Vasa Expression in the Development of Somatic Cells and in Tumorigenesis

Developmental Biology 415 (2016) 24–32

Contents lists available at ScienceDirect

Developmental Biology

journal homepage: www.elsevier.com/locate/developmentalbiology

An unregulated regulator: Vasa expression in the development of somatic cells and in tumorigenesis

Jessica Poon, Gary M. Wessel, Mamiko Yajima n

MCB Department, Brown University, 185 Meeting Street, BOX-GL173, Providence, RI 02912, USA article info abstract

Article history: Growing evidence in diverse organisms shows that genes originally thought to function uniquely in the Received 5 December 2015 germ line may also function in somatic cells, and in some cases even contribute to tumorigenesis. Here Received in revised form we review the somatic functions of Vasa, one of the most conserved “germ line” factors among me- 9 May 2016 tazoans. Vasa expression in somatic cells is tightly regulated and often transient during normal devel- Accepted 11 May 2016 opment, and appears to play essential roles in regulation of embryonic cells and regenerative tissues. Its Available online 11 May 2016 dysregulation, however, is believed to be an important element of tumorigenic cell regulation. In this Keywords: perspectives paper, we propose how some conserved functions of Vasa may be selected for somatic cell Vasa regulation, including its potential impact on efﬁcient and localized translational activities and in some DDX4 cases on cellular malfunctioning and tumorigenesis. Germ line factors & 2016 Elsevier Inc. All rights reserved. Tumorigenesis Translation

1. Introduction and Ashburner, 1988) as a polar granule component consisting of cytoplasmic granules inherited by posterior pole cells, which di- More than 40 genes that are normally expressed selectively in rects the fate of the cell to the germ line (Illmensee and Mahowald, the germ line of many model organisms are also expressed in 1974). It is a member of the DEAD-box RNA helicase family (for the various tumor types in humans (Simpson et al., 2005). Re- molecular classification and characterization of Vasa, see reviews capitulating functions of a germ line program thus has been hy- of Raz (2000), Linder (2006), Gustafson and Wessel (2010), and pothesized to contribute to the characteristic features of neoplastic Lasko (2013)), similar in sequence to eukaryotic initiation factor 4A diseases, including immortality, hypomethylation of DNA, and (eIF4A) (Hay et al., 1988; Lasko and Ashburner, 1988). Vasa is metastatic migrations (Hanahan and Weinberg, 2011). Indeed, in considered to function as a translational regulator in the germ line Caenorhabditis elegans, mutations of the retinoblastoma (Rb) tu- (Hay et al., 1988; Lasko and Ashburner, 1988; Linder et al., 1989; mor suppressor complex induce somatic cells to express germ line Sengoku et al., 2006). Follow-up reports in Drosophila suggest that genes, leading cells to revert to patterns of gene expression nor- Vasa associates with eIF5B, an essential translation initiation factor mally restricted to germ cells (Wang et al., 2005). Further, C. ele- required for joining of ribosomal subunits (Carrera et al., 2000; gans mutants with decreased insulin-like signaling (e.g. daf-2, the Pestova et al., 2000), and is involved in translation of gurken in insulin-like receptor, and age-1, the downstream PI3K mutant oocytes and mei-P26 mRNA in germ line stem cells (Johnstone and strains) cause mis-expression of the germ line factors pie-1 and pgl Lasko, 2004; Liu et al., 2010). A recent report in insect ovarian cells in the somatic cells. These altered somatic cells are then more also suggests that Vasa binds to mRNAs with its DEAD-box domain resistant to stress-induced damage and have increased cellular and functions in the generation of germ line-specific, Piwi-inter- lifespans (or negligible senescence) (Curran et al., 2009). This re- acting RNAs (piRNAs) that protect animal genomes against trans- sult is particularly noteworthy considering C. elegans somatic cells posons and are essential for fertility (Xiol et al., 2014). Vasa thus are genetically programmed to cease cell divisions after the animal appears to have broad functions, including translational regulation reaches adulthood. These observations suggest that the acquisition of specific mRNAs and piRNA generation, which contribute to cell of germ line elements may contribute to somatic phenotypes, and fate determination within the germ line. even of tumorgenic cells. Vasa is one of the most conserved germ line factors in various fi fi Vasa was rst identi ed in Drosophila (Hay et al., 1988; Lasko metazoan organisms, based on sequence, expression patterns, and functions in the germ line. Its knockdown in major model organ- n Corresponding author. isms compromises the animal's reproductive capabilities with E-mail address: [email protected] (M. Yajima). varying severity between different organisms, or even between http://dx.doi.org/10.1016/j.ydbio.2016.05.012 0012-1606/& 2016 Elsevier Inc. All rights reserved. J. Poon et al. / Developmental Biology 415 (2016) 24–32 25 sexes of the same species. For instance, Vasa knockdown in Dro- condensation and segregation (Pek and Kai, 2011). These reports sophila results in sterility in females but not in males (Styhler et al., suggest a conserved function of Vasa as a mitotic regulator both in 1998; Lasko and Ashburner, 1990; Renault, 2012), whereas in mice the germ line and in the soma (Fig. 1). it causes sterility in males but not in females (Tanaka et al., 2000). Detailed functional contributions of Vasa in mitotic regulation In C. elegans, GLH1, one of four Vasa homologs in this organism, is of somatic cells are yet to be identified but it may “simply” function important both for germ cell development and for gamete ma- to increase translation locally. A recent report in the sea urchin turation (Roussell and Bennett, 1993; Gruidl et al., 1996; Kuznicki embryo suggests that Vasa interacts with most mRNAs in the cell et al., 2000; Spike et al., 2008). In zebrafish, maternal Vasa is stable and its knockdown results in inhibition of overall protein synthesis throughout early development and is necessary for initial germ down to approximately 20% that of the normal embryo (Yajima cell formation, and Vasa produced embryonically is required for and Wessel, 2015). Vasa may thus function by contributing to germ cell maintenance (Braat et al., 2001; Hartung et al., 2014). In translation