InteractiveFly: GeneBrief

wunen and wunen-2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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).

REGULATION

Characterization of fly and mammalian lipid phosphate phosphatase substrate specificity

Wunen, a homolog of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad-range lipid phosphatase activities in vitro, with little apparent specificity between them. This study shows that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ-cell-specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans (Burnett, 2003).

The human LPPs are grouped into three isoforms, 1, 2 and 3, and the human genes also have splice variants (Waggoner, 1999). Sequence alignments of Wun and Wun2 were analyzed with mammalian LPPs, and it was concluded that both proteins have the greatest homology with human LPP3 (Burnett, 2003).

Past work has indicated that the three known LPP isoforms can dephosphorylate a broad range of lipid phosphates, notably lysophosphatidic acid (LPA), phosphatidic acid (PA), ceramide-1-phosphate (C(1)P), diacylglycerol pyrophosphate (DGPP) and sphingosine-1-phosphate (S(1)P), with relatively little apparent specificity (Dillon, 1997; Jasinska, 1999; Kai, 1997; Roberts, 1998; Waggoner, 1996). Apart from Wun and Wun2, little is known about the biological functions of these proteins. Dri42 (differentially expressed in rat intestine 42), the rat homolog of LPP3, is upregulated during differentiation of the crypt cells in the small intestine (Barila, 1996), whereas the human splice-variant LPP1-1 is downregulated in human colon-tumor tissue (Leung, 1998). In 2000, Zhang reported a homozygous null mutation in murine Lpp2 that produced viable, fertile mice with no detectable phenotype. In addition, Lpp2 is expressed at lower levels than the Lpp1 and Lpp3 isoforms, leading to the proposal that Lpp2 functions redundantly with them (Zhang, 2000b). Starz-Gaiano (2001) reported that null mutations in either wun or wun2 also present no detectable phenotype, with embryonic development and PGC migration occurring normally. By contrast, removal of both genes results in highly perturbed PGC migration, with PGCs scattering widely on exiting the midgut at stage 10. This led to the suggestion that these LPPs function redundantly (Burnett, 2003).

Given the remarkable bioactivity of the potential lipid substrates for these enzymes, and the specificity of some of their receptors (Takuwa, 2002), it was curious that the separate isoforms present such broad-range activity in biochemical assays and seem to be functionally redundant in vivo. This study examines specificity in substrate recognition among the fly and mammalian isoforms in vitro for a range of known substrates, and in vivo by investigating their ability to dephosphorylate the PGC-specific guidance molecule that functions as a substrate for Wun. It is shown that there are marked differences in relative activity between immunopurified Wun and the mammalian isoforms in a biochemical phosphate-release assay, with Wun showing negligible activity for two of the substrates. Transgenic flies were created expressing mouse Lpp1 and human LPP3; although active on all of the tested substrates in vitro, mouse Lpp1 is completely inactive in the germ-cell migration bioassay. Conversely, overexpression of human LPP3 results in aberrant PGC migration and death, with a remarkably similar phenotype to Wun overexpression. This demonstrates a distinct difference in bioactivity between the isoforms for the first time, and may point towards an underlying signalling system that is conserved between flies and humans (Burnett, 2003).

Although detailed kinetic analyses was not performed, the fly LPP Wun and the mammalian LPP1 and LPP3 isoforms differ in their relative activities on lysophosphatidic acid (LPA), phosphatidic acid (PA), ceramide-1-phosphate (C(1)P) in a PiPer® phosphate-release assay. Although Wun can dephosphorylate LPA with a similar efficiency to mouse Lpp1, it shows negligible activity on both PA and C(1)P. This indicates that there are distinct differences in substrate preference between the fly and mammalian enzymes, with Wun showing a narrower activity range in this assay than was previously reported for the mammalian LPPs. Both mouse Lpp1 and human LPP3 are active on all three substrates. Mouse Lpp1 has a 1.7-fold higher activity on C(1)P than human LPP3 in this assay. Human LPP3, however, had a 1.6-fold higher activity than mouse Lpp1 on PA (Burnett, 2003).

