which suggests that there is "crosstalk" between H3K27 methylation and H3K9 methylation, which rescues the de-regulated chromatin structure (168). A similar system may operate in migrating PGCs, in which hypo-methylated regions for H3K9me2 might be "sensed and rescued" by H3K27me3.
7.4.2. Potential significance of epigenetic reprogramming in migrating PGCs
What could be the potential biological significance of epigenetic reprogramming in migrating PGCs? Essentially, migrating PGCs convert their genome-wide repressive modifications from DNA methylation and H3K9me2 to H3K27me3 (Figure 3). It has been reported that DNA methylation and H3K9me2 are critical for the stable maintenance of the repressed genes (169, 170). In contrast, PRC2-mediated H3K27me3 is, for example, enriched in pluripotent ES cells and functions to repress developmentally regulated lineage-specific genes (171, 172). Many of these developmentally regulated genes bear bi-valent modifications with H3K27me3 and H3K4me3, which ensure that these repressed genes can be expressed quickly upon the induction of differentiation.
DNA methylation and H3K9me2 are therefore considered to play critical roles in stably maintaining the repressed state of unused genes during cell fate specification, whereas H3K27me3 may participate in a more plastic repression of the lineage-specific genes in pluripotent cells. Since PGCs bear significant levels of genome-wide H3K4me3, specific up-regulation of H3K27me3 in PGCs may contribute to the creation of an ES cell-like genome organization. Indeed, it is possible to derive pluripotent EG cells from PGCs (104, 105) (see section 7.3). This fact has been the basis for the notion that PGCs possess potential pluripotency. Epigenetic reprogramming and specific expression/re-acquisition of the key pluripotency genes Oct3/4, Sox2, and Nanog in PGCs (see section 6.4) may thus be critical for their potential pluripotency (97, 173). It might be the case that genes such as Blimp1 are important to prevent PGCs from overtly reverting to a pluripotent state in vivo. To understand the significance of genome-wide epigenetic reprogramming in migrating PGCs, it is critical to determine the target sequences from which DNA methylation and H3K9me2 are removed and upon which H3K27me3 is imposed.
Soon after PGCs enter the genital ridges, they undergo further DNA demethylation, including the erasure of parental imprints between E9.5 and E12.5 (156). Repetitive elements such as IAP (intracisternal A particle elements, classified as LTRs, ~ 1,000 copies per mouse genome) and LINE1 (the autonomous long interspersed nucleotide element-like element, ~10,000-100,000 copies per genome) are highly methylated in migrating PGCs and somatic cells. Post-migrating PGCs demethylate the CpG methylation on LINE1 elements, while most of the CpG cites on the IAP elements remain highly methylated (156, 174). The female PGCs, which initially undergo random X-chromosome inactivation as in the somatic cells, reactivate the inactive X (157), which most likely reflects the global epigenetic reprogramming process in gonadal PGCs. A recent report showed that X reactivation may start in PGCs as early as E7.75 (159). The precise analysis of genome-wide epigenetic reprogramming in migrating PGCs will be important for understanding the events occurring in PGCs in the genital ridges.
- PGC SPECIFICATION/DEVELOPMENT AND INDUCED PLURIPOTENCY IN SOMATIC CELLS
Remarkably, a series of recent studies has shown that retroviral transduction of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, can reprogram adult mouse fibroblast cells to an undifferentiated state similar to the state of ES cells, albeit at a low frequency (~1/104-~1/105 per transduced cells), and these cells have been termed induced pluripotent stem (iPS) cells (7-9). Subsequently, human iPS cells were also generated using two different sets of transcription factors (OCT4, SOX2, KLF4 with or without c-MYC, or OCT4, SOX2, NANOG, LIN28) (10-12, 175), creating an unprecedented opportunity to produce patient-specific stem cells for the study of the diseased state in culture. Moreover, iPS cells were shown to be generated by direct reprogramming of lineage-committed somatic cells (hepatocytes and gastric epithelial cells), and retroviral integration into specific sites was not required (176).
As has been discussed, PGC specification involves re-activation of Sox2 and maintains Oct4 and Nanog. Consequently, the specified PGCs are the only cells that express all three of the key pluripotency regulators Oct4, Sox2 and Nanog after gastrulation, and they also express Klfs and N-Myc (M.S., unpublished observation), although they repress c-Myc upon their specification (97). Accordingly, PGCs, which are basically unipotent in vivo, can be reprogrammed into pluripotent EG cells in the presence of LIF, SCF, and bFGF at a ratio of approximately one EG cell colony generated from one out of ~50 PGCs ( (104, 105, 148) and M.S., unpublished observation). This ratio is clearly higher than the ratio of iPS cell induction from somatic cells, although this depends considerably on their developmental stages (104, 105, 148).
These two lines of evidences raise a question as to how a cell that expresses these key genes becomes a pluripotent stem cell. Although the precise mechanisms of iPS cell induction, which appear to require at least several cell division cycles, are currently unknown, it is conceivable that the epigenetic state of a cell regulates the phenotype conferred by the transcriptional activity of these potential pluripotency inducing factors. Under normal ES cell conditions, Oct4, Sox2, and Nanog co-occupy many target genes, many of which are developmentally important transcriptional regulators (172, 177). The genome-wide epigenetic reprogramming that seems integral to and ensues PGC specification may thus create a more permissive state for the generation of pluripotent stem cells under an appropriate condition. Further studies on germ cell development and the mechanisms of iPS cell induction would lead to a more integrated understanding of the potency of a cell in general.