of a large number of mRNAs, which allows cells to Medaka, on the other hand, Vasa protein synthesized in the em- produce sufficient amounts of proteins for rapid cell replication bryo is important for germ cell migration but not for survival (Li and differentiation during embryogenesis, especially in a large cell et al., 2009). The mechanism of these variable Vasa inputs among with rapid cell fate transitions. different species and sexes is unclear. It was recently proposed that Vasa might have multiple different functions in each cell type or organism by using different partner proteins (Dehghani and 3. Vasa's function in the soma during regeneration and Lasko, 2015). The identity of such distinct interactors though, re- tumorigenesis mains unclear. Recent reports in more diverse organisms suggest that Vasa Vasa also appears to function within the soma during re- might also function outside of the germ line. These somatic generation and somatic tumorigenesis. In planarian regenerative functions of Vasa include various types of embryonic cells (Yajima tissues, Vasa expression is upregulated (Wagner et al., 2012), as it and Wessel, 2011a; Schwager et al., 2014), regenerative tissues is in Drosophila brain tumor cells (Janic et al., 2010), and even in (Wagner et al., 2012; Yajima and Wessel, 2015), and even in tu- human cancer cells (Hashimoto et al., 2008; Nguyen et al., 2011; morigenic cells (Janic et al., 2010). These reports cast potential Kim et al., 2014). In planarians, transcriptional profiling identified additional functions of Vasa and/or of Vasa's interacting proteins that expression of several germ line genes are upregulated in adult in the soma. In this Perspectives paper, we speculate on the proliferating neoblasts during regeneration. Among those genes, functional impact of Vasa in somatic cells as well as on potential the vasa-like gene, vasa-1 was identified to be essential for pro- contributions during tumorigenesis. liferative expansion to promote the process of differentiation in neoblast descendants, suggesting its functional role in stem cell regulation (Wagner et al., 2012). 2. Vasa's contributions as a mitotic factor during When a malignant brain tumor was induced in Drosophila (by embryogenesis inactivation of a polycomb repressor gene, lethal (3) malignant brain tumor [l(3)mbt]; Gateff et al., 1993), 102 genes were found to Although expression and activities of Vasa are most prominent increase transcript levels specific to this mutant (Janic et al., 2010). in the germ line, Vasa protein and/or mRNA is often widely ex- Among these genes, a quarter of them (26 of 102) are known pressed in somatic lineages during embryogenesis of many or- factors required for the Drosophila germ line. In addition to vasa, ganisms (summarized in Table 1). Proteins and mRNAs are often these genes included, for instance, nanos, necessary for pole plasm stored maternally in the oocyte and embryo, and thus Vasa pre- development (Wong et al., 2005) and piwi and aubergine, involved sence in the resultant somatic cells does not necessarily indicate a in the biogenesis of Piwi interacting RNAs (piRNAs) that protect functional role. Initial observations in Drosophila showed that germ line cells against transposable elements and viruses (Klat- maternal Vasa contributes to the formation of both the germ cells tenhoff and Theurkauf, 2008; Patil and Kai, 2010). Importantly, not and the embryonic abdominal segments (Schupbach and Wie- only were these genes expressed in the tumor cells, but inhibition schaus, 1986a, 1986b; Hay et al., 1990), implying a broad impact on of vasa, piwi, aubergine,ornanos halted tumor growth, suggesting embryogenesis. Flies mutant for Vasa embryonically, however, are that the expression of these factors in the somatic tumor had an viable as homozygotes, suggesting no essential zygotic roles in essential function. In this process of tumorigenesis regulation, somatic development of Drosophila. Recent studies in the sea interestingly, vasa was not upregulated at the mRNA level but its urchin and spider, on the other hand, demonstrated that Vasa does protein expression was increased. This is distinct from other germ function in somatic cells during embryogenesis. In the sea urchin, line factors such as piwi and aubergine, which were up-regulated Vasa was found to localize to the nuclear periphery at S-phase of both at the mRNA and protein levels during tumorigenesis. This the cell cycle in blastomeres and accumulate on the mitotic ap- finding suggests that vasa mRNA (and potentially Vasa protein as paratus at M-phase of every blastomere during early embryogen- well) is expressed at low levels during normal soma development, esis. The knockdown of Vasa in these embryos resulted in defects and can be differentially regulated and utilized upon appropriate of proper spindle organization and chromosome segregation, cell stimulation. This post-transcriptional regulation of Vasa is a cycle delays and often death of the embryo (Yajima and Wessel, common theme (e.g. Wagner et al., 2012; Yajima and Wessel, 2015) 2011a, 2011b). In spider embryogenesis, Vasa is localized to small likely important for this gene function. puncta ubiquitously distributed throughout the cortex before PGCs DDX4 (the Vasa ortholog in humans) expression was also found emerged at the late germ band stage and its knockdown by RNA in various types of cancer tissues in humans. In ovarian cancer interference resulted in defects of mitotic progression in the em- cells, DDX4 expression was significantly increased together with bryo (Schwager et al., 2014). Importantly, this function of Vasa as a CD133, a cancer stem cell marker, whereas almost all CD133-ne- mitotic regulator was also reported in Drosophila germ line stem gative cells had no DDX4 expression, implying that DDX4 may play cells (Pek and Kai, 2011). In those cells, Vasa concentrated in the an important role in cancer stem cells (Kim et al., 2014). Further, in perinuclear region during S-phase and to the spindle and chro- the SKOV-3 (epithelial ovarian cancer) cell line, artificially forced- mosome region during M-phase. Its functionality was highlighted expression of DDX4 resulted in up-regulation of 10 proteins and in mitotic progression by regulating the localization of chromo- down-regulation of 6 proteins. Among these proteins altered by some-associated proteins, which in turn mediated chromosome DDX4, 14-3-3s protein was down-regulated but its mRNA level 26

