DEAD box family proteins in Drosophila

The "DEAD box" family of proteins contains the conserved motif Asp-Glu-Ala-Asp, and is typified by the eukaryotic translation initiation factor eIF4A. Its members are believed to share the functional property of ATP-dependent RNA unwinding, the helicase activity. In addition to vasa, a second Drosophila gene has been isolated that shows an mRNA expression pattern somewhat similar to that of vasa and also encodes a DEAD box protein. This gene is called ME31B to reflect its maternal (ovarian germ-line) expression and its location within the 31B chromosome region. Comparison with the other members of this family reveals that although ME31B is most like the protein Tif1/Tif2, which probably represents the Saccharomyces cerevisiae version of eIF4A, it is unlikely that ME31B represents the Drosophila eIF4A protein per se (de Valoir, 1991).

Vasa homologs in insects

Vasa is a widely conserved germline marker, both in vertebrates and invertebrates. A vasa orthologue, Sgvasa, has been identified and it has been used to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early oogenesis, but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most cleavage energids (zygotic cells that reach the egg surface) as these cells reach the surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after gastrulation, Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, it is suggested that the Vasa-stained cells in the abdominal margin are germ cells, and not cardioblasts, as has been proposed by others (Chang, 2002).

Germ cell development in the silkworm Bombyx mori is interesting in that the species has no recognizable germ plasm, and its germ cells appear first on the ventral side of the embryo, not on the posterior pole as in Drosophila. A vasa homologue (BmVLG) from B. mori has been identified and the specific expression of transcript in the germ cells has been revealed. Consistent with the lack of recognizable germ plasm, BmVLG is not localized in freshly laid eggs, and its specific expression is first detectable several hours after energids penetrate the periplasm. This is in contrast to D. melanogaster, where germ cell lineage can be traced with anti-vasa antibody just after the formation of pole cells as they sequester vasa-positive germ (pole) plasm during cellularization. It is also revealed that, within the first few hours of their appearance when extensive cell movement does not seem to occur, stained cells are sometimes widely dispersed along the midline, which eventually may lead to the formation of ectopic germ cells. The implications of these results for germ cell development are discussed (Nakao, 2006).

There seem to be two explanations for this unusual distribution pattern of the stained cells: in situ differentiation and cell migration. The former possibility is supported if a static nature of the cells within the first few hours after their appearance is postulated. This stage is several hours earlier than when morphological change of the germband begins. If this interpretation is correct, it may imply that cells in the vicinity of the midline (thus along the dorsal midline of the egg) have the potential to generate the germ cells, although their appearance is usually restricted to the posterior position. For the latter possibility, it is interesting to note that the germ cell movement would be directional along the midline, since dispersal toward the lateral sides has never been observed. This finding will lead to further understanding of Bombyx germ cell formation processes (Nikao, 2006).

The correct specification of germ cells during embryogenesis is a fundamental step in life that ensures the existence of the next generation. Although different species display various cellular modes of generating germ cells, the product of the vasa gene proves to be a reliable marker of primordial germ cells in metazoans. The vasa ortholog from the red flour beetle Tribolium castaneum, named Tc-vasa, and is described in this study, along with its genomic organisation and its expression pattern during early embryogenesis. Unlike in Drosophila where vasa messenger RNA (mRNA) is ubiquitously distributed in the egg, Tc-vasa mRNA gradually accumulates at the posterior egg pole during blastoderm formation. Shortly before gastrulation, Tc-vasa mRNA marks a group of intra-blastodermal cells at the posterior pole. In the germ rudiment, a ball-like group of vasa-positive cells adheres to the growth zone at the posterior end of the embryo. These vasa-positive cells likely represent the primordial germ cells that have not been described in Tribolium prior to gonad formation. At the beginning of germ growth, a small band of vasa-positive cells starts to migrate along the dorsal side of the growth zone. vasa transcription ceases during further germ band extension. In contrast to Drosophila, Tc-vasa transcripts cannot be detected in the germ cells within the gonadal anlage after segmentation is completed (Schröder, 2006).

Vass homologs in other invertebrates

The vasa (vas)-related genes are members of the DEAD box protein family and are involved in germ cell formation in higher metazoans. In the present study, the vas-related genes as well as the PL10-related genes, other members of the DEAD box protein family, were cloned from lower metazoans: sponge, Hydra and planaria. The phylogenetic analysis suggested that the vas-related genes arose by duplication of a PL10-related gene before the appearance of sponges but after the diversion of fungi and plants. The vas-related genes in Hydra, Cnvas1 and Cnvas2 are strongly expressed in germline cells and less strongly expressed in multipotent interstitial stem cells and ectodermal epithelial cells. These results suggest that the vas-related genes occur universally among metazoans and that their expression in germline cells was established at least before cnidarian evolution (Mochizuki, 2001).

