Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering. G erm-line cells are responsible for transmitting genetic information and for reproducing totipotency from generation to generation. Pluripotent stem cell lines, embryonic stem (ES) cells, and embryonic germ cells are established from cells of the germ cell lineage. Therefore, germ cell specification must be linked to the maintenance of pluripotency, as well as to cell fate commitment leading to gametogenesis.Unlike many animal species, in which the germ line is predetermined by maternal factors, germ cell specification in mammals takes place at the onset of gastrulation, after implantation of the embryo. In the mouse, primordial germ cells (PGCs) are first distinguished at the base of the allantois in gastrulating embryos at embryonic day (E) 7.25 (1). Lineage studies of epiblast cells show that mouse PGCs are specified by inductive interactions at the onset of gastrulation (2, 3). Genetic analyses using targeted mutations have revealed that bone morphogenic protein (BMP) 4 and -8b, soluble growth factors belonging to the transforming growth factor ␤ superfamily that are produced by extraembryonic ectoderm close to the boundary with the proximal epiblast, are required for the generation of PGCs from epiblast cells (4,5). Moreover, primary cultures of epiblast fragments from embryos at E5.5-E6.0 generate migrating PGCs when they are cocultured with extraembryonic ectoderm (6), and culturing of whole epiblasts from E6.0 embryos on feeder cells expressing both BMP4 and BMP8b gives rise to PGCs (7). These results reveal that BMPs derived from the extraembryonic ectoderm play crucial roles in PGC determination in the proximal epiblast.Despite such developments, it is not yet known how the founder population of PGCs is segregated from other pluripotent epiblast cells that form somatic cells. To approach this question, we examined the production of germ cells by an established pluripotent ES cell line. ES cells can form all cell lineages when introduced into host blastocysts and give rise to various somatic cell lineages in culture. However, it is not known whether they can generate the germ cell lineage in culture in the absence of the morphogenetic events associated with gastrulation. It has been difficult to addr...

Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.

SUMMARY MitoPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini, the Drosophila homolog of MitoPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MitoPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MitoPLD is involved in piRNA biogenesis, we generated MitoPLD mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MitoPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MitoPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.

In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh tnscripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.In mice, germ cell precursors, termed primordial germ cells (PGCs), are generated in the epiblast. They consist of putative pluripotential cells that can be first identified as a small cluster of alkaline phosphatase-positive cells in the extraembryonic mesoderm at 7.25 days postcoitum (dpc). Then they move down to the embryonic mesoderm at the posterior end of the primitive streak (8.0 dpc), migrate through the hindgut endoderm, and colonize the developing genital ridges (10.5-11.5 dpc), in which they are destined to form functional germ cells (1, 2). Despite these detailed morphological observations, molecular mechanisms regulating this developmental pathway still remain unclear.Unlike mammals, it has been well established that several maternal factors are involved in germ cell determination of many animal species. In particular, in Drosophila it has been shown that the pole plasm localized at the posterior pole ofthe oocyte contains determining substances for the abdomen and the germ lineage. After fertilization, only the nuclei that migrate into the pole plasm are destined to form the germ-line progenitors (pole cells). Genetic identification ofgenes whose function is required for pole cell formation has revealed at least eight maternally active genes, cappucino, spire, staufen, oskar, vasa, valois, mago-nashi, and tudor (3, 4). Among these genes, vasa (vas) is one of the best characterized. Homozygous mutant vasa females produce no eggs. In ectopic formation of pole cells induced by mislocalization of oskar mRNA to the anterior pole of the oocyte, only vasa and tudor were required for ectopic pole cell formation (5-10). The gene vas encodes a DEAD (Asp-Glu-Ala-Asp)-family protein of putative RNA helicases, which is found to be present as a component of both the polar granules at the posterior end of the oocyte and the nuage structure in the germ cells, and zygotic expression is also restricted to the ge...

Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor, that function in posterior and germ-cell determination. We report here that a targeted mutation in Tudor domain containing 1͞mouse tudor repeat 1 (Tdrd1͞Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1͞MTR-1 predominantly localizes to nuage͞ germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue͞DEAD box polypeptide 4 (Mvh͞Ddx4), similar to Drosophila vasa-tudor. Tdrd1͞Mtr-1 mutants lack, and Mvh͞Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1͞Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and͞or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh-Tdrd1 in mouse spermatogenic cells and vasa-tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages͞sexes of the germ line.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI-and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.Supplemental material is available at http://www.genesdev.org.

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.