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Soma-Germ Line Competition for Lipid Phosphate Uptake Regulates Germ Cell Migration and Survival

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Science  24 Sep 2004:
Vol. 305, Issue 5692, pp. 1963-1966
DOI: 10.1126/science.1102421

Abstract

Lipid phosphates can act as signaling molecules to influence cell division, apoptosis, and migration. wunen and wunen2 encode Drosophila lipid phosphate phosphohydrolases, integral membrane enzymes that dephosphorylate extracellular lipid phosphates. wun and wun2 act redundantly in somatic tissues to repel migrating germ cells, although the mechanism by which germ cells respond is unclear. Here, we report that wun2 also functions in germ cells, enabling them to perceive the wun/wun2–related signal from the soma. Upon Wun2 expression, cultured insect cells dephosphorylate and internalize exogenously supplied lipid phosphate. We propose that Drosophila germ cell migration and survival are controlled by competition for hydrolysis of a lipid phosphate between germ cells and soma.

Extracellular lipid phosphates influence proliferation, programmed cell death, and the migration of various cell types. For example, 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 (1, 2). Signaling by means of such lipid phosphates not only is vital to normal development (3, 4) but also contributes to the progression of diseases such as tumorigenesis and atherosclerosis (5, 6).

In Drosophila, extracellular lipid phosphates have been implicated in guiding germ cell migration (7, 8). 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 (9). 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).

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 (fig. S1A) (7, 8). In embryos with no wun and wun2 in somatic tissues, most germ cells fail to reach the SGPs and instead scatter throughout the embryo (7, 8). In contrast, overexpression of either wun or wun2 in somatic tissues results in germ cell death (7).

wun and wun2 encode lipid phosphate phosphohydrolases (LPPs), membrane enzymes that dephosphorylate extracellular lipid phosphates. There are three mammalian LPPs (10), and human LPP3 (hLPP3), but not mouse LPP1 (mLPP1), is able to kill Drosophila germ cells when it is overexpressed in the soma (11). Although in vivo substrates for LPPs are yet to be confirmed, in vitro substrates include the bioactive lipids S1P, LPA, phosphatidic acid (PA), and ceramide 1-phosphate (10).

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) S1P1-5 and LPA1-4, respectively (12). Although LPPs are found in vertebrates, insects, and worms, these GPCRs seem to be restricted to vertebrates (13), raising the possibility that additional lipid phosphate signaling pathways exist. While screening for such pathways in Drosophila germ cells, we noticed that wun2 RNA, but not wun, is expressed in early germ cells (14). This expression is presumably due to selective stabilization of the maternal RNA, because early germ cells are transcriptionally inactive (15).

To test whether maternal wun2 expression is necessary for germ cell formation, migration, or survival, we generated a wun2-null allele (16) (fig. S1B). We examined embryos laid by wun2-null females, which cannot supply wun2 expression to the germ cells, and used the paternal chromosome to supply zygotic wun and wun2 expression to the soma. Such embryos formed normal numbers of germ cells, but their numbers dropped from more than 30 to 9, on average, by late embryogenesis (Fig. 1, A, C, and F). The death phenotype is nonapoptotic and results specifically from the lack of maternal wun2 and not wun (see SOM Text).

Fig. 1.

Wun2 is required in germ cells for their survival. (A) Average number of germ cells per embryo at different developmental stages laid by wun2-null mothers [EP2650ex34 in trans to Df(2R)wunGL, which removes wun and wun2] mated to males carrying Df(2R)wunGL in trans to a lacZ marked wild-type chromosome. wun2-null germ cells die in embryos that inherit a wild-type chromosome from the male [LacZ-positive embryos (C) and (F) and blue bars in (A)], whose soma expresses wun/wun2 zygotically. Some germ cells survive and reach the gonad, but this survival does not depend on SGPs (see SOM Text). However, wun2-null germ cells survive but mismigrate in embryos that inherit the Df(2R)wunGL chromosome from the male [LacZ-negative embryos (D) and (G) and white bars in (A)], whose soma cannot express wun/wun2 zygotically. (B to M) Drosophila embryos (anterior left) with Vasa-stained germ cells (brown). (B and E) Wild-type embryos. (C, D, F, and G) Embryos from cross described for (A), also stained for lacZ (blue). (B to D) Stage 10 lateral view. (E to G) Stage 15 dorsal view. (H to M) Stage 16 embryos (dorsal view) laid by wun2-null mothers also containing the germ cell driver nanos::GAL4VP16 mated to UAS::lacZ (H), UAS::wun (I), UAS::wun2 (J), UAS::wun2 H326K (K), UAS::mLPP1 (L), and UAS::hLPP3 (M) males.

To determine whether the germ cell death caused by lack of maternal wun2 reflects a requirement for wun2 in germ cells or soma, we used the nanos::GAL4VP16 driver (17) to express wun2 specifically in germ cells. We found that germ cell expression of wun2 was sufficient to rescue germ cell death in embryos laid by wun2-null mothers (Fig. 1J). Overexpression of wun2 in germ cells using this driver in a wild-type background caused slight migration defects (7), explaining the imperfect migration observed in the rescued embryos. In addition, we found that germ cell expression of wun2 Y225W (7) (which contains a control substitution in a nonconserved residue), wun, and hLPP3 was able to rescue the death but that expression of wun2 H326K (a catalytically dead mutant form of wun2) or mLPP1 was not. This result indicates that the ability to function in germ cells parallels the ability to act in the soma (Fig. 1, H to M). We conclude that catalytically active wun2 is required in germ cells for their survival and that wun and hLPP3 can substitute for its function.