9. PERSPECTIVE
In the last decade, our understanding of the mechanism for the specification of the germ cell lineage in mice has considerably increased at the molecular level. However, there are still many gaps in our knowledge. Specifically, for example, do the signaling molecules for PGC specification directly up-regulate the expression of Blimp1? Why does only a subset of proximal epiblast cells acquire germ cell fate? What is the mechanism of Bmp8b function? Are there any other key transcriptional regulators critical for PGC specification? How precisely do PGCs undergo genome-wide epigenetic reprogramming? Does epigenetic reprogramming in migrating PGCs have any implications in further reprogramming of PGCs in the genital ridges, including erasure of parental imprints? Studies specifically targeting each of these questions will be needed for a conceptually more integrated and a more comprehensive understanding of the PGC specification in mice. Such endeavors will lead to a more precise reconstruction of germ cell fate in vitro, which will have a profound impact on reproductive and regenerative medicine.
10. ACKNOWLEDGEMENTS
I thank all the members of our laboratory for their discussion of this study. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a PRESTO project grant from the Japan Science and Technology Agency.
11. REFERENCES
1. Extavour C. G., M. Akam: Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130, 5869-5884 (2003)
doi:10.1242/dev.00804
2. Johnson A. D., B. Crother, M. E. White, R. Patient, R. F. Bachvarova, M. Drum, T. Masi: Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage. Philos Trans R Soc Lond B Biol Sci 358, 1371-1379 (2003)
doi:10.1098/rstb.2003.1331
3. Seydoux G., R. E. Braun: Pathway to totipotency: lessons from germ cells. Cell 127, 891-904 (2006)
doi:10.1016/j.cell.2006.11.016
4. Ohinata Y., Y. Seki, B. Payer, D. O'Carroll, M. A. Surani, M. Saitou: Germline recruitment in mice: a genetic program for epigenetic reprogramming. Ernst Schering Res Found Workshop, 143-174 (2006)
5. Hayashi K., S. M. de Sousa Lopes, M. A. Surani: Germ cell specification in mice. Science 316, 394-396 (2007)
doi:10.1126/science.1137545
6. Strome S., R. Lehmann: Germ versus soma decisions: lessons from flies and worms. Science 316, 392-393 (2007)
doi:10.1126/science.1140846
7. Takahashi K., S. Yamanaka: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006)
doi:10.1016/j.cell.2006.07.024
8. Okita K., T. Ichisaka, S. Yamanaka: Generation of germline-competent induced pluripotent stem cells. Nature 448, 313-317 (2007)
doi:10.1038/nature05934
9. Wernig M., A. Meissner, R. Foreman, T. Brambrink, M. Ku, K. Hochedlinger, B. E. Bernstein, R. Jaenisch: In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 (2007)
doi:10.1038/nature05944
10. Takahashi K., K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, S. Yamanaka: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007)
doi:10.1016/j.cell.2007.11.019
11. Yu J., M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, Slukvin, II, J. A. Thomson: Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 (2007)
doi:10.1126/science.1151526
12. Park I. H., R. Zhao, J. A. West, A. Yabuuchi, H. Huo, T. A. Ince, P. H. Lerou, M. W. Lensch, G. Q. Daley: Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141-146 (2008)
doi:10.1038/nature06534
13. Mayer W., A. Niveleau, J. Walter, R. Fundele, T. Haaf: Demethylation of the zygotic paternal genome. Nature 403, 501-502. (2000)
doi:10.1038/35000656
14. Oswald J., S. Engemann, N. Lane, W. Mayer, A. Olek, R. Fundele, W. Dean, W. Reik, J. Walter: Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10, 475-478 (2000)
doi:10.1016/S0960-9822(00)00448-6
15. Morgan H. D., F. Santos, K. Green, W. Dean, W. Reik: Epigenetic reprogramming in mammals. Hum Mol Genet 14 Spec No 1, R47-58 (2005)
16. Reik W.: Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425-432 (2007)
doi:10.1038/nature05918
17. Dean W., F. Santos, M. Stojkovic, V. Zakhartchenko, J. Walter, E. Wolf, W. Reik: Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 98, 13734-13738 (2001)
doi:10.1073/pnas.241522698
18. Santos F., A. H. Peters, A. P. Otte, W. Reik, W. Dean: Dynamic chromatin modifications characterise the first cell cycle in mouse embryos. Dev Biol 280, 225-236 (2005)
doi:10.1016/j.ydbio.2005.01.025
19. Hamatani T., M. G. Carter, A. A. Sharov, M. S. Ko: Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell 6, 117-131 (2004)
doi:10.1016/S1534-5807(03)00373-3
20. Evsikov A. V., J. H. Graber, J. M. Brockman, A. Hampl, A. E. Holbrook, P. Singh, J. J. Eppig, D. Solter, B. B. Knowles: Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo. Genes Dev 20, 2713-2727 (2006)
doi:10.1101/gad.1471006
21. Johnson M. H., C. A. Ziomek: The foundation of two distinct cell lineages within the mouse morula. Cell 24, 71-80 (1981)
doi:10.1016/0092-8674(81)90502-X
22. Strumpf D., C. A. Mao, Y. Yamanaka, A. Ralston, K. Chawengsaksophak, F. Beck, J. Rossant: Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132, 2093-2102 (2005)
doi:10.1242/dev.01801
