In many animals, primordial germ cells (PGCs) migrate through the embryo toward the future gonad, a process guided by attractive and repulsive cues provided from surrounding somatic cells. In Drosophila, the two related lipid phosphate phosphatases (LPPs), Wunen (Wun) and Wun2, are thought to degrade extracellular attractive substrates and to act redundantly in somatic cells to provide a repulsive environment to steer the migration of PGCs, or pole cells. Wun and Wun2 also affect the viability of pole cells, because overexpression of either one in somatic cells causes pole cell death. However, the means by which they regulate pole cell migration and survival remains elusive. Wun2 has a maternal function required for the survival of pole cells during their migration to the gonad. Maternal wun2 RNA was found to be concentrated in pole cells and pole cell-specific expression of wun2 rescues the pole cell death phenotype of the maternal wun2 mutant, suggesting that wun2 activity in pole cells is required for their survival. Furthermore, genetic evidence was obtained that pole cell survival requires a proper balance of LPP activity in pole cells and somatic cells. In somatic cells, Wun and Wun2 may provide a repulsive environment for pole cell migration by depleting this extracellular, attractive substrate. Upon Wun2 expression, cultured insect cells dephosphorylate and internalize exogenously supplied lipid phosphate. It is proposed that Wun2 in pole cells competes with somatic Wun and Wun2 for a common lipid phosphate substrate, which is required by pole cells to produce their survival signal (Renault, 2004; Hanyu-Nakamura, 2004).

Extracellular lipid phosphates influence proliferation, programmed cell death, and the migration of various cell types. For example, in mammals lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are secreted by stimulated platelet cells leading to migratory and proliferative effects on smooth muscle cells, endothelial cells, and white blood cells. Signaling by means of such lipid phosphates not only is vital to normal development but also contributes to the progression of diseases such as tumorigenesis and atherosclerosis (Renault, 2004 and references therein).

In Drosophila, extracellular lipid phosphates have been implicated in guiding germ cell migration (Starz-Gaiano, 2001; Zhang, 1997). As in most organisms, Drosophila germ cells form spatially and temporally separate from the somatic cells of the gonad and must migrate through the embryo to associate with them. Drosophila germ cells form at the syncytial blastoderm stage, and during gastrulation they are carried into the posterior midgut pocket where they actively migrate through the midgut epithelium. Once on the basal side of the midgut, they reorient dorsally, which is important for the subsequent migration into the mesoderm, where they associate with the somatic gonadal precursors (SGPs) (Renault, 2004 and references therein).

The dorsal reorientation after crossing the gut results from the repellent activity of two redundant genes, wunen (wun) and wunen2 (wun2), which are zygotically expressed in regions of the midgut that germ cells avoid (Starz-Gaiano, 2001, Zhang, 1997). In embryos with no wun and wun2 in somatic tissues, most germ cells fail to reach the SGPs and instead scatter throughout the embryo (Starz-Gaiano, 2001, Zhang, 1997). In contrast, overexpression of either wun or wun2 in somatic tissues (Starz-Gaiano, 2001) results in germ cell death (Renault, 2004 and references therein).

wun and wun2 encode lipid phosphate phosphohydrolases (LPPs), membrane enzymes that dephosphorylate extracellular lipid phosphates. There are three mammalian LPPs (Roberts, 1998), and human LPP3 (hLPP3), but not mouse LPP1 (mLPP1); mammalian LPPs able to kill Drosophila germ cells when they are overexpressed in the soma (Burnett, 2003). Although in vivo substrates for LPPs have yet to be confirmed, in vitro substrates include the bioactive lipids S1P, LPA, phosphatidic acid (PA), and ceramide 1-phosphate (Roberts, 1998; Renault, 2004).

Key to understanding the effects of these lipids is the identification of their receptors and downstream pathways. Mammalian cells respond to S1P and LPA through the G protein-coupled receptors (GPCRs) S1P_1-5 and LPA_1-4, respectively (Moolenaar, 1999). Although LPPs are found in vertebrates, insects, and worms, these GPCRs seem to be restricted to vertebrates (Chun, 2002), raising the possibility that additional lipid phosphate signaling pathways exist. While screening for such pathways in Drosophila germ cells, it was noticed that wun2 RNA, but not wun, is expressed in early germ cells (Renault, 2002). This expression is presumably due to selective stabilization of the maternal RNA, because early germ cells are transcriptionally inactive (Renault, 2004).