Planarians are known for their strong regenerative ability. This ability has been considered to reside in the totipotent somatic stem cell called the 'neoblast'. Neoblasts contain a unique cytoplasmic structure called the 'chromatoid body', which has similar characteristics to the germline granules of germline cells of other animals. The chromatoid bodies decrease in number and size during cytodifferentiation and disappear in completely differentiated cells during regeneration. However, germ cells maintain the chromatoid body during their differentiation from neoblasts. These observations suggest that the chromatoid body is concerned with the totipotency of cells. To understand the molecular nature of the chromatoid body in the neoblast, a focus was placed on vasa (vas)-related genes, since VAS and VAS-related proteins are known to be components of the germline granules in Drosophila and Caenorhabditis elegans. By PCR, two vas-related genes (Dugesia japonica vasa-like gene, DjvlgA and DjvlgB) were isolated, and shown to be expressed in germ cells. Interestingly, DjvlgA is also expressed in a number of somatic cells in the mesenchymal space. In regenerating planarians, accumulation of DjvlgA-expressing cells is observed in both the blastema and the blastema-proximal region. In X-ray-irradiated planarians, which have lost regenerative capacity, the number of DjvlgA-expressing cells decreases drastically. These results suggest that the product of DjvlgA may be a component of the chromatoid body and may be involved in the totipotency of the neoblast (Shibata, 1999).

The GLHs (germline RNA helicases), homologs of Drosophila Vasa, are constitutive components of the germline-specific P granules in the nematode C. elegans and are essential for fertility, yet how GLH proteins are regulated remains unknown. KGB-1 and CSN-5 are both GLH binding partners, previously identified by two-hybrid interactions. KGB-1 is a MAP kinase in the Jun N-terminal kinase (JNK) subfamily, whereas CSN-5 is a subunit of the COP9 signalosome. Intriguingly, although loss of either KGB-1 or CSN-5 results in sterility, their phenotypes are strikingly different. Whereas csn-5 RNA interference (RNAi) results in under-proliferated germlines, similar to glh-1/glh-4(RNAi), the kgb-1(um3) loss-of-function mutant exhibits germline over-proliferation. When kgb-1(um3) mutants are compared with wild-type C. elegans, GLH-1 protein levels are as much as 6-fold elevated and the organization of GLH-1 in P granules is grossly disrupted. A series of additional in vivo and in vitro tests indicates that KGB-1 and CSN-5 regulate GLH-1 levels, with GLH-1 targeted for proteosomal degradation by KGB-1 and stabilized by CSN-5. It is proposed the 'good cop: bad cop' team of CSN-5 and KGB-1 imposes a balance on GLH-1 levels, resulting in germline homeostasis. In addition, both KGB-1 and CSN-5 bind Vasa, a Drosophila germ granule component; therefore, similar regulatory mechanisms might be conserved from worms to flies (Orsborn, 2007).

Vasa is a broadly conserved ATP-dependent RNA helicase that functions in the germ line of organisms from cnidarians to mammals. Curiously, Vasa is also present in the somatic cells of many animals and functions as a regulator of multipotent cells. This study reports a mitotic function of Vasa revealed in the sea urchin embryo. Vasa protein is present in all blastomeres of the early embryo and that its abundance oscillates with the cell cycle. Vasa associates with the spindle and the separating sister chromatids at metaphase, and then quickly disappears after telophase. Inhibition of Vasa protein synthesis interferes with proper chromosome segregation, arrests cells at M-phase, and delays overall cell cycle progression. Cdk activity is necessary for the proper localization of Vasa, implying that Vasa is involved in the cyclin-dependent cell cycle network, and Vasa is required for the efficient translation of cyclinB mRNA. These results suggest an evolutionarily conserved role of Vasa that is independent of its function in germ line determination (Yajima, 2011).

The Vasa homolog RDE-12 engages target mRNA and multiple Argonaute proteins to promote RNAi in C. elegans

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi, was immunoprecipitated. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs [RDE-1 (Drosophila homolog: Ago1) and ERGO-1], where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing (Shirayama, 2014).

MUT-14 and SMUT-1 DEAD Box RNA helicases have overlapping roles in germline RNAi and endogenous siRNA formation

More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. This study shows that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of mut-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs (Phillips, 2014).

Germ-Granule Components Prevent Somatic Development in the C. elegans Germline

Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans. In Drosophila, germ granules have been implicated in germline determination. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination, but their components are still needed for fertility. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 (Drosophila homolog: Held out wings) or upon neuronal induction by CHE-1 (Drosophila homolog: Glass). However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, P granules were severly compromised by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization [GLH-1 (see Drosophila Vasa) and GLH-4] and an investigation was carried out to see whether this causes germ cells to lose totipotency and initiate somatic reprogramming. It was found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate (Updike, 2014).

Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. This study shows that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, these data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells (Wu, 2011).

Vertebrate Vasa homologs

Although the zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, the zebrafish homolog of the Drosophila vasa gene, a germ line specific protein, was cloned. The predicted amino acid sequence is 52.8% homologous to mouse and 40.9% homologous to Drosophila Vasa protein. Northern blotting reveals that zebrafish vasa homolog (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, the expression pattern of vas mRNA was examined in zebrafish embryos from the 1-cell stage to 10 days of development. vas mRNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. vas mRNA remains localized to exactly four cells through the 1000-cell stage; in embryos of the 4000 cell stage, RNA is detected in four to twelve cells per embryo and is no longer subcellularly localized in clumps but appears to fill the cytoplasm (Yoon, 1997).

Vasa-related genes have been isolated from several metazoa, including the nematode, frog and species of mammals. In order to gain insight into the early events in vertebrate germline development, zebrafish was chosen as a model. Two zebrafish vasa-related genes were isolated, pl10a and vlg. The pl10a gene is widely expressed during embryogenesis. The vlg gene and vasa belong to the same subfamily of RNA helicase encoding genes. Putative maternal vlg transcripts are detected shortly after fertilization; from the blastula stage onward, expression is restricted to migratory cells, most likely to be primordial germ cells (Olsen, 1997).

Described here is the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity to microtubules. Its translocation to the distal furrows is microtubule dependent. It is proposed that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment (Knaut, 2000).

It was of interest to determine why the number of presumptive primordial germ cells (PGCs) remains constant during initial cleavages while somatic cells increase dramatically in number. There are two possibilities to account for the lack in PGC proliferation until the sphere stage (cell cycle 13): either germ plasm-containing cells do not divide during early embryogenesis, as has been shown for Drosophila, or germ plasm is segregated asymmetrically to one daughter cell during cell division, as has been reported for Caenorhabditis and Xenopus and as is suggested in zebrafish. To address this question, germ plasm-positive cells were followed during early embryogenesis. In the 512-cell stage embryos, dividing cells segregate the germ plasm with one of the two daughter nuclei, whereas in sphere stage (cell cycle 13) embryos, germ plasm is inherited by both daughter cells. In 1,000-cell stage embryos, germ plasm is still seen as a tight structure restricted to one part of the cell. Three-dimensional analysis shows that the germ plasm is punctate and tightly localized between the nucleus and the vegetal-most membrane, often forming a cuplike structure. Beginning at the sphere stage (cell cycle 12-13) the germ plasm disintegrates and seems to spread into the cytoplasm. Shortly before gastrulation (30% epiboly), the germ plasm is fully disintegrated and fills the cytoplasm evenly in little patches. Germ plasm is localized to one side of the division plane during early cell divisions. Most frequently, the axis of cellular division to the animal-vegetal axis is seen with the germ plasm segregated to the vegetal spindle pole. Therefore, the observed low number of vasa-positive cells in early embryogenesis is due to asymmetric localization of germ plasm to one daughter cell, which is a process that is discontinued at the beginning of the sphere stage (cell cycle 12-13) when germ plasm fills the cytoplasm. In addition, changes in the germ plasm segregation pattern are observed before the beginning of Vasa protein localization and zygotic vasa transcription, suggesting that this change is independent of Vasa protein function (Knaut, 2000).

An important mechanism for the specification and development of the animal germ line is the localization of specific molecules to the germ plasm. Restriction of these molecules to the germ line is considered to be critical for proper development of the germ line as well as the soma. Cytoplasmic localization alone, however, may not be sufficient to achieve germ line-specific expression. While zebrafish vasa mRNA is localized to the germ plasm, the Vasa protein is initially distributed uniformly in the embryo, and its expression becomes restricted to the PGCs only later in development. In addition to vasa RNA localization, multiple cell type-specific posttranscriptional mechanisms act on vasa mRNA and Vasa protein. The portion of the maternal vasa mRNA that is partitioned to somatic cells is rapidly degraded, whereas vasa RNA is stabilized in the PGCs in a process that is mediated by cis-acting elements within the molecule. Similarly, the Vasa protein is highly unstable in somatic cells, but not in the PGCs. Finally, subcellular localization of Vasa protein involves cis-acting domains within the protein. In conclusion, this study shows that posttranscriptional degradation-protection mechanisms acting on RNA and protein function in vertebrates enrich for specific molecules in the PGCs (Wolke, 2002).