23. Niwa H., Y. Toyooka, D. Shimosato, D. Strumpf, K. Takahashi, R. Yagi, J. Rossant: Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917-929 (2005)
doi:10.1016/j.cell.2005.08.040
24. Yagi R., M. J. Kohn, I. Karavanova, K. J. Kaneko, D. Vullhorst, M. L. Depamphilis, A. Buonanno: Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 134, 3827-3836 (2007)
doi:10.1242/dev.010223
25. Nishioka Y., S. Yamamoto, H. Kiyonari, H. Sato, A. Sawada, M. Ota, K. Nakao, H. Sasaki: Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech Dev doi:10.1016/j.mod.2007.11.002 (2008)
doi:10.1016/j.mod.2007.11.002
26. Gardner R. L., J. Rossant: Investigation of the fate of 4-5 day post-coitum mouse inner cell mass cells by blastocyst injection. J Embryol Exp Morphol 52, 141-152 (1979)
27. Gardner R. L., M. F. Lyon, E. P. Evans, M. D. Burtenshaw: Clonal analysis of X-chromosome inactivation and the origin of the germ line in the mouse embryo. J Embryol Exp Morphol 88, 349-363 (1985)
28. Monk M., M. Boubelik, S. Lehnert: Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371-382 (1987)
29. Rougier N., D. Bourc'his, D. M. Gomes, A. Niveleau, M. Plachot, A. Paldi, E. Viegas-Pequignot: Chromosome methylation patterns during mammalian preimplantation development. Genes Dev 12, 2108-2113 (1998)
doi:10.1101/gad.12.14.2108
30. Okano M., D. W. Bell, D. A. Haber, E. Li: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257 (1999)
doi:10.1016/S0092-8674(00)81656-6
31. Li E.: Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3, 662-673 (2002)
doi:10.1038/nrg887
32. Liu H., J. M. Kim, F. Aoki: Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos. Development 131, 2269-2280 (2004)
doi:10.1242/dev.01116
33. Tam P. P., D. A. Loebel: Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet 8, 368-381 (2007)
doi:10.1038/nrg2084
34. Coucouvanis E., G. R. Martin: Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83, 279-287 (1995)
doi:10.1016/0092-8674(95)90169-8
35. Chiquoine A. D.: The identification, origin and migration of the primordial germ cells in the mouse embryo. Anat. Rec. 118, 135-146 (1954)
doi:10.1002/ar.1091180202
36. Ginsburg M., M. H. Snow, A. McLaren: Primordial germ cells in the mouse embryo during gastrulation. Development 110, 521-528. (1990)
37. Lawson K. A., W. J. Hage: Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found Symp 182, 68-84 (1994)
doi:10.1002/9780470514573.ch5
38. Tam P. P., S. X. Zhou: The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Dev Biol 178, 124-132. (1996)
doi:10.1006/dbio.1996.0203
39. Zhao G. Q.: Consequences of knocking out BMP signaling in the mouse. Genesis 35, 43-56 (2003)
doi:10.1002/gene.10167
40. Chang H., C. W. Brown, M. M. Matzuk: Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr Rev 23, 787-823 (2002)
doi:10.1210/er.2002-0003
41. Lawson K. A., N. R. Dunn, B. A. Roelen, L. M. Zeinstra, A. M. Davis, C. V. Wright, J. P. Korving, B. L. Hogan: Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 13, 424-436. (1999)
doi:10.1101/gad.13.4.424
42. Fujiwara T., N. R. Dunn, B. L. Hogan: Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse. Proc Natl Acad Sci U S A 98, 13739-13744 (2001)
doi:10.1073/pnas.241508898
43. Ying Y., X. M. Liu, A. Marble, K. A. Lawson, G. Q. Zhao: Requirement of Bmp8b for the generation of primordial germ cells in the mouse. Mol Endocrinol 14, 1053-1063 (2000)
doi:10.1210/me.14.7.1053
44. Ying Y., G. Q. Zhao: Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev Biol 232, 484-492 (2001)
doi:10.1006/dbio.2001.0173
45. Shi Y., J. Massague: Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685-700 (2003)
doi:10.1016/S0092-8674(03)00432-X
46. Massague J., J. Seoane, D. Wotton: Smad transcription factors. Genes Dev 19, 2783-2810 (2005)
doi:10.1101/gad.1350705
47. Beppu H., M. Kawabata, T. Hamamoto, A. Chytil, O. Minowa, T. Noda, K. Miyazono: BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev Biol 221, 249-258 (2000)
doi:10.1006/dbio.2000.9670
48. Mishina Y., A. Suzuki, N. Ueno, R. R. Behringer: Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev 9, 3027-3037 (1995)
doi:10.1101/gad.9.24.3027
49. Gu Z., E. M. Reynolds, J. Song, H. Lei, A. Feijen, L. Yu, W. He, D. T. MacLaughlin, J. van den Eijnden-van Raaij, P. K. Donahoe, E. Li: The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo. Development 126, 2551-2561 (1999)
50. Mishina Y., R. Crombie, A. Bradley, R. R. Behringer: Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis. Dev Biol 213, 314-326 (1999)
doi:10.1006/dbio.1999.9378
51. de Sousa Lopes S. M., B. A. Roelen, R. M. Monteiro, R. Emmens, H. Y. Lin, E. Li, K. A. Lawson, C. L. Mummery: BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes Dev 18, 1838-1849 (2004)
doi:10.1101/gad.294004
52. Tremblay K. D., N. R. Dunn, E. J. Robertson: Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development 128, 3609-3621 (2001)
53. Chang H., M. M. Matzuk: Smad5 is required for mouse primordial germ cell development. Mech Dev 104, 61-67 (2001)
doi:10.1016/S0925-4773(01)00367-7
54. Hayashi K., T. Kobayashi, T. Umino, R. Goitsuka, Y. Matsui, D. Kitamura: SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast. Mech Dev 118, 99-109 (2002)