Table 1 Summary of Vasa expression and functional contributions in the soma.

Organism mRNA Protein Functional contribution References

Fly (Drosophila melanogaster) Uniformly distributed during syncytial blastoderm Uniformly distributed during syncytial blastoderm Abdominal segment formation. Schupbach and Wieschaus, 1986; stage. stage. Lasko and Ashburner, 1988, 1990; Hay et al., 1988

Spider (Parasteatoda tepidariorum) Uniformly distributed in the early embryo, and be- Uniformly distributed in the early embryo, and be- Early embryonic mitotic divisions and Schwager et al., 2014 24 (2016) 415 Biology Developmental / al. et Poon J. comes highly expressed in mesoderm and ectoderm comes undetectable after germ band formation. spindle integrity. after germ band formation. Planarians (Dugesia japonica and Expressed at the lower level in the neoblasts and ND Neoblast proliferation and differ- Shibata et al., 1999; Wagner et al., Schmidtea mediterranea) highly expressed in the growing blastema during entiation during regeneration. 2012 regeneration. Hydra (Hydra magnipapillata) Expressed in multipotent stem cells (i-cells) and ec- ND ND Mochizuki et al., 2001 todermal epithelial cells. C. elegans Expressed in all cells of young embryos and reduced Background level ND Gruidl et al., 1996; Kuznicki et al., after the 8- to 10-cell stage. 2000 Polychaete (Platynereis dumerilii Uniformly distributed until the four cell stage, and Distributed in the yolk free cytoplasm of all blas- ND Rebscher et al., 2007; Dill and and Capitella sp. I) becomes conﬁned to the ventral plate and the meso- tomeres at 4-cell stage, and becomes enriched to Seaver, 2008 dermal bands at 15 pf and to mesodermal posterior secondary mesoblast cells and MPGZ during larval growth zone (MPGZ) during larval and adult stages. and adult stages. Sea urchin (Strongylocentrotus Uniformly distributed in every cell until gastrula stage, Enriched on the mitotic apparatus of every early Early embryonic mitotic cell cycle Yajima and Wessel, 2011b, 2015 purpuratus) and transiently expressed in the cells of the adult embryonic cell, and transiently expressed in the adult progression, wound-healing, and de- precursor (rudiment). precursor cells and wound-healing tissues. velopmental reprograming. –

Ascidians (Ciona intestinalis) Uniformly distributed in each cell until 16-cell stage Concentrated at the postplasm at 64-cell and en- ND Shirae-Kurabayashi et al., 2006 32 and widely expressed in the postplasm and the trunk riched in the posterior-most cells (the B7.6 cells) and region. theDistributed trunk region. throughout the embryo during ND Yoon et al., 1997; Braat et al., 1999, Zebraﬁsh Localizedcleavage to planes the distal at 4-cell ends stage. of the ﬁrst and second embryogenesis. 2000; Kosaka et al., 2007 Frog (Xenopus laevis) Expressed during early embryogenesis by RT-PCR Uniformly expressed until tailbud stage, and becomes ND Ikenishi et al., 1996; Li et al., 2010 analysis. enriched in the mesoderm during early tadpole stage. Chick ND Localized in the basal cleavage furrow in the early ND Tsunekawa et al., 2000 embryo, and becomes scattered in the hypoblast layer after stage X.