A DEAD box family protein has been isolated from Xenopus. The mRNA of XVLG1 (Xenopus vasa-like gene) is specifically expressed in adult testis and ovary. XVLG1 is somewhat homologous to that of Drosophila Vasa. XVLG1 protein is expressed exclusively in adult testis and ovary. Immunocytological study shows that XVLG1 protein is expressed in oogonia, oocytes, spermatogonia, spermatocytes, and also in primordial germ cells in tadpoles (Komiya, 1994).

In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization. To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, different vasa homologs were tested for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish. These results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline (Knaut, 2002).

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, a chicken homolog (Cvh) to vasa gene has been isolated. The germline-specific expression of CVH protein throughout all stages of development is demonstrated. Immunohistochemical analyses using specific antibody raised against CVH protein indicate that CVH protein is localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells are scattered in the central zone of the area pellucida at stage X; later, 45-60 cells are found in the hypoblast layer and subsequently 200-250 positive cells are found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein is predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein-enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm (Tsunekawa, 2000).

Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins. These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans. While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes. p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin(GAP-43) and neurogranin (RC3). p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner. Both phosphorylation and calmodulin binding inhibit p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain (Buelt, 1994).

DEAD-box genes are found throughout evolution and encode RNA-binding proteins. Such proteins include eukaryotic initiation factor-4A, which is essential for protein translation; Vasa, which is essential for germ line development, and a number of nuclear and mitochondrial RNA splicing factors. Transcription of a human DEAD-box gene, DDX1, is elevated in two retinoblastoma cell lines as a result of amplification of the immediate chromosomal region surrounding it, suggesting an important role for this gene in control of cell growth and division. A Drosophila melanogaster (Dm) homolog (Ddx1) of DDX1 has been isolated that is strikingly similar to the human gene. The similarity (58.3% amino acid (aa) identity over 720 aa) extends beyond regions conserved in all DEAD-box proteins and covers the entire lengths of the proteins. The 2.7-kb Dm Ddx1 mRNA is expressed throughout development, but its levels are elevated in early embryos. There is a high level of expression during oogenesis with storage of the Ddx1 mRNA in oocytes. Ddx1 maps to polytene chromosome band 79D4 on the left arm of Dm chromosome 3 (Rafti, 1996).

A mouse homolog to vasa is an essential maternal determining factor for the formation of mouse germ cell precursors. Mvh encodes a DEAD-family protein that shows a much higher degree of similarity with the product of the Drosophila vasa gene than previously reported mouse genes. In adult tissues, Mvh transcripts are exclusively detected in testicular germ cells, in which Mvh protein is found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein is also expressed in germ cells colonized in embryonic gonads but is not detected in pluripotential embryonic cells such as stem cells and germ cells. This suggests the possibility that Mvh protein may play an important role in the determination events of mouse germ cells, as is the case with Vasa in Drosophila (Fujiwara, 1994).

To demonstrate the cellular and subcellular localization of mouse vasa homolog protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrate that MVH protein is 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 indicates inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein is localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure. Such structures include polar granules that contain Vasa protein in Drosophila (Toyooka, 2000).

A conserved feature of germ cells in many animal species is the presence of perinuclear electron-dense material called the 'nuage' that is believed to be a precursor of germinal (or polar or P) granules. In Xenopus oogenesis the nuage is first observed near the nuclear envelope and subsequently in close contact with mitochondria, at which stage it is called the mitochondrial cement. In Xenopus pre-stage I and stage I oocytes, nuage and mitochondrial cement contain the spliceosomal Sm proteins, Xcat2 mRNA, and DEAD-box RNA helicase XVLG1 (Vasa like gene). Other components of Cajal bodies or splicing machinery such as coilin, SMN protein, and snRNAs are absent from the nuage and mitochondrial cement. It is suggested that Xenopus Sm proteins have adapted to a role independent of pre-mRNA splicing and that instead of binding to their traditional spliceosomal partner such as snRNA, they bind mRNAs that are the components of germinal granules (i.e., Xcat2 mRNA) and facilitate the transport of these mRNAs from the nucleus to the nuage that is a precursor of germinal granules. In addition, the presence of Vasa-like DEAD-box helicase in Xenopus nuage suggests involvement of nuage in the microRNA and/or RNAi pathway, similar to the role of nuage in Drosophila (Bilinski, 2004).

Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice

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. A targeted mutation in Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), similar to Drosophila vasa-tudor. Tdrd1/Mtr-1 mutants lack, and Mvh/Ddx4 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 Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or 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 stages/sexes of the germ line (Chuma, 2006; full text of article).

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice

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. 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 (Hosokawa, 2007).

vasa: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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