doi:10.1016/S0925-4773(02)00237-X
55. Arnold S. J., S. Maretto, A. Islam, E. K. Bikoff, E. J. Robertson: Dose-dependent Smad1, Smad5 and Smad8 signaling in the early mouse embryo. Dev Biol 296, 104-118 (2006)
doi:10.1016/j.ydbio.2006.04.442
56. Chu G. C., N. R. Dunn, D. C. Anderson, L. Oxburgh, E. J. Robertson: Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo. Development 131, 3501-3512 (2004)
doi:10.1242/dev.01248
57. Aubin J., A. Davy, P. Soriano: In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis. Genes Dev 18, 1482-1494 (2004)
doi:10.1101/gad.1202604
58. Sapkota G., C. Alarcon, F. M. Spagnoli, A. H. Brivanlou, J. Massague: Balancing BMP signaling through integrated inputs into the Smad1 linker. Mol Cell 25, 441-454 (2007)
doi:10.1016/j.molcel.2007.01.006
59. Fuentealba L. C., E. Eivers, A. Ikeda, C. Hurtado, H. Kuroda, E. M. Pera, E. M. De Robertis: Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell 131, 980-993 (2007)
doi:10.1016/j.cell.2007.09.027
60. Waldrip W. R., E. K. Bikoff, P. A. Hoodless, J. L. Wrana, E. J. Robertson: Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo. Cell 92, 797-808 (1998)
doi:10.1016/S0092-8674(00)81407-5
61. Nomura M., E. Li: Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393, 786-790 (1998)
doi:10.1038/31693
62. Beddington R. S., E. J. Robertson: Axis development and early asymmetry in mammals. Cell 96, 195-209 (1999)
doi:10.1016/S0092-8674(00)80560-7
63. Saitou M., S. C. Barton, M. A. Surani: A molecular programme for the specification of germ cell fate in mice. Nature 418, 293-300 (2002)
doi:10.1038/nature00927
64. Tanaka S. S., Y. Matsui: Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells. Mech Dev 119 Suppl 1, S261-267 (2002)
doi:10.1016/S0925-4773(03)00126-6
65. Tanaka S. S., Y. L. Yamaguchi, B. Tsoi, H. Lickert, P. P. Tam: IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion. Dev Cell 9, 745-756 (2005)
doi:10.1016/j.devcel.2005.10.010
66. Sato M., T. Kimura, K. Kurokawa, Y. Fujita, K. Abe, M. Masuhara, T. Yasunaga, A. Ryo, M. Yamamoto, T. Nakano: Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech Dev 113, 91-94. (2002)
doi:10.1016/S0925-4773(02)00002-3
67. Bortvin A., K. Eggan, H. Skaletsky, H. Akutsu, D. L. Berry, R. Yanagimachi, D. C. Page, R. Jaenisch: Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673-1680 (2003)
doi:10.1242/dev.00366
68. Payer B., M. Saitou, S. C. Barton, R. Thresher, J. P. Dixon, D. Zahn, W. H. Colledge, M. B. Carlton, T. Nakano, M. A. Surani: Stella is a maternal effect gene required for normal early development in mice. Curr Biol 13, 2110-2117 (2003)
doi:10.1016/j.cub.2003.11.026
69. Nakamura T., Y. Arai, H. Umehara, M. Masuhara, T. Kimura, H. Taniguchi, T. Sekimoto, M. Ikawa, Y. Yoneda, M. Okabe, S. Tanaka, K. Shiota, T. Nakano: PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol 9, 64-71 (2007)
doi:10.1038/ncb1519
70. Ohinata Y., B. Payer, D. O'Carroll, K. Ancelin, Y. Ono, M. Sano, S. C. Barton, T. Obukhanych, M. Nussenzweig, A. Tarakhovsky, M. Saitou, M. A. Surani: Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436, 207-213 (2005)
doi:10.1038/nature03813
71. Turner C. A., Jr., D. H. Mack, M. M. Davis: Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77, 297-306 (1994)
doi:10.1016/0092-8674(94)90321-2
72. Shaffer A. L., K. I. Lin, T. C. Kuo, X. Yu, E. M. Hurt, A. Rosenwald, J. M. Giltnane, L. Yang, H. Zhao, K. Calame, L. M. Staudt: Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 17, 51-62 (2002)
doi:10.1016/S1074-7613(02)00335-7
73. Shapiro-Shelef M., K. I. Lin, L. J. McHeyzer-Williams, J. Liao, M. G. McHeyzer-Williams, K. Calame: Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19, 607-620 (2003)
doi:10.1016/S1074-7613(03)00267-X
74. Calame K. L., K. I. Lin, C. Tunyaplin: Regulatory mechanisms that determine the development and function of plasma cells. Annu Rev Immunol 21, 205-230 (2003)
doi:10.1146/annurev.immunol.21.120601.141138
75. Saitou M., B. Payer, D. O'Carroll, Y. Ohinata, M. A. Surani: Blimp1 and the emergence of the germ line during development in the mouse. Cell Cycle 4, 1736-1740 (2005)
76. Vincent S. D., N. R. Dunn, R. Sciammas, M. Shapiro-Shalef, M. M. Davis, K. Calame, E. K. Bikoff, E. J. Robertson: The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development 132, 1315-1325 (2005)
doi:10.1242/dev.01711
77. Robertson E. J., I. Charatsi, C. J. Joyner, C. H. Koonce, M. Morgan, A. Islam, C. Paterson, E. Lejsek, S. J. Arnold, A. Kallies, S. L. Nutt, E. K. Bikoff: Blimp1 regulates development of the posterior forelimb, caudal pharyngeal arches, heart and sensory vibrissae in mice. Development 134, 4335-4345 (2007)
doi:10.1242/dev.012047
78. Ren B., K. J. Chee, T. H. Kim, T. Maniatis: PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins. Genes Dev 13, 125-137 (1999)
doi:10.1101/gad.13.1.125
79. Yu J., C. Angelin-Duclos, J. Greenwood, J. Liao, K. Calame: Transcriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol Cell Biol 20, 2592-2603 (2000)
doi:10.1128/MCB.20.7.2592-2603.2000