Human LPP3 is highly active when ectopically expressed in Drosophila embryos, as assayed by the disruption of PGC migration and survival, which results in a phenotype similar to the ectopic expression of Wun or Wun2. That human LPP3 shows the same phenotype in a bioassay as Drosophila Wun suggests a conserved signalling pathway that regulates germ-cell migration and survival from flies to humans. Conversely, although active in the biochemical assay, mouse Lpp1 is completely inactive in vivo, and has no apparent effect on PGC migration or survival. This shows an absolute difference in functional bioactivity between the mammalian LPP isoforms. That the LPP isoforms present different functional outputs when assayed in vivo has a number of implications. It may be that the Lpp1 isoform simply cannot recognize or catalyse the dephosphorylation of the specific factor acted on by Wun and LPP3. This would demonstrate specificity in substrate choice between the isoforms, and may indicate that the germ-cell-specific factor is not PA, LPA or C(1)P, on which mouse Lpp1 is active in vitro, particularly as Wun shows negligible activity on PA and C(1)P in the same assay. Alternatively, there may be specific components of the pathway that are required for selection and recognition of the factor. It is possible that as yet unidentified conformational or structural differences in mouse Lpp1 preclude its association with these factors, thus inhibiting its enzymatic function in this system. Thus, LPA could be the factor, and the unnatural presentation and high concentration of LPA in the biochemical assay may override the specific selection mechanisms used to regulate activity in vivo, allowing mouse Lpp1 access to this otherwise inaccessible substrate. Primary sequence analyses have not identified any immediate candidates for residues conferring such a difference. It is speculated that the observed differences in substrate preference may be related to the proteins' structure, which are as yet unsolved. Barila has studied the properties of the internal sequences of Dri42 in its trafficking to the cell surface (Barila, 1996). The contributions of the termini to biological and biochemical properties are yet to be reported in any detail (Burnett, 2003).

In conclusion, evidence is presented of differences in relative activity between the mammalian and fly LPP isoforms on LPA, PA and C(1)P in a biochemical assay. These results indicate that the fly LPP Wun has a narrower activity range than the mammalian LPPs in this assay. Evidence is also presented to show that, although active in vitro, mouse Lpp1 cannot dephosphorylate the same endogenous germ-cell-specific factor as Wun in vivo, whereas human LPP3 seems to do so. This demonstrates that despite broad-range activity in Triton-micelle assays, the mammalian LPP isoforms do show distinct differences in bioactivity when assayed in a physiological context. It is expected that a combination of biochemical and biological data, such as those presented here, will in time help to identify the physiological substrate for Wun, which is, presumably, a stem-cell control factor (Burnett, 2003).

Protein Interactions

Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, thereby modifying or attenuating their activities. Wunen, a Drosophila LPP homolog, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. This possibility was explored to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity. Evidence is presented that Wunen dimerises and that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. Murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally, addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo. It is concluded that LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations seen are highly specific and occur only between monomers of the same protein (Burnett, 2004).

DEVELOPMENTAL BIOLOGY

Spatially restricted activity of a Drosophila lipid phosphatase guides migrating germ cells

Temporal and spatial controls of cell migration are crucial during normal development and in disease. The mechanisms that guide cells along a specific migratory path remain largely unclear. wunen 2 has been identified as a repellant for migrating primordial germ cells. wunen 2 maps next to and acts redundantly with the previously characterized gene wunen, and known wunen mutants affect both transcripts. Both genes encode Drosophila homologs of mammalian phosphatidic acid phosphatase. This work demonstrates that the catalytic residues of Wunen 2 are necessary for its repellant effect and that Wunen 2 can affect germ cell survival. It is proposed that spatially restricted phospholipid hydrolysis creates a gradient of signal necessary and specific for the migration and survival of germ cells (Starz-Gaiano, 2001).

wun and wun2 map 5 kb apart, are transcribed in opposite orientations, and have identical expression patterns. The previously identified 'wun' mutations affect expression of both wun and wun2. Furthermore mutations in either wun or wun2 do not cause a 'wun' germ cell migration phenotype, suggesting that the two genes act redundantly. wun and wun2 mRNAs are expressed in those regions of the gut that the germ cells avoid during their migration. Furthermore, ectopic expression of either gene repels the germ cells from the sites of expression and ultimately causes germ cell loss. These and previous data have led to the hypothesis that Wun and Wun2 produce a repellant signal for migrating germ cells, and that spatially restricted expression of wun and wun2 in the midgut is required to orient germ cells towards the mesoderm (Starz-Gaiano, 2001).