To test whether maternal wun2 expression is necessary for germ cell formation, migration, or survival, a wun2-null allele was generated. Embryos laid by wun2-null females, which cannot supply wun2 expression to the germ cells, were examined and the paternal chromosome was used to supply zygotic wun and wun2 expression to the soma. Such embryos formed normal numbers of germ cells, but germ cell numbers dropped from more than 30 to 9, on average, by late embryogenesis. The death phenotype is nonapoptotic and results specifically from the lack of maternal wun2 and not wun (Renault, 2004).

To determine whether the germ cell death caused by lack of maternal wun2 reflects a requirement for wun2 in germ cells or soma, the nanos::GAL4VP16 driver was used to express wun2 specifically in germ cells. Germ cell expression of wun2 is sufficient to rescue germ cell death in embryos laid by wun2-null mothers. Overexpression of wun2 in germ cells using this driver in a wild-type background causes slight migration defects (Starz-Gaiano, 2001), explaining the imperfect migration observed in the rescued embryos. In addition, germ cell expression of wun2 Y225W (Starz-Gaiano, 2001) (which contains a control substitution in a nonconserved residue), wun, and hLPP3 is able to rescue the death, but expression of wun2 H326K (a catalytically dead mutant form of wun2) or mLPP1 is not. This result indicates that the ability to function in germ cells parallels the ability to act in the soma. It is concluded that catalytically active wun2 is required in germ cells for their survival and that wun and hLPP3 can substitute for its function (Renault, 2004).

To test how the requirement of wun2 in germ cells relates to the function of wun/wun2 in the soma, the behavior of wun2-null germ cells was examined in embryos lacking somatic expression of wun/wun2. In such embryos, the germ cells showed only a slight reduction in number, comparable to the normal decrease seen in wild-type embryos. Although some germ cells migrated to the gonad, most were scattered, resembling the zygotic wun/wun2 loss of function phenotype in which germ cells scatter but survive (Zhang, 1997). Thus, death of wun2-null germ cells can be rescued by a reduction in the somatic expression of wun/wun2. To further examine the relationship between somatic and germ cell Wunens, an examination was performed to see whether the germ cell death resulting from somatic overexpression of wun2 could be suppressed. Germ cell expression of wun, wun2, wun2 Y225W, and hLPP3, but not wun2 H326K or mLPP1, can suppress the death from wun2 overexpression in the soma (Renault, 2004).

The data show that the same molecule has opposite effects on germ cell survival: Wun2 in germ cells protects them from death, whereas Wun/Wun2 in somatic cells repels and kills germ cells. In both germ and somatic cells, the effect of Wun2 on germ cell survival requires its phosphatase activity. Furthermore, there is a direct and dose sensitive relationship between somatic and germ cell Wunens: Germ cell death resulting from lack of germ cell wun2 can be rescued by reducing somatic wun/wun2, and germ cell death resulting from somatic overexpression of wun2 can be suppressed by increasing germ cell wun2 expression. These data strongly argue that germ cell wun2 and somatic wun/wun2 share the same function and are consistent with a model in which the soma and the germ cells compete for a common wun/wun2 substrate that is required to allow germ cells to survive (Renault, 2004).

To explore how Wun2 might be acting in germ cells to regulate their survival, Wun2 biochemical activity was examined. Wun2 was expressed in insect Hi5 cells and, using membrane fractions, it was determined that Wun2 dephosphorylates PA and LPA in vitro, similar to hLPP1 and Wun (Burnett, 2003). The predicted catalytically null Wun2 mutant forms, H274K or H326K, exhibited no phosphatase activity, whereas the non-conserved substitution, Y225W, retained high phosphatase activity. The fate of such lipids was analyzed using intact Hi5 cells and a PA analog, 1-Oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycerol-3-Phosphate (NBD-PA), that is fluorescently labeled on its lipid moiety. Cell-associated fluorescence increased by a factor of 3 to 4 in Wun2 wild-type or Wun2 Y225W-expressing cells compared with control. Expression of Wun2 H274K or H326K did not result in any increase in cell-associated fluorescence compared with the control. The localization of the internalized lipid was analyzed and it was found that Wun2 promotes rapid lipid accumulation in the cytoplasm, similar to hLPP1. Wun2 therefore confers the ability of cells to internalize lipid substrates concurrent with dephosphorylation (Renault, 2004).