80. Tachibana M., K. Sugimoto, M. Nozaki, J. Ueda, T. Ohta, M. Ohki, M. Fukuda, N. Takeda, H. Niida, H. Kato, Y. Shinkai: G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 16, 1779-1791 (2002)
doi:10.1101/gad.989402
81. Gyory I., J. Wu, G. Fejer, E. Seto, K. L. Wright: PRDI-BF1 recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat Immunol 5, 299-308 (2004)
doi:10.1038/ni1046
82. Sciammas R., M. M. Davis: Modular nature of Blimp-1 in the regulation of gene expression during B cell maturation. J Immunol 172, 5427-5440 (2004)
83. Baxendale S., C. Davison, C. Muxworthy, C. Wolff, P. W. Ingham, S. Roy: The B-cell maturation factor Blimp-1 specifies vertebrate slow-twitch muscle fiber identity in response to Hedgehog signaling. Nat Genet 36, 88-93 (2004)
doi:10.1038/ng1280
84. Roy S., T. Ng: Blimp-1 specifies neural crest and sensory neuron progenitors in the zebrafish embryo. Curr Biol 14, 1772-1777 (2004)
doi:10.1016/j.cub.2004.09.046
85. Hernandez-Lagunas L., I. F. Choi, T. Kaji, P. Simpson, C. Hershey, Y. Zhou, L. Zon, M. Mercola, K. B. Artinger: Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification. Dev Biol 278, 347-357 (2005)
doi:10.1016/j.ydbio.2004.11.014
86. de Souza F. S., V. Gawantka, A. P. Gomez, H. Delius, S. L. Ang, C. Niehrs: The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate in Spemann's organizer. Embo J 18, 6062-6072 (1999)
doi:10.1093/emboj/18.21.6062
87. Wilm T. P., L. Solnica-Krezel: Essential roles of a zebrafish prdm1/blimp1 homolog in embryo patterning and organogenesis. Development 132, 393-404 (2005)
doi:10.1242/dev.01572
88. Chang D. H., G. Cattoretti, K. L. Calame: The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development. Mech Dev 117, 305-309 (2002)
doi:10.1016/S0925-4773(02)00189-2
89. Horsley V., D. O'Carroll, R. Tooze, Y. Ohinata, M. Saitou, T. Obukhanych, M. Nussenzweig, A. Tarakhovsky, E. Fuchs: Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126, 597-609 (2006)
doi:10.1016/j.cell.2006.06.048
90. Magnusdottir E., S. Kalachikov, K. Mizukoshi, D. Savitsky, A. Ishida-Yamamoto, A. A. Panteleyev, K. Calame: Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1. Proc Natl Acad Sci U S A 104, 14988-14993 (2007)
doi:10.1073/pnas.0707323104
91. Kallies A., E. D. Hawkins, G. T. Belz, D. Metcalf, M. Hommel, L. M. Corcoran, P. D. Hodgkin, S. L. Nutt: Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance. Nat Immunol 7, 466-474 (2006)
doi:10.1038/ni1321
92. Martins G. A., L. Cimmino, M. Shapiro-Shelef, M. Szabolcs, A. Herron, E. Magnusdottir, K. Calame: Transcriptional repressor Blimp-1 regulates T cell homeostasis and function. Nat Immunol 7, 457-465 (2006)
doi:10.1038/ni1320
93. Ancelin K., U. C. Lange, P. Hajkova, R. Schneider, A. J. Bannister, T. Kouzarides, M. A. Surani: Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat Cell Biol 8, 623-630 (2006)
doi:10.1038/ncb1413
94. Bedford M. T., S. Richard: Arginine methylation an emerging regulator of protein function. Mol Cell 18, 263-272 (2005)
doi:10.1016/j.molcel.2005.04.003
95. Gonsalvez G. B., T. K. Rajendra, L. Tian, A. G. Matera: The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance. Curr Biol 16, 1077-1089 (2006)
doi:10.1016/j.cub.2006.04.037
96. Anne J., R. Ollo, A. Ephrussi, B. M. Mechler: Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134, 137-146 (2007)
doi:10.1242/dev.02687
97. Yabuta Y., K. Kurimoto, Y. Ohinata, Y. Seki, M. Saitou: Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling. Biol Reprod 75, 705-716 (2006)
doi:10.1095/biolreprod.106.053686
98. Scholer H. R., G. R. Dressler, R. Balling, H. Rohdewohld, P. Gruss: Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. Embo J 9, 2185-2195 (1990)
99. Okazawa H., K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, H. Hamada: The oct3 gene, a gene for an embryonic transcription factor, is controlled by a retinoic acid repressible enhancer. Embo J 10, 2997-3005 (1991)
100. Nichols J., B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Scholer, A. Smith: Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379-391. (1998)
doi:10.1016/S0092-8674(00)81769-9
101. Niwa H., J. Miyazaki, A. G. Smith: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-376 (2000)
doi:10.1038/74199
102. Avilion A. A., S. K. Nicolis, L. H. Pevny, L. Perez, N. Vivian, R. Lovell-Badge: Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17, 126-140 (2003)
doi:10.1101/gad.224503
103. Masui S., Y. Nakatake, Y. Toyooka, D. Shimosato, R. Yagi, K. Takahashi, H. Okochi, A. Okuda, R. Matoba, A. A. Sharov, M. S. Ko, H. Niwa: Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9, 625-635 (2007)
doi:10.1038/ncb1589
104. Matsui Y., K. Zsebo, B. L. Hogan: Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841-847 (1992)
doi:10.1016/0092-8674(92)90317-6
105. Labosky P. A., D. P. Barlow, B. L. Hogan: Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 120, 3197-3204 (1994)
106. Tam P. P., M. H. Snow: Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. J Embryol Exp Morphol 64, 133-147 (1981)
107. Anderson R., T. K. Copeland, H. Scholer, J. Heasman, C. Wylie: The onset of germ cell migration in the mouse embryo. Mech Dev 91, 61-68 (2000)