ND, no data. J. Poon et al. / Developmental Biology 415 (2016) 24–32 27

Fig. 1. Vasa is involved in mitotic progression. A. Vasa localizes on the mitotic apparatus and is essential for cell cycle progression of sea urchin embryos (Adapted from Yajima and Wessel (2011a)) and Drosophila germ line stem cells (Adapted from Pek and Kai (2011)). Vasa is enriched in peri-nuclear regions prior to mitotic entry (Inter- phase), localizes on the mitotic apparatus during M-phase, and disappears at the end of mitosis, followed by peri-nuclear localization at the beginning of next mitotic entry. Vasa knockdown (right panels) in those cells result in defects of spindle organization and chromosome segregation. The knockdown image of sea urchin is an embryo at 16- cell stage, and that of Drosophila is a germ line stem cell at Anaphase. B. A cartoon diagram of Vasa protein (red) localization in the sea urchin embryonic cells (left panel) and Drosophila germline stem cells (middle panel). Both in sea urchin embryonic cells and Drosophila germ line stem cells, Vasa is evenly distributed over the spindle (green) and DNA (blue) during mitosis, and its knockdown (right panel) results in cell division defects, including abnormal chromosomal segregation. Further, in the sea urchin, Vasa becomes more enriched toward the vegetal blastomere (a future germ line) of the spindle at 16-cell stage by an unknown mechanism. was not changed, suggesting that DDX4 may be involved in post- et al., 2000; Osada et al., 2002; Akahira et al., 2004). 14-3-3s is transcriptional regulation of 14-3-3s (Hashimoto et al., 2008). known to sequester the mitotic initiation complex (cdk1-cyclin B1) Importantly, 14-3-3s is frequently suppressed in human breast, to the cytoplasm in response to the DNA damage, and induces cell gastric, lung and ovarian cancers (Ferguson et al., 2000; Iwata cycle arrest for the G2 checkpoint (Chan et al., 1999). With the 28 J. Poon et al. / Developmental Biology 415 (2016) 24–32 forced expression of DDX4 in which 14-3-3s is down-regulated, other embryonic mRNAs (Liu et al., 2009; Yajima and Wessel, the SKOV-3 ovarian cancer cells appeared to ignore the G2 2015). This may result in over expression of embryonic proteins checkpoint and underwent rapid cell cycle progression even in the and a recapitulation of the embryonic molecular status in the cells, presence of DNA damage (Hashimoto et al., 2008). In these cells, but only in an “unregulated” manner. This could lead to un- DDX4 may have precociously nucleated the mitotic initiation controlled cellular activities, which may in some cases enable the complex and induced pre-mature mitotic entry by down-regulat- retention of continuously proliferating tumorgenic cells. ing 14-3-3s activities. If this speculation is correct, DDX4 over- expressing cells could then continue to proliferate with damaged genetic materials, which can eventually result in tumor formation. 4. Vasa function is tightly regulated by post-transcriptional These speculations need to be tested through further experiments. mechanisms One could test, for instance, if forced expression of 14-3-3s would rescue DDX4-overexpression phenotypes, slow down cell cycle Knockdown or overexpression of genes of interest has been a progression and enable repair of DNA damage in the genome. The gold standard to allow researchers to identify the gene's functional SKOV cell line used here is derived from ovarian carcinomas whose contributions as well as its surrounding molecular network during molecular status and cellular dynamics may be quite different development. Vasa over-expression, however, often results in no from normal cells or even non-ovarian cancer cells. Another im- dramatic phenotype in an organism's normal development (Ike- portant experiment is to test the protein content of cells with or nishi and Yamakita, 2003; Orsborn et al., 2007; Kugler et al., 2010; without DDX4 present to determine if the proteome varies by Gustafson et al., 2011), suggesting again that the presence of Vasa DDX4's functionality in non-ovarian cells. Since Vasa protein level mRNA and/or protein in the soma does not immediately indicate in the cell is thought to be regulated by the Ubiquitin proteasome- its function in those cells, and that Vasa function is tightly regu- dependent protein degradation mechanism (Liu et al., 2003; lated by post-transcriptional mechanisms. In Drosophila embryos, Styhler et al., 1998, 2002; Kugler et al., 2010; Woo et al., 2006a, it is thought that Vasa protein is actively degraded in the soma 2006b), introducing constitutively active forms of DDX4 into the through E3 Ubiquitin ligase pathways, resulting in embryos with cell would be important to identify its functional roles. For these Vasa enriched only in the germ line (Liu et al., 2003; Styhler et al., experiments, one could mutate putative protein degradation re- 1998, 2002; Kugler et al., 2010; Woo et al., 2006a, 2006b). The cognition sites, or may target DDX4 expression to an ectopic sub- knockdown of these Ubiquitin pathways in the embryo, however, cellular region where ‘Vasa-regulators’ may be absent. These ex- resulted only in higher expression of Vasa in the germ line but did periments both in non-oncogenic and in non-ovarian-cancer cells not appear to induce Vasa accumulation in the soma even when its will potentially reveal actual and conserved contributions of DDX4 mRNA was present. These facts suggest that a differential me- to cancer cell regulation. chanism of Vasa regulation in the germ line and the soma. In the These recent results in tumorgenic and cancerous cells suggest sea urchin embryo, similarly, Vasa protein over-expression did not that somatic cells that have been considered Vasa-null in their have a significant phenotype (Gustafson et al., 2011). Therefore, normal state could potentially induce Vasa expression through introducing excessive amounts of Vasa protein usually “simply” post-transcriptional regulation under certain stimuli. Indeed, in results in extra degradation of Vasa protein at each cell cycle, and the sea urchin embryo, Vasa mRNA and protein appears to be no developmental defects in these embryos. It should be noted consistently expressed at low levels in various types of somatic that Vasa does not appear to be sufficient for translational activity cells during normal development, but the protein can be upon its own; it may be rate limiting in some cells, and/or be part of a regulated ‘transiently’ from the same amount of mRNA during the complex of translational regulators that are upregulated upon formation of adult precursor tissues, wound-healing, or develop- stimuli that leads to increased translation of cellular mRNAs. Fur- mental re-programming, processes which require cells to undergo ther, a recent report in Drosophila with a series of deletions/mu- active proliferation and differentiation (Voronina et al., 2008; Ya- tations in the vasa gene demonstrated that multiple domains in jima and Wessel, 2015). During these regulatory processes, im- the N- and C-terminal regions of Vasa protein are important for its portantly, the number of cells that entered into M-phase was in- localization in the germ line and for its functions in transposon creased 2-fold (Yajima and Wessel, 2015). Vasa might thus assist repression, posterior patterning, and pole cell specification. No- cells to temporarily up-regulate translation of molecules essential tably, some of these engineered mutant -Vasa proteins resulted in for cell proliferation. In somatic lineages, therefore, Vasa might be the increase or decrease of Vasa protein accumulation in the germ essential for ‘a transient induction’ of active cell proliferation line but not in the somatic cells, suggesting again a simple mu- through its translational activities that normally occur in the germ tation of the gene or inhibition of degradation pathway is in- line. sufficient to induce Vasa accumulation in the soma (Dehghani and In the germ line and gonads, on the other hand, Vasa protein is Lasko, 2015). Embryos thus appear to have a high fidelity system to enriched and functional, yet these cells typically divide slowly. keep titrated levels of Vasa protein, especially in the somatic cells. This may be because of different binding partners of Vasa or other This strong post-transcriptional and -translational regulation of more dominating factors that have inhibitory effects on the Vasa's Vasa makes it difficult to artificially over-express Vasa proteins to function or cell cycle control in these cells. For instance, Nanos, the level where a phenotype may be apparent. another germ line factor, is known to inhibit cyclin-B mRNA Further, modifications of Vasa also appear critical for its dif- translation and functions as a cell cycle repressor, preventing the ferential activities in some cases. In Drosophila oogenesis, it has germ cells from cell cycle progression (Barker et al., 1992; Asaoka been reported that phosphorylated-Vasa activity is required for et al., 1999; Kadyrova et al., 2007; Juliano et al., 2010). Whether the translation of Gurken (Grk), which is responsible for axis specifi- somatic cells described above also recover this regulatory me- cation of oocytes (Ghabrial and Schupbach, 1999; Klattenhoff et al., chanism ‘transiently’ to manipulate the level of Vasa activities for 2007). In the DNA-repair mutants (e.g. spindle-B/DMC1/RAD51- proper embryogenesis or regeneration has yet to be tested. An- like, spindle-C and okra/Rad54) or the repeat-associated small other possibility is that Vasa needs to be modified in order to be interfering RNA (rasiRNA) pathway mutants (e.g. armitage, spin- functional in some somatic cells (as described in the next section). dle-E, aubergine), Vasa protein appears to migrate distinctively on In tumorigenic cells, however, this regulatory mechanism may be a poly-acrylamide gel relative to wild-type ovary samples. This inactivated. Vasa over-expression will then not adequately limit retardation was reversed after phosphatase treatment, indicating the continual translation of Vasa's interactors such as cyclin-B and that the retarded mobility resulted from a phosphorylated form of J. Poon et al. / Developmental Biology 415 (2016) 24–32 29