It is suggested that Wun2 function in germ cells is to uptake a lipid by dephosphorylation and that this lipid, or a metabolite, is responsible for the survival of germ cells by binding an intracellular or membrane-bound target. Germ cells are unique in requiring this lipid for their survival, whereas the somatic cells do not (See supporting online material). It is further proposed that through their restricted expression pattern, the function of somatic wun/wun2 is to create a gradient of lipid phosphate that provides directional cues to the germ cells. Regions of high wun/wun2 expression correlate with lowest levels of lipid phosphate and are therefore unfavorable for germ cell survival. Germ cells follow the lipid phosphate gradient and migrate away from wun/wun2-expressing somatic cells. For wun2-null germ cells, if the soma expresses wun/wun2, the common phospholipid pool is depleted and germ cells die. If, on the other hand, the soma lacks wun/wun2, lipid phosphate levels remain high throughout the embryo and germ cells survive but mismigrate as a result of the loss of a gradient, which provided the spatial cues needed for correct migration. The fact that wun2-null germ cells survive in the absence of somatic wun/wun2 further suggests that at high phospholipid levels alternate mechanisms for lipid uptake may exist (Renault, 2004).

It is proposed that germ cell survival is controlled through competition between somatic and germ cell Wunens for an extracellular lipid phosphate. Because the same genes have opposite effects on germ cell survival when expressed in the germ line and soma, these observations represent a novel paradigm for cell survival and migration. It had been assumed that all lipid phosphate signaling occurs through GPCRs, but the data suggest an alternate or parallel pathway through which lipid phosphates can signal, namely by means of internalization through dephosphorylation by LPPs. This pathway may be conserved in vertebrates because the mitogenic responses of some mammalian cells to LPA are inconsistent with GPCR receptor activation (Hooks, 2001). In Drosophila, this pathway shows remarkable specificity for germ cell survival, because somatic cells seem to be insensitive to wun/wun2 levels. Although it is clear that wun and wun2 are critical in controlling germ cell migration and survival, their function is likely to be redundant with other pathways such as Hmgcr (Van Doren, 1998), as demonstrated by the ability of some germ cells to reach the gonad even in the absence of wun/wun2 signaling (Renault, 2004).

Recent studies have revealed striking similarities between the guidance cues regulating germ cell migration in Drosophila and vertebrates (Santos, 2004). Mouse germ cells also express an LPP, and analysis of its function may reveal further parallels between early germ cell behavior in flies and mice (Renault, 2004).

GENE STRUCTURE

cDNA clone length - 1594 for wun, 1982 for wun2

Bases in 5' UTR - 203 for wun, 717 for wun2

Exons - 6 for wun, 6 for wun-2

Bases in 3' UTR - 488 for wun, 212 for wun2

PROTEIN STRUCTURE

Amino Acids - 379 for Wun, 350 for Wun-2

Structural Domains

Comparison of the wun2 cDNA sequence with the database revealed that although the predicted protein has 80 additional residues at the N terminus, the remainder of the protein is 50% identical and 67% similar to Wun. In addition, genomic Southern blot analysis and partial sequencing revealed that the transcriptional start sites of the two genes are approximately 5 kb apart and that they are transcribed in opposite orientations. Both genes are Drosophila homologs of the mammalian protein phosphatidic acid phosphatase type 2 alpha2 (PAP 2alpha2, also known as lipid phosphate phosphohydrolase 1 - LPP1). wun2 is 38% identical and 52% similar to human PAP 2alpha2 over the core catalytic and transmembrane domains (Starz-Gaiano, 2001).

date revised: 20 October 2004

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