doi:10.1016/S0925-4773(99)00271-3
108. Gomperts M., M. Garcia-Castro, C. Wylie, J. Heasman: Interactions between primordial germ cells play a role in their migration in mouse embryos. Development 120, 135-141 (1994)
109. Molyneaux K. A., J. Stallock, K. Schaible, C. Wylie: Time-lapse analysis of living mouse germ cell migration. Dev Biol 240, 488-498 (2001)
doi:10.1006/dbio.2001.0436
110. Seki Y., M. Yamaji, Y. Yabuta, M. Sano, M. Shigeta, Y. Matsui, Y. Saga, M. Tachibana, Y. Shinkai, M. Saitou: Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice. Development (2007)
111. Mintz B., E. S. Russell: Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp Zool 134, 207-237 (1957)
doi:10.1002/jez.1401340202
112. McCoshen J. A., D. J. McCallion: A study of the primordial germ cells during their migratory phase in Steel mutant mice. Experientia 31, 589-590 (1975)
doi:10.1007/BF01932475
113. Bernstein A., B. Chabot, P. Dubreuil, A. Reith, K. Nocka, S. Majumder, P. Ray, P. Besmer: The mouse W/c-kit locus. Ciba Found Symp 148, 158-166; discussion 166-172 (1990)
114. Besmer P., K. Manova, R. Duttlinger, E. J. Huang, A. Packer, C. Gyssler, R. F. Bachvarova: The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev Suppl, 125-137 (1993)
115. Buehr M., A. McLaren, A. Bartley, S. Darling: Proliferation and migration of primordial germ cells in We/We mouse embryos. Dev Dyn 198, 182-189 (1993)
116. Dolci S., D. E. Williams, M. K. Ernst, J. L. Resnick, C. I. Brannan, L. F. Lock, S. D. Lyman, H. S. Boswell, P. J. Donovan: Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 352, 809-811 (1991)
doi:10.1038/352809a0
117. Matsui Y., D. Toksoz, S. Nishikawa, D. Williams, K. Zsebo, B. L. Hogan: Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 353, 750-752 (1991)
doi:10.1038/353750a0
118. Pesce M., M. G. Farrace, M. Piacentini, S. Dolci, M. De Felici: Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development 118, 1089-1094 (1993)
119. Runyan C., K. Schaible, K. Molyneaux, Z. Wang, L. Levin, C. Wylie: Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration. Development 133, 4861-4869 (2006)
doi:10.1242/dev.02688
120. De Miguel M. P., L. Cheng, E. C. Holland, M. J. Federspiel, P. J. Donovan: Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer. Proc Natl Acad Sci U S A 99, 10458-10463 (2002)
doi:10.1073/pnas.122249399
121. Stallock J., K. Molyneaux, K. Schaible, C. M. Knudson, C. Wylie: The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo. Development 130, 6589-6597 (2003)
doi:10.1242/dev.00898
122. Manova K., R. F. Bachvarova: Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts. Dev Biol 146, 312-324 (1991)
doi:10.1016/0012-1606(91)90233-S
123. Yeom Y. I., G. Fuhrmann, C. E. Ovitt, A. Brehm, K. Ohbo, M. Gross, K. Hubner, H. R. Scholer: Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881-894. (1996)
124. Kehler J., E. Tolkunova, B. Koschorz, M. Pesce, L. Gentile, M. Boiani, H. Lomeli, A. Nagy, K. J. McLaughlin, H. R. Scholer, A. Tomilin: Oct4 is required for primordial germ cell survival. EMBO Rep 5, 1078-1083 (2004)
doi:10.1038/sj.embor.7400279
125. Stevens L. C.: A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. J Natl Cancer Inst 50, 235-242 (1973)
126. Stevens L. C.: Spontaneous and experimentally induced testicular teratomas in mice. Cell Differ 15, 69-74 (1984)
doi:10.1016/0045-6039(84)90054-X
127. Noguchi T., M. Noguchi: A recessive mutation (ter) causing germ cell deficiency and a high incidence of congenital testicular teratomas in 129/Sv-ter mice. J Natl Cancer Inst 75, 385-392 (1985)
128. Sakurai T., Iguchi, T., Moriwaki, K., and Noguchi, M.: The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation. Develop. Growth Differ. 37, 293-302 (1995)
doi:10.1046/j.1440-169X.1995.t01-2-00007.x
129. Weidinger G., J. Stebler, K. Slanchev, K. Dumstrei, C. Wise, R. Lovell-Badge, C. Thisse, B. Thisse, E. Raz: dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13, 1429-1434 (2003)
doi:10.1016/S0960-9822(03)00537-2
130. Youngren K. K., D. Coveney, X. Peng, C. Bhattacharya, L. S. Schmidt, M. L. Nickerson, B. T. Lamb, J. M. Deng, R. R. Behringer, B. Capel, E. M. Rubin, J. H. Nadeau, A. Matin: The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours. Nature 435, 360-364 (2005)
doi:10.1038/nature03595
131. Mehta A., M. T. Kinter, N. E. Sherman, D. M. Driscoll: Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol Cell Biol 20, 1846-1854 (2000)
doi:10.1128/MCB.20.5.1846-1854.2000
132. Wang C., R. Lehmann: Nanos is the localized posterior determinant in Drosophila. Cell 66, 637-647 (1991)
doi:10.1016/0092-8674(91)90110-K
133. Subramaniam K., G. Seydoux: nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Development 126, 4861-4871 (1999)
134. Tsuda M., Y. Sasaoka, M. Kiso, K. Abe, S. Haraguchi, S. Kobayashi, Y. Saga: Conserved role of nanos proteins in germ cell development. Science 301, 1239-1241 (2003)
doi:10.1126/science.1085222
135. Kobayashi S., M. Yamada, M. Asaoka, T. Kitamura: Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature 380, 708-711 (1996)