Vasa. This retardation was also restored by an additional mutation Blower et al., 2007), suggesting potential translational activities on in the double-strand-break signaling (e.g. mei-41, mnk), which is the mitotic apparatus during M-phases. The large cells of 100– activated in the presence of DNA-damage or -double-strand 1000 mm such as eggs require distinct mechanisms of molecular breaks. Therefore, in the DNA-repair and rasiRNA mutants, per- regulation; simple expression and diffusion of molecules may not sistence of double-strand break activates the DNA-damage accomplish sufficient distribution of molecules to each daughter checkpoint pathway, which results in phosphorylation of Vasa and cell during rapid cell divisions of a large-sized cell (e.g. every 30– then blocks efficient translation of Grk, leading to axial patterning 40 min per cell division for Xenopus and sea urchin embryos). defects. These results suggest that Vasa may be differentially Localized translation on the mitotic apparatus is therefore con- modified in each lineage or developmental stage for its differential sidered to provide a mechanism important for efficient and precise activities. In support of this idea, recent efforts in Drosophila to protein production necessary for rapid cell divisions and differ- identify functional regions of Vasa in vivo (Dehghani and Lasko, ential cell fate determinations during embryogenesis. In these 2015) demonstrated that many of the Vasa mutations that would embryonic cells, Vasa may recruit various mRNAs to the mitotic be expected to abrogate DEAD-box helicase function localized and apparatus and help their interactions with translational regulators functioned appropriately in oogenesis, but mis-functioned in em- during M-phase. By doing so, Vasa could regulate translation in a bryonic patterning, pole cell specification, grk activation, or temporally and spatially controlled-manner during embryogen- transposon repression. Therefore, Vasa uses different domains and esis. This hypothesis has to be tested by analyzing the subcellular modifications for each of its differential activities during devel- function of Vasa on the mitotic apparatus in developing embryos. opment. This fact implies that post-transcriptional modifications This can be accomplished by recruiting Vasa localization to the of Vasa are essential for its differential activities in each lineage ectopic sub-cellular region to sequester Vasa from the mitotic and/or role. Vasa (DDX4) protein also appears phosphorylated in apparatus. One could then analyze if the sequestered Vasa induces sea urchins (Guo et al., 2015), and potentially in mice and humans ectopic localized translation, and how that could result in changes based on the search in the PhosphoSite proteomic database of overall translation efficiency, cell divisions, and embryonic de- (http://www.phosphosite.org; Gustafson and Wessel, 2010), yet no velopment. This sub-cellular level of analysis will be essential in detailed analysis has been provided especially in somatic cells. In the future to delineate the potential functions of Vasa as a loca- the future, accomplishing the phosphor-proteomics analysis in the lized translational regulator during embryogenesis. germ line, embryonic cells, regenerative tissues, and in tumor cells These actively dividing embryonic cells as well as tumorgenic will likely reveal if Vasa undergoes differential modifications for cells are also known to use distinct mechanisms of energy meta- each cellular process and for each development stage. The iden- bolism. Differentiated cells with slower cell cycling typically use tified Vasa phosphorylation sites could then be mutated to test if oxidative phosphorylation for energy generation, which produces any induce differential functions in each cell type. These detailed 32–38 mol ATP/mol glucose. Rapidly dividing embryonic cells, on functional assays will be essential to reveal differential functional the other hand, have altered metabolisms that rely more on mechanisms of Vasa. anaerobic glycolysis (Warburg-like metabolism; Warburg, 1930, 1956a, 1956b; Kondoh et al., 2007; Hsu and Sabatini, 2008), which produces only 2 mol ATP/mol glucose. This altered pathway is 5. Hypothesis: potential functions of Vasa in the soma hypothesized to allow the diversion of glycolytic intermediates into various biosynthetic pathways, including those generating Both in germ line and somatic lineages, Vasa is sometimes nucleotides and amino acids, facilitating the biosynthesis of the found in granule-like aggregations. Recent work in the insect macromolecules and organelles required for assembly of new cell ovarian cell line suggests that Vasa functions as a transient am- proliferation (DeBerardinis et al., 2008; Vander Heiden et al., plifier complex that mediates biogenesis of secondary piRNAs with 2009). Perhaps, in the environment of anaerobic glycolysis with Piwi proteins (Xiol et al., 2014). Vasa forms a tight clamp (inter- low-ATP production, Vasa may be essential for efficient translation action) on the RNA to anchor the amplifier onto transposon tran- using minimal amounts of ATP to meet the required level of pro- scripts to inhibit their activities in the germ line at the perinuclear tein synthesis during rapid embryonic cell divisions. A recent re- region. Although this RNA binding activity of Vasa has not been port in Drosophila demonstrated a functional involvement of germ tested in the soma, members of this amplifier complex, Piwi pro- line factors in the glycolytic pathway; glycolytic enzymes (e.g. teins, have been also recently proposed to work in somatic stem Aldolase, GAPDH, Phosphoglycerate Kinase, Enolase, and Pyruvate cells (Ross et al., 2014). This casts an interesting insight that Vasa Kinase) were found to be components of germ plasm. Importantly, and Piwi may function in a similar manner for quality control of GAPDH was enriched with a germ line specific Tudor/Vasa com- the genomes in stem cells. In the sea urchin embryo, similarly, it plex, and Pyruvate Kinase directly interacted with Tudor in germ was found that Vasa is essential for the clustering of various cells. Further, knockdown of Enolase or Pyruvate Kinase resulted in mRNAs to the nuclear envelope and to the mitotic apparatus. reduction of piRNAs, and compromised germ cell formation. Further, Vasa knockdown results in 80% reduction of total pro- Through the interaction with germ line factors, therefore, glyco- tein synthesis in these embryos (Yajima and Wessel, 2015). These lytic pathway components appear to function in protecting the results suggest again that Vasa's primary function may be in genome from transposable elements and in germ cell formation clustering of various mRNAs to a proper sub-cellular localization (Gao et al., 2015). Since Tudor and Vasa are reported to function (e.g. mitotic apparatus) for precise and efficient translation. This is together in the germ cells (Arkov et al., 2006), it will be intriguing likely essential for the quality control of embryonic development to test if Vasa is involved in this process of metabolic pathway. in which each blastomere in a single embryo undergoes robust but Further, it is important to identify if Vasa is in any way associated different translation for differential cell fate determination. Indeed, with a particular metabolic state of the cell (e.g. glucose con- temporally and spatially controlled mRNA translation on the mi- sumption rate, and generation of lactate and ATP) during regula- totic apparatus has been proposed as an important biological tion of embryonic and tumorgenic cells. This can be measured by event for proper embryonic cell regulation (Raff et al., 1990; Gross conventional HPLC analysis and enzymatic assays, or by Mass and Fry, 1966; Blower et al., 2007). spectrometry-based metabolomics that can provide broad in- In Xenopus and sea urchin embryos, ribosomes and translation formation on the chemical composition of complex (metabolomes) factors are reported to be tightly associated to the mitotic appa- in cell extracts. The outcomes of these experiments may reflect ratus (Suprenant, 1993; Liska et al., 2004; Mitchison et al., 2004; Vasa's “employment” during minimal ATP-generation, perhaps for 30 J. Poon et al. / Developmental Biology 415 (2016) 24–32 more efficient translational contributions during embryogenesis tumorigenesis as an important translation factor when expressed and tumorigenesis. in the context of other translation factors of the cancer cell phenotype. Considering that Vasa's post-transcriptional modifications are critical to development of various somatic cells, it is important 6. Additional future questions to rely on a protein level database for analyzing Vasa function in cancer cells. Other germ line factors may also be resolved with Many cells and organisms appear to have a tightly regulated such an approach. In concert with metabolomics studies, one system for Vasa expression, especially in the soma, implying a could then potentially predict even more close associations with broad biological importance. In tumorgenic cells where cellular dynamics are often altered, on the other hand, this tight control tumorigenesis. over Vasa may be lacking, allowing cells to accumulate Vasa and In conclusion, most cells of the body do not have detectable its target factors (e.g. cell cycle materials) more effectively to level of Vasa, but when Vasa protein is expressed in somatic and/ contribute to a part of tumorigenic processes. Investigating the or tumorgenic cells, Vasa is essential for cellular regulation. We mechanisms of Vasa regulation in naturally Vasa-expressing tu- think of this as a Vasa addiction that perhaps alters the cellular morgenic cells may be thus helpful to understand the nature of metabolism, including translational regulation. Were Vasa protein this molecule function in the cells. Further, identifying “upstream altered in those addicted cells, the cell then crashes and burns. regulator(s)” of Vasa in these tumorigenic cells could also decipher Such theoretical compensatory regulation within these cells will how this molecule may be tightly regulated for its transient inform us as to how Vasa fundamentally functions in the cell. function during normal development, especially in the soma. Further study of the determinants of Vasa localization and activity DDX4 (Vasa ortholog in humans) mutations in the cancer cell in various somatic cells including cancerous cells should help genome have been reported so far in 55 cancer cell types, which elucidate why ectopic Vasa expression can be detrimental. include prostate, ovarian, breast, glioma, melanoma, uterine, and many other cancer cell lines or tissues (Fig. 2, Identified by the cBioPortal search; Gao et al., 2013; Cerami et al., 2012). Through this genomic mutation-based search, however, the cause or con- Acknowledgements sequence of these DDX4 mutations in cancer cells appears rather random; some cancer cell lines such as prostate cancer cells lack ddx4, whereas other cells such as breast cancer cell lines duplicate This work was supported by the American Heart Association ddx4 genes in the genome. It is thus unlikely that DDX4 is a cause Scientist Development Grant-14SDG18350021 to M.Y. and by the of cancer initiation nor is consistently involved in a major step National Institutes of Health-2R01HD028152 and the National of cancer cell development, yet rather might contribute to Science Foundation- IOS-1120972. grants to G.M.W.