To test how the requirement of wun2 in germ cells relates to the function of wun/wun2 in the soma, we examined the behavior of wun2-null germ cells 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 (Fig. 1, A, D, and G). 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 (8). Thus, death of wun2-null germ cells can be rescued by a reduction in the somatic expression of wun/wun2 (compare Fig. 1, F and G). To further examine the relationship between somatic and germ cell Wunens, we examined whether we could suppress the germ cell death resulting from somatic overexpression of wun2. We found that germ cell expression of wun, wun2, wun2 Y225W, and hLPP3, but not wun2 H326K or mLPP1, could suppress the death from wun2 overexpression in the soma (Fig. 2).

Fig. 2.

Suppression of germ cell death due to wun2 overexpression in soma. Constitutively overexpressed somatic wun2 (from tubulin::wun2 transgene) kills germ cells. (A) Graph showing average number of germ cells in stage 15 and 16 wild-type (white bar) and tubulin::wun2 embryos misexpressing various genes (as UAS lines) in germ cells using the nanos::GAL4VP16 driver (shaded bars). Significant differences relative to the control UAS::CD8-GFP [analysis of variance (ANOVA), P < 0.01] are indicated with an asterisk. Note the partial rescue of number of germ cells when wun, wun2, and hLPP3, but not mLPP1 or wun2 H326K, are misexpressed in germ cells. (B to D) Stage 16 embryos with germ cells in brown (dorsal view, anterior left). (B) Wild-type embryo. Expression of wun2 (D) compared with the control CD8-GFP (C) in germ cells partially suppresses the germ cell death in tubulin::wun2 embryos. Germ cells are found singly (arrowhead) or clumped together (arrow), often associated with the gut. Not all germ cells are in the focal plane shown.

Our 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 dosesensitive 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 (Fig. 1, A, F, and G), and germ cell death resulting from somatic overexpression of wun2 can be suppressed by increasing germ cell wun2 expression (Fig. 2). 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.

To explore how Wun2 might be acting in germ cells to regulate their survival, we examined Wun2 biochemical activity. We expressed Wun2 in insect Hi5 cells and, using membrane fractions, determined that Wun2 dephosphorylates PA and LPA in vitro, similar to hLPP1 (table S1) and Wun (11). The predicted catalytically null Wun2 mutant forms, H274K or H326K, exhibited no phosphatase activity, whereas the non-conserved substitution, Y225W, retained high phosphatase activity (table S1). We then analyzed the fate of such lipids 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 (16). We found that cell-associated fluorescence increased by a factor of 3 to 4 in Wun2 wild-type or Wun2 Y225W–expressing cells compared with control (Fig. 3). Expression of Wun2 H274K or H326K did not result in any increase in cell-associated fluorescence compared with the control (Fig. 3). We analyzed the localization of the internalized lipid and found that Wun2 promotes its rapid accumulation in the cytoplasm, similar to hLPP1 (fig. S3, C and E). Wun2 therefore confers the ability of cells to internalize lipid substrates concurrent with dephosphorylation.

Fig. 3.

Catalytically active Wun2 confers lipid uptake ability. Relative fluorescence of Hi5 cells after exposure to the fluorescent PA analog NBD-PA, expressing a control protein, hLPP1, wild-type or mutant forms of Wun2, including H274K and H326K (catalytically dead), or Y225W (catalytically active).

We suggest 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 (Fig. 4A). Germ cells are unique in requiring this lipid for their survival, whereas the somatic cells do not (18). We further propose 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 (Fig. 4A) (18). For wun2-null germ cells, if the soma expresses wun/wun2, the common phospholipid pool is depleted and germ cells die (Fig. 4C). 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 (Fig. 4D). 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.

Fig. 4.

Germ line–soma competition model for wun2 function in germ cell migration and survival. In wild-type embryos (A), a gradient of a lipid phosphate (blue) attractant is established by restricted expression of somatic Wun/Wun2 through dephosphorylation. Germ cells express Wun2 and compete with the soma for this lipid phosphate. Germ cell Wun2 perceives lipid phosphate levels by dephosphorylation-dependent internalization of the lipid moiety (light blue) activating an intracellular pathway (open half moon). The germ cells migrate toward higher concentrations of lipid phosphate (arrow). In embryos with wild-type germ cells and wun/wun2 mutant soma (B), germ cells mismigrate due to lack of lipid phosphate gradient. In embryos with wun2-null germ cells and wild-type soma (C), germ cells die because they are unable to compete with the soma, which is depleting lipid phosphate levels. In embryos with wun2-null germ cells and wun/wun2 mutant soma (D), germ cells survive because the soma is no longer depleting lipid phosphate levels. The loss of directional cues from the lipid phosphate gradient leads to germ cell scattering. The mechanism by which germ cells perceive lipid phosphate levels independent of wun2 is not known.

We propose 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, our observations represent a novel paradigm for cell survival and migration. It had been assumed that all lipid phosphate signaling occurs through GPCRs, but our 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 (19). In Drosophila, this pathway shows remarkable specificity for germ cell survival, because somatic cells seem to be insensitive to wun/wun2 levels (18). 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 (20), as demonstrated by the ability of some germ cells to reach the gonad even in the absence of wun/wun2 signaling.

Recent studies have revealed striking similarities between the guidance cues regulating germ cell migration in Drosophila and vertebrates (9). Mouse germ cells also express an LPP (21), and analysis of its function may reveal further parallels between early germ cell behavior in flies and mice.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1102421/DC1

Materials and Methods

SOM Text

Figs. S1 to S4

Table S1

References

References and Notes

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