doi:10.1038/380708a0
136. Hayashi Y., M. Hayashi, S. Kobayashi: Nanos suppresses somatic cell fate in Drosophila germ line. Proc Natl Acad Sci U S A 101, 10338-10342 (2004)
doi:10.1073/pnas.0401647101
137. Beck A. R., I. J. Miller, P. Anderson, M. Streuli: RNA-binding protein TIAR is essential for primordial germ cell development. Proc Natl Acad Sci U S A 95, 2331-2336 (1998)
doi:10.1073/pnas.95.5.2331
138. Kawakami A., Q. Tian, X. Duan, M. Streuli, S. F. Schlossman, P. Anderson: Identification and functional characterization of a TIA-1-related nucleolysin. Proc Natl Acad Sci U S A 89, 8681-8685 (1992)
doi:10.1073/pnas.89.18.8681
139. Beck A. R., Q. G. Medley, S. O'Brien, P. Anderson, M. Streuli: Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res 24, 3829-3835 (1996)
doi:10.1093/nar/24.19.3829
140. Anderson P., N. Kedersha: Stressful initiations. J Cell Sci 115, 3227-3234 (2002)
141. Mazan-Mamczarz K., A. Lal, J. L. Martindale, T. Kawai, M. Gorospe: Translational repression by RNA-binding protein TIAR. Mol Cell Biol 26, 2716-2727 (2006)
doi:10.1128/MCB.26.7.2716-2727.2006
142. Covello K. L., J. Kehler, H. Yu, J. D. Gordan, A. M. Arsham, C. J. Hu, P. A. Labosky, M. C. Simon, B. Keith: HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev 20, 557-570 (2006)
doi:10.1101/gad.1399906
143. Semenza G. L.: HIF-1 and human disease: one highly involved factor. Genes Dev 14, 1983-1991 (2000)
144. Keith B., M. C. Simon: Hypoxia-inducible factors, stem cells, and cancer. Cell 129, 465-472 (2007)
doi:10.1016/j.cell.2007.04.019
145. Stevens L. C.: Origin of testicular teratomas from primordial germ cells in mice. J Natl Cancer Inst 38, 549-552 (1967)
146. Durcova-Hills G., I. R. Adams, S. C. Barton, M. A. Surani, A. McLaren: The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 24, 1441-1449 (2006)
doi:10.1634/stemcells.2005-0424
147. Kimura T., A. Suzuki, Y. Fujita, K. Yomogida, H. Lomeli, N. Asada, M. Ikeuchi, A. Nagy, T. W. Mak, T. Nakano: Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production. Development 130, 1691-1700 (2003)
doi:10.1242/dev.00392
148. Kimura T., M. Tomooka, N. Yamano, K. Murayama, S. Matoba, H. Umehara, Y. Kanai, T. Nakano: AKT signaling promotes derivation of embryonic germ cells from primordial germ cells. Development 135, 869-879 (2008)
doi:10.1242/dev.013474
149. Bird A.: DNA methylation patterns and epigenetic memory. Genes Dev 16, 6-21 (2002)
doi:10.1101/gad.947102
150. Peters A. H., D. Schubeler: Methylation of histones: playing memory with DNA. Curr Opin Cell Biol 17, 230-238 (2005)
doi:10.1016/j.ceb.2005.02.006
151. Martin C., Y. Zhang: The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 6, 838-849 (2005)
doi:10.1038/nrm1761
152. Bernstein B. E., A. Meissner, E. S. Lander: The mammalian epigenome. Cell 128, 669-681 (2007)
doi:10.1016/j.cell.2007.01.033
153. Misteli T.: Beyond the sequence: cellular organization of genome function. Cell 128, 787-800 (2007)
doi:10.1016/j.cell.2007.01.028
154. Kafri T., M. Ariel, M. Brandeis, R. Shemer, L. Urven, J. McCarrey, H. Cedar, A. Razin: Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev 6, 705-714 (1992)
doi:10.1101/gad.6.5.705
155. Surani M. A.: Reprogramming of genome function through epigenetic inheritance. Nature 414, 122-128. (2001)
doi:10.1038/35102186
156. Hajkova P., S. Erhardt, N. Lane, T. Haaf, O. El-Maarri, W. Reik, J. Walter, M. A. Surani: Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117, 15-23 (2002)
doi:10.1016/S0925-4773(02)00181-8
157. Tam P. P., S. X. Zhou, S. S. Tan: X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene. Development 120, 2925-2932 (1994)
158. Seki Y., K. Hayashi, K. Itoh, M. Mizugaki, M. Saitou, Y. Matsui: Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol 278, 440-458 (2005)
doi:10.1016/j.ydbio.2004.11.025
159. Sugimoto M., K. Abe: X chromosome reactivation initiates in nascent primordial germ cells in mice. PLoS Genet 3, e116 (2007)
doi:10.1371/journal.pgen.0030116
160. Tachibana M., J. Ueda, M. Fukuda, N. Takeda, T. Ohta, H. Iwanari, T. Sakihama, T. Kodama, T. Hamakubo, Y. Shinkai: Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev 19, 815-826 (2005)
doi:10.1101/gad.1284005
161. Klose R. J., E. M. Kallin, Y. Zhang: JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet 7, 715-727 (2006)
doi:10.1038/nrg1945
162. Shi Y.: Histone lysine demethylases: emerging roles in development, physiology and disease. Nat Rev Genet 8, 829-833 (2007)
doi:10.1038/nrg2218
163. Fodor B. D., S. Kubicek, M. Yonezawa, R. J. O'Sullivan, R. Sengupta, L. Perez-Burgos, S. Opravil, K. Mechtler, G. Schotta, T. Jenuwein: Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells. Genes Dev 20, 1557-1562 (2006)
doi:10.1101/gad.388206
164. Stewart M. D., J. Sommerville, J. Wong: Dynamic regulation of histone modifications in Xenopus oocytes through histone exchange. Mol Cell Biol 26, 6890-6901 (2006)
doi:10.1128/MCB.00948-06
165. Okada Y., G. Scott, M. K. Ray, Y. Mishina, Y. Zhang: Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature (2007)
166. Cao R., Y. Zhang: The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr Opin Genet Dev 14, 155-164 (2004)