Fig. 2. The cBioPortal (http://www.cbioportal.org/) database search result. Cancer cell types that contain mutations in the DDX4 (Vasa) gene in the genome are listed. X-axis lists cancer type, and Y-axis indicates the frequency of the alterations in the cancers. Mutations of DDX4 in the genome is indicated in green, deletion in blue, ampliﬁcation in red, and multiple alterations in gray colored balls indicate cancer cell types. The text in parenthesis indicates the original data source of each cancer cell. J. Poon et al. / Developmental Biology 415 (2016) 24–32 31

References cell differentiation and maintenance. Mol. Reprod. Dev. 81 (10), 946–961. Hashimoto, H., Sudo, T., Mikami, Y., Otani, M., Takano, M., Tsuda, H., Itamochi, H., Katabuchi, H., Ito, M., Nishimura, R., 2008. Germ cell specific protein VASA is Akahira, J., Sugihashi, Y., Suzuki, T., Ito, K., Niikura, H., Moriya, T., Nitta, M., Okamura, over-expressed in epithelial ovarian cancer and disrupts DNA damage-induced H., Inoue, S., Sasano, H., Okamura, K., Yaegashi, N., 2004. Decreased expression G2 checkpoint. Gynecol. Oncol. 111, 312–319. of 14-3-3 sigma is associated with advanced disease in human epithelial Hay, B., Jan, L.Y., Jan, Y.N., 1988. A protein component of Drosophila polar granules is ovarian cancer: its correlation with aberrant DNA methylation. Clin. Cancer Res. encoded by vasa and has extensive sequence similarity to ATP-dependent he- – 10, 2687 2693. licases. Cell 55, 577–587. Arkov, A.L., Wang, J.Y., Ramos, A., Lehmann, R., 2006. The role of Tudor domains in Hay, B., Jan, L.Y., Jan, Y.N., 1990. Localization of vasa, a component of Drosophila germline development and polar granule architecture. Development 133, polar granules, in maternal-effect mutants that alter embryonic anteroposterior – 4053 4062. polarity. Development 109, 425–433. Asaoka, M.T., Yamada, M., Nakamura, A., Hanyu, K., Kobayashi, S., 1999. Maternal Hsu, P.P., Sabatini, D.M., 2008. Cancer cell metabolism: Warburg and beyond. Cell Pumilio acts together with Nanos in germline development in Drosophila 134, 703–707. – embryos. Nat. Cell Biol. 1, 431 437. Ikenishi, K., Yamakita, S., 2003. A trial for induction of supernumerary primordial Barker, D.D., Wang, C., Moore, J., Dickinson, L.K., Lehmann, R., 1992. Pumilio is es- germ cells in Xenopustadpoles by injecting RNA of Xenopus vasa homologue sential for function but not for distribution of the Drosophila abdominal de- into germline cells of 32-cell embryos. Dev. Growth Differ. 45 (5–6), 417–426. – terminant Nanos. Genes Dev. 6, 2312 2326. Ikenishi, K., Tanaka, T., Komiya, T., 1996. Spatio-temporal distribution of the protein Blower, M., Feric, E., Weis, K., Heald, R., 2007. Genome-wide analysis demonstrates of the Xenopus vasa homologue (Xenopus vasalike gene 1, XVLG1) in embryos. conserved localization of messenger RNAs to mitotic microtubules. J. Cell Biol. Dev. Growth Differ. 38, 527–535. – 179, 1365 1373. Illmensee, K., Mahowald, A.P., 1974. Transplantation of posterior polar plasm in Braat, A., Zandbergen, T., van de Water, S., Goos, H., Zivkovic, D., 1999. Character- Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Natl. fi ization of zebra sh primordial germ cells: morphology and early distribution of Acad. Sci. USA 71 (4), 1016–1020. – vasa RNA. Dev. Dyn. 216, 153 167. Iwata, N., Yamamoto, H., Sasaki, S., Itoh, F., Suzuki, H., Kikuchi, T., Kaneto, H., Iku, S., fi Braat, A.K., van de Water, S., Korving, J., Zivkovic, D., 2001. A zebra sh vasa mor- Ozeki, I., Karino, Y., Satoh, T., Toyota, J., Satoh, M., Endo, T., Imai, K., 2000. Fre- phant abolishes vasa protein but does not affect the establishment of the quent hypermethylation of CpG islands and loss of expression of the 14-3-3 – germline. Genesis 30, 183 185. sigma gene in human hepatocellular carcinoma. Oncogene 19, 5298–5302. Braat, A.K., van de Water, S., Goos, H., Bogerd, J., Zivkovic, D., 2000. Vasa protein Janic, A., Mendizabal, L., Llamazares, S., Rossell, D., Gonzalez, C., 2010. Ectopic ex- expression and localization in the zebrafish. Mech. Dev. 95 (1–2), 271–274. pression of germline genes drives malignant brain tumor growth in Drosophila. ̈ Carrera, P., Johnstone, O., Nakamura, A., Casanova, J., Jackle, H., Lasko, P., 2000. Vasa Science 330, 1824–1827. mediates translation through interaction with a Drosophila yIF2 homolog. Mol. Johnstone, O., Lasko, P., 2004. Interaction with eIF5B is essential for Vasa function Cell 5, 181–187. during development. Development 131, 4167–4178. Cerami, E., Gao, J., Dogrusoz, U., Gross, B.E., Sumer, S.O., Aksoy, B.A., Jacobsen, A., Juliano, C.E., Yajima, M., Wessel, G.M., 2010. Nanos functions to maintain the fate of Byrne, C.J., Heuer, M.L., Larsson, E., Antipin, Y., Reva, B., Goldberg, A.P., Sander, the small micromere lineage in the sea urchin embryo. Dev. Biol. 337, 220–232. C., Schultz, N., 2012. The cBio cancer genomics portal: an open platform for Kadyrova, L.Y., Habara, Y., Lee, T.H., Wharton, R.P., 2007. Translational control of exploring multidimensional cancergenomics data. Cancer Discov. 2 (5), maternal Cyclin B mRNA by Nanos in the Drosophila germline. Development 401–404. 134 (8), 1519–1527. Chan, T.A., Hermeking, H., Lengauer, C., Kinzler, K.W., Vogelstein, B., 1999. 14-3- Kim, K.H., Kang, Y.J., Jo, J.O., Ock, M.S., Moon, S.H., Suh, D.S., Yoon, M.S., Park, E.S., 3sigma is required to prevent mitotic catastrophe after DNA damage. Nature Jeong, N., Eo, W.K., Kim, H.Y., Cha, H.J., 2014. DDX4 (DEAD box polypeptide 4) 401, 616–620. colocalizes with cancer stem cell marker CD133 in ovarian cancers. Biochem. Curran, S.P., Wu, X., Riedel, C.G., Ruvkun, G., 2009. A soma-to-germline transfor- Biophys. Res. Commun. 447, 315–322. mation in long-lived Caenorhabditis elegans mutants. Nature 459, 1079–1084. Klattenhoff, C., Theurkauf, W., 2008. Biogenesis and germline functions of piRNAs. DeBerardinis, R.J., Lum, J.J., Hatzivassiliou, G., Thompson, C.B., 2008. The biology of Development 135, 3–9. cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Klattenhoff, C., Bratu, D.P., McGinnis-Schultz, N., Koppetsch, B.S., Cook, H.A., Metab. 7, 11–20. Theurkauf, W.E., 2007. Drosophila rasiRNA pathway mutations disrupt em- Dehghani, M., Lasko, P., 2015. In vivo mapping of the functional regions of the bryonic axis specification through activation of an ATR/Chk2 DNA damage re- DEAD-box helicase Vasa. Biol. Open 4 (4), 450–462. sponse. Dev. Cell 2 (1), 45–55. Dill, K.K., Seaver, E.C., 2008. Vasa and nanos are coexpressed in somatic and germ Kondoh, H., Lleonart, M.E., Nakashima, Y., Yokode, M., Tanaka, M., Bernard, D., Gil, J., line tissue from early embryonic cleavage stages through adulthood in the Beach, D., 2007. A high glycolytic flux supports the proliferative potential of polychaete Capitella sp. I. Dev. Genes Evol. 218, 453–463. murine embryonic stem cells. Antioxid. Redox Signal. 9, 293–299. Ferguson, A.T., Evron, E., Umbricht, C.B., Pandita, T.K., Chan, T.A., Hermeking, H., Kosaka, K., Kawakami, K., Sakamoto, H., Inoue, K., 2007. Spatiotemporal localization Marks, J.R., Lambers, A.R., Futreal, P.A., Stampfer, M.R., Sukumar, S., 2000. High of germ plasm RNAs during zebrafish oogenesis. Mech. Dev. 124 (4), 279–289. frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silen- Kugler, J.M., Woo, J.S., Oh, B.H., Lasko, P., 2010. Regulation of Drosophila Vasa in vivo cing in breast cancer. Proc. Natl. Acad. Sci. USA 97, 6049–6054. through paralogous Cullin-RING E3 ligase specificity receptors. Mol. Cell Biol. Gao, J., Aksoy, B.A., Dogrusoz, U., Dresdner, G., Gross, B., Sumer, S.O., Sun, Y., Ja- 30, 1769–1782. cobsen, A., Sinha, R., Larsson, E., Cerami, E., Sander, C., Schultz, N., 2013. In- Kuznicki, K.A., Smith, P.A., Leung-Chiu, W.M., Estevez, A.O., Scott, H.C., Bennett, K.L., tegrative analysis of complex cancer genomics and clinical profiles using the 2000. Combinatorial RNA interference indicates GLH-4 can compensate for cBioPortal. Sci. Signal. 6 (269), pl1. GLH-1; these two P granule components are critical for fertility in C. elegans. Gao, M., Thomson, T.C., Creed, T.M., Tu, S., Loganathan, S.N., Jackson, C.A., McClus- Development 127, 2907–2916. key, P., Lin, Y., Collier, S.E., Weng, Z., Lasko, P., Ohi, M.D., Arkov, A.L., 2015. Lasko, P.F., Ashburner, M., 1988. The product of the Drosophila gene vasa is very Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ similar to eukaryotic initiation factor-4A. Nature 335, 611–617. cells, bind Tudor and protect from transposable elements. EMBO Rep. 16, Lasko, P.F., Ashburner, M., 1990. Posterior localization of vasa protein correlates 379–386. with, but is not sufficient for, pole cell development. Genes Dev. 4, 905–921. Gateff, E., Löffler, T., Wismar, J., 1993. A temperature-sensitive brain tumor sup- Lasko, P., 2013. The DEAD-box helicase Vasa: evidence for a multiplicity offunctions pressor mutation of Drosophila melanogaster: developmental studies and mo- in RNA processes and developmental biology. Biochem. Biophys. Acta 1829, lecular localization of the gene. Mech. Dev. 41 (1), 15–31. 810–816. Ghabrial, A., Schüpbach, T., 1999. Activation of a meiotic checkpoint regulates Li, C.J., Liu, L., Chen, X.H., Zhang, T., Gan, F., Cheng, B.L., 2010. Identification of a vasa translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1 (6), homologue gene in grass carp and its expression pattern in tissues and during 354–357. embryogenesis. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 157 (2), 159–166. Gross, P.R., Fry, B.J., 1966. Continuity of protein synthesis through cleavage meta- Li, M., Hong, N., Xu, H., Yi, M., Li, C., Guia, J., Hong, Y., 2009. Medaka vasa is required phase. Science 153 (3737), 749–751. for migration but not survival of primordial germ cells. Mech. Dev. 126, Gruidl, M.E., Smith, P.A., Kuznicki, K.A., McCrone, J.S., Kirchner, J., Roussell, D.L., 366–381 (å). Strome, S., Bennett, K.L., 1996. Multiple potential germline helicases are com- Linder, P., 2006. Dead-box proteins: a family affair–active and passive players in ponents of the germ-line-specific P granules of Caenorhabditis elegans. Proc. RNP-remodeling. Nucleic Acids Res. 34, 4168–4180. Natl. Acad. Sci. USA 93, 13837–13842. Linder, P., Lasko, P.F., Ashburner, M., Leroy, P., Nielsen, P.J., Nishi, K., Schnier, J., Guo, H., Garcia-Vedrenne, A.E., Isserlin, R., Lugowski, A., Morada, A., Sun, A., Miao, Slonimski, P.P., 1989. Birth of the D-E-A-D box. Nature 337, 121–122. Y., Kuzmanov, U., Wan, C., Ma, H., Foltz, K., Emili, A., 2015. Phosphoproteomic Liska, A.J., Popov, A.V., Sunyaev, S., Coughlin, P., Habermann, B., Shevchenko, A., network analysis in the sea urchin Strongylocentrotus purpuratus reveals new Bork, P., Karsenti, E., Shevchenko, A., 2004. Homology-based functional pro- candidates in egg activation. Proteomics 15 (23–24), 4080–4095. teomics by mass spectrometry: application to the Xenopus microtubule asso- Gustafson, E.A., Wessel, G.M., 2010. DEAD-box helicases: posttranslational regula- ciated proteome. Proteomics 4, 2707–2721. tion and function. Biochem. Biophys. Res. Commun. 395, 1–6. Liu, N., Dansereau, D.A., Lasko, P., 2003. Fat facets interacts with vasa in the Dro- Gustafson, E.A., Yajima, M., Juliano, C.E., Wessel, G.M., 2011. Post-translational sophila pole plasm and protects it from degradation. Curr. Biol. 13, 1905–1909. regulation by gustavus contributes to selective Vasa protein accumulation in Liu, N., Han, H., Lasko, P., 2009. Vasa promotes Drosophila germline stem cell dif- multipotent cells during embryogenesis. Dev. Biol. 349, 440–450. ferentiation by activating mei-P26 translation by directly interacting with a Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell (U)-rich motif in its 3′ UTR. Genes Dev. 23, 2742–2752. 144, 646–674. Mitchison, T.J., Maddox, P., Groen, A., Cameron, L., Perlman, Z., Ohi, R., Desai, R., Hartung, O., Forbes, M.M., Marlow, F.L., 2014. Zebrafish vasa is required for germ Salmon, E.D., Kapoor, T.M., 2004. Bipolarization and poleward flux correlate 32 J. Poon et al. / Developmental Biology 415 (2016) 24–32