doi:10.1016/j.gde.2004.02.001
167. Cao R., Y. Zhang: SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 15, 57-67 (2004)
doi:10.1016/j.molcel.2004.06.020
168. Peters A. H., S. Kubicek, K. Mechtler, R. J. O'Sullivan, A. A. Derijck, L. Perez-Burgos, A. Kohlmaier, S. Opravil, M. Tachibana, Y. Shinkai, J. H. Martens, T. Jenuwein: Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. Mol Cell 12, 1577-1589. (2003)
doi:10.1016/S1097-2765(03)00477-5
169. Feldman N., A. Gerson, J. Fang, E. Li, Y. Zhang, Y. Shinkai, H. Cedar, Y. Bergman: G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat Cell Biol 8, 188-194 (2006)
doi:10.1038/ncb1353
170. Feng Y. Q., R. Desprat, H. Fu, E. Olivier, C. M. Lin, A. Lobell, S. N. Gowda, M. I. Aladjem, E. E. Bouhassira: DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLoS Genet 2, e65 (2006)
doi:10.1371/journal.pgen.0020065
171. Bernstein B. E., T. S. Mikkelsen, X. Xie, M. Kamal, D. J. Huebert, J. Cuff, B. Fry, A. Meissner, M. Wernig, K. Plath, R. Jaenisch, A. Wagschal, R. Feil, S. L. Schreiber, E. S. Lander: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315-326 (2006)
doi:10.1016/j.cell.2006.02.041
172. Lee T. I., R. G. Jenner, L. A. Boyer, M. G. Guenther, S. S. Levine, R. M. Kumar, B. Chevalier, S. E. Johnstone, M. F. Cole, K. Isono, H. Koseki, T. Fuchikami, K. Abe, H. L. Murray, J. P. Zucker, B. Yuan, G. W. Bell, E. Herbolsheimer, N. M. Hannett, K. Sun, D. T. Odom, A. P. Otte, T. L. Volkert, D. P. Bartel, D. A. Melton, D. K. Gifford, R. Jaenisch, R. A. Young: Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301-313 (2006)
doi:10.1016/j.cell.2006.02.043
173. Yamaguchi S., H. Kimura, M. Tada, N. Nakatsuji, T. Tada: Nanog expression in mouse germ cell development. Gene Expr Patterns 5, 639-646 (2005)
doi:10.1016/j.modgep.2005.03.001
174. Lane N., W. Dean, S. Erhardt, P. Hajkova, A. Surani, J. Walter, W. Reik: Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse. Genesis 35, 88-93 (2003)
doi:10.1002/gene.10168
175. Nakagawa M., M. Koyanagi, K. Tanabe, K. Takahashi, T. Ichisaka, T. Aoi, K. Okita, Y. Mochiduki, N. Takizawa, S. Yamanaka: Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26, 101-106 (2008)
doi:10.1038/nbt1374
176. Aoi T., K. Yae, M. Nakagawa, T. Ichisaka, K. Okita, K. Takahashi, T. Chiba, S. Yamanaka: Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science (2008)
177. Boyer L. A., T. I. Lee, M. F. Cole, S. E. Johnstone, S. S. Levine, J. P. Zucker, M. G. Guenther, R. M. Kumar, H. L. Murray, R. G. Jenner, D. K. Gifford, D. A. Melton, R. Jaenisch, R. A. Young: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956 (2005)
doi:10.1016/j.cell.2005.08.020
178. Winnier G., M. Blessing, P. A. Labosky, B. L. Hogan: Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 9, 2105-2116 (1995)
doi:10.1101/gad.9.17.2105
179. Zhao G. Q., K. Deng, P. A. Labosky, L. Liaw, B. L. Hogan: The gene encoding bone morphogenetic protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev 10, 1657-1669 (1996)
doi:10.1101/gad.10.13.1657
180. Zhang H., A. Bradley: Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 2977-2986 (1996)
181. Chang H., D. Huylebroeck, K. Verschueren, Q. Guo, M. M. Matzuk, A. Zwijsen: Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects. Development 126, 1631-1642 (1999)
182. Sirard C., J. L. de la Pompa, A. Elia, A. Itie, C. Mirtsos, A. Cheung, S. Hahn, A. Wakeham, L. Schwartz, S. E. Kern, J. Rossant, T. W. Mak: The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev 12, 107-119 (1998)
doi:10.1101/gad.12.1.107
183. Yang X., C. Li, X. Xu, C. Deng: The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice. Proc Natl Acad Sci U S A 95, 3667-3672 (1998)
doi:10.1073/pnas.95.7.3667
184. Motro B., D. van der Kooy, J. Rossant, A. Reith, A. Bernstein: Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci. Development 113, 1207-1221 (1991)
185. Matsui Y., K. M. Zsebo, B. L. Hogan: Embryonic expression of a haematopoietic growth factor encoded by the Sl locus and the ligand for c-kit. Nature 347, 667-669 (1990)
doi:10.1038/347667a0
186. Flamme I., T. Frohlich, M. von Reutern, A. Kappel, A. Damert, W. Risau: HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels. Mech Dev 63, 51-60 (1997)
doi:10.1016/S0925-4773(97)00674-6
187. Ema M., S. Taya, N. Yokotani, K. Sogawa, Y. Matsuda, Y. Fujii-Kuriyama: A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A 94, 4273-4278 (1997)
doi:10.1073/pnas.94.9.4273
188. Tian H., S. L. McKnight, D. W. Russell: Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev 11, 72-82 (1997)
doi:10.1101/gad.11.1.72
189. Tian H., R. E. Hammer, A. M. Matsumoto, D. W. Russell, S. L. McKnight: The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development. Genes Dev 12, 3320-3324 (1998)
doi:10.1101/gad.12.21.3320
Key Words: Primordial germ cells, Specification, Bmp signaling, Blimp1, Epigenetic reprogramming, Pluripotency, Review
Send correspondence to: Mitinori Saitou, Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan, Tel: 81-78-306-3376, Fax: 81-78-306-3377, E-mail:saitou@cdb.riken.jp