during Xenopus extract spindle assembly. Mol. Biol. Cell 15, 5603–5615. Spike, C., Meyer, N., Racen, E., Orsborn, A., Kirchner, J., Kuznicki, K., Yee, C., Bennett, Mochizuki, K., Nishimiya-Fujisawa, C., Fujisawa, T., 2001. Universal occurrence of K., Strome, S., 2008. Genetic analysis of the Caenorhabditis elegans GLH family the vasa-related genes among metazoans and their germline expression in of P-granule proteins. Genetics 178, 1973–1987. Hydra. Dev. Genes Evol. 211 (6), 299–308. Styhler, S., Nakamura, A., Lasko, P., 2002. VASA localization requires the SPRY-do- Nguyen, L.B., Diskin, S.J., Capasso, M., Wang, K., Diamond, M.A., Glessner, J., Kim, C., main and SOCS-box containing protein. GUSTAVUS. Dev. Cell 3, 865–876. Attiyeh, E.F., Mosse, Y.P., Cole, K., Iolascon, A., Devoto, M., Hakonarson, H., Li, H. Styhler, S., Nakamura, A., Swan, A., Suter, B., Lasko, P., 1998. vasa is required for K., Maris, J.M., 2011. Phenotype restricted genome-wide association study using GURKEN accumulation in the oocyte, and is involved in oocyte differentiation a gene-centric approach identifies three low-risk neuroblastoma susceptibility and germline cyst development. Development 125, 1569–1578. Loci. PLoS Genet. 7, e1002026. Suprenant, K.A., 1993. Microtubules, ribosomes, and RNA: evidence for cytoplasmic Orsborn, A.M., Li, W., McEwen, T.J., Mizuno, T., Kuzmin, E., Matsumoto, K., Bennett, localization and translational regulation. Cell Motil. Cytoskelet. 25 (1), 1–9. K.L., 2007. GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB- Tanaka, S.S., Toyooka, Y., Akasu, R., Katoh-Fukui, Y., Nakahara, Y., Suzuki, R., Yo- 1 and by the COP9 subunit CSN-5. Development 134 (18), 3383–3392. koyama, M., Noce, T., 2000. The mouse homolog of Drosophila Vasa is required Osada, H., Tatematsu, Y., Yatabe, Y., Nakagawa, T., Konishi, H., Harano, T., Tezel, E., for the development of male germ cells. Genes Dev. 14, 841–853. Takada, M., Takahashi, T., 2002. Frequent and histological type-specific in- Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., Noce, T., 2000. Isolation of chicken activation of 14-3-3sigma in human lung cancers. Oncogene 21, 2418–2424. vasa homolog gene and tracing the origin of primordial germ cells. Develop- Patil, V.S., Kai, T., 2010. Repression of retroelements in Drosophila germline via ment 127, 2741–2750. piRNA pathway by the Tudor domain protein Tejas. Curr. Biol. 20, 724–730. Vander Heiden, M.G., Cantley, L.C., Thompson, C.B., 2009. Understanding the Pek, J.W., Kai, T., 2011. A role for vasa in regulating mitotic chromosome con- Warburg effect: the metabolic requirements of cell proliferation. Science 324, densation in Drosophila. Curr. Biol. 21, 39–44. 1029–1033. Pestova, T.V., Lomakin, I.B., Lee, J.H., Choi, S.K., Dever, T.E., Hellen, C.U.T., 2000. The Voronina, E., Lopez, M., Juliano, C.E., Gustafson, E., Song, J.L., Extavour, C., George, S., joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, Oliveri, P., McClay, D., Wessel, G.M., 2008. Vasa protein expression is restricted 332–335. to the small micromeres of the sea urchin, but is inducible in other lineages Raff, J.W., Whitfield, W.G., Glover, D.M., 1990. Two distinct mechanisms localize early in development. Dev. Biol. 314, 276–286. cyclin B transcripts in syncytial Drosophila embryos. Development 110, Wagner, D.E., Ho, J.J., Reddien, P.W., 2012. Genetic regulators of a pluripotent adult 1249–1261. stem cell system in planarians identified by RNAi and clonal analysis. Cell Stem Raz, E., 2000. The function and regulation of vasa-like genes in germ-cell devel- Cell 10, 299–311. opment. Genome Biol. 1 (3), Reviews 1017.1. Wang, D., Kennedy, S., Conte, D.J., Kim, J.K., Gabel, H.W., Kamath, R.S., Mello, C.C., Rebscher, N., Zelada-Gonzalez,́ F., Banisch, T.U., Raible, F., Arendt, D., 2007. Vasa Ruvkun, G., 2005. Somatic misexpression of germline P granules and enhanced unveils a common origin of germ cells and of somatic stem cells from the RNA interference in retinoblastoma pathway mutants. Nature 436, 593–597. posterior growth zone in the polychaete Platynereis dumerilii. Dev. Biol. 306, Warburg, O.H., 1956a. On the origin of cancer cells. Science 123, 309–314. 599–611. Warburg, O.H., 1956b. On respiratory impairment in cancer cells. Science 124, Renault, A.D., 2012. vasa is expressed in somatic cells of the embryonic gonad in a 269–270. sex-specificmanner in Drosophila melanogaster. Biol. Open 1 (10), 1043–1048. Warburg, O.H., 1930. The Metabolism of Tumours: Investigations from the Kaiser Ross, R.J., Weiner, M.M., Lin, H., 2014. PIWI proteins and PIWI-interacting RNAs in Wilhelm Institute for Biology, Berlin-Dahlem, London, UK, Arnold Constable. the soma. Nature 505, 353–359. Wong, M.D., Jin, X., Xie, T., 2005. Molecular mechanisms of germline stem cell Roussell, D.L., Bennett, K.L., 1993. glh-1, a germ-line putative RNA helicase from regulation. Annu. Rev. Genet. 39, 173–195. Caenorhabditis, has four zinc fingers. Proc. Natl. Acad. Sci. USA 90, 9300–9304. Woo, J.S., Suh, H.Y., Park, S.Y., Oh, B.H., 2006b. Structural basis for protein re- Schupbach, T., Wieschaus, E., 1986. Maternal-effect mutations altering the anterior- cognition by B30.2/SPRY domains. Mol. Cell 24, 967–976. posterior pattern of the Drosophila embryo. Dev. Biol. 113, 443–448. Woo, J.S., Imm, J.H., Min, C.K., Kim, K.J., Cha, S.S., Oh, B.H., 2006a. Structural and Schwager, E.E., Meng, Y., Extavour, C.G., 2014. Vasa and piwi are required for mitotic functional insights into the B30.2/SPRY domain. EMBO J. 25, 1353–1363. integrity in early embryogenesis in the spider Parasteatoda tepidariorum. Dev. Xiol, J., Spinelli, P., Laussmann, M.A., Homolka, D., Yang, Z., Cora, E., Couté, Y., Conn, Biol. 402 (2), 276–290. S., Kadlec, J., Sachidanandam, R., Kaksonen, M., Cusack, S., Ephrussi, A., Pillai, R. Sengoku, T., Nureki, O., Nakamura, A., Kobayashi, S., Yokoyama, S., 2006. Structural S., 2014. RNA clamping by Vasa assembles a piRNA amplifier complex on basis for RNA unwinding by the DEAD-box protein Drosophila Vasa. Cell 125, transposon transcripts. Cell 157, 1698–1711. 287–300. Yajima, M., Wessel, G.M., 2011a. The DEAD-box RNA helicase Vasa functions in Shibata, N., Umesono, Y., Orii, H., Sakurai, T., Watanabe, K., Agata, K., 1999. Ex- embryonic mitotic progression in the sea urchin. Development 138, 2217–2222. pression of vasa(vas)-related genes in germline cells and totipotent somatic Yajima, M., Wessel, G.M., 2011b. The multiple hats of Vasa: its functions in the stem cells of planarians. Dev. Biol. 206, 73–87. germline and in cell cycle progression. Mol. Reprod. Dev. 78, 861–867. Shirae-Kurabayashi, M., Nishikata, T., Takamura, K., Tanaka, K.J., Nakamoto, C., Na- Yajima, M., Wessel, G.M., 2015. The germ line factor Vasa functions broadly in so- kamura, A., 2006. Dynamic redistribution of vasa homolog and exclusion of matic cells: mRNA clustering, translational regulation, and wound healing. somatic cell determinants during germ cell specification in Ciona intestinalis. Development 142 (11), 1960–1970. Development 133, 2683–2693. Yoon, C., Kawakami, K., Hopkins, N., 1997. Zebrafish vasa homologue RNA is loca- Simpson, A.J., Caballero, O.L., Jungbluth, A., Chen, Y.T., Old, L.J., 2005. Cancer/testis lized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in antigens, gametogenesis and cancer. Nat. Rev. Cancer 5, 615–625 (Review). the primordial germ cells. Development 124, 3157–3165.