Wun and Wun2 belong to a conserved family of phosphatidic acid phosphatases. Human PAP 2a is thought to have its catalytic domains exposed on the cell surface (Jasinska, 1999) and has been shown in vitro to hydrolyze a variety of phospholipid substrates, including lysophosphatidic acid (LPA), phosphatidic acid (PA), sphingosine 1- phosphate (S-1-P) and ceramide-1-phosphate (C-1-P; Roberts, 1998). In support of the idea that Wun and Wun2 are functional homologs of mammalian PAP 2a, Wun2 protein is shown to localize to the cell membrane and its catalytic activity is required for its non-cell-autonomous effects on the germ cells. Little is known about the in vivo function of PAP 2a in mammals, although in Drosophila, Wun and Wun2 seem to have a rather specific effect on germ cell migration and survival (Starz-Gaiano, 2001).

Several models have been proposed to explain how germ cells are repelled by Wun (in the following, the activities encoded by both the wun and wun2 genes is referred to as 'Wun activity'). A model is favored in which either the extracellular products or substrates of Wun activity are directly received on the surface of the germ cells and initiate a signaling cascade to direct these cells. For example, a dephosphorylated lipid may act as a repellant. Alternatively, a phospholipid may normally act as a diffusible attractant, and this signal may be destroyed by Wun activity. In this manner, germ cells would show no preference for sites of Wun activity and would instead be guided towards higher concentration of the attractive phospholipid. The putative attractant on which Wun acts, however, is most likely provided generally, or by the midgut itself, and not by the gonadal mesoderm, because in embryos with no mesoderm, such as twist, snail double mutants, the germ cells still move correctly to the top of the gut. Interestingly, phospholipid substrates for PAP 2a have been shown to promote a variety of cell responses in mammalian in vitro culture systems including cell migration, differentiation and apoptosis. Some of these responses are mediated by activation of G-protein coupled receptors of the epithelial differentiation gene (Edg) family. In addition, a S-1-P receptor has recently been shown to be required for cell migration in zebrafish. Thus, the activation of a G-protein-coupled receptor in germ cells may mediate the migratory response to Wun activity (Starz-Gaiano, 2001 and references therein).

In addition to affecting germ cell migration, overexpression of wun and wun2 during development leads to striking germ cell loss. This reduction in germ cell number seems to require that high levels of Wun activity are present early during embryogenesis. This effect is specific to germ cells, since no general effects are seen on pattern formation, other cell migrations, or cell survival in these embryos. At this point, there is no evidence that links this reduction in germ cell number to activation of the apoptotic pathway in germ cells. Markers for germ cells seem to be lost quickly in dying cells. During the normal development of many organisms, some germ cells fail to find their way to the gonad, and it is probably important to prevent the survival of these lost germ cells, since they could differentiate into other tissues and potentially produce tumors. In Drosophila, the expression domains of wun and wun2 in the endoderm and ectoderm border the germ cell migratory path. A lipid substrate for Wun activity could act as a survival factor for germ cells, while this signal would be removed in areas with high levels of Wun activity and the germ cells would die. Interestingly, overexpression of a mammalian S-1-P phosphatase has been recently shown to promote cell death (Mandala, 2000). Mutants that lack wun and wun2 transcripts, though, still have the wild-type decline in germ cell numbers. Thus, these genes may not normally act to control germ cell survival, or germ cell death may require very high levels of Wun activity. Alternatively, some residual lipid phosphatase activity may be present in the mutant embryos. Indeed, there are six other highly conserved wun-like genes in the Drosophila genome some of which could act to control germ cell number. No candidate phospholipid receptor has yet been identified in Drosophila that could mediate the guidance and survival cues to the germ cells. Ongoing maternal effect screens, as well as germ cell specific misexpression screens should lead to the identification of such molecules (Starz-Gaiano, 2001).

Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2

In most organisms, primordial germ cells (PGCs) arise far from the region where somatic gonadal precursors (SGPs) are specified. Although PGCs in general originate as a single cluster of cells, the somatic parts of the gonad form on each site of the embryo. Thus, to reach the gonad, PGCs not only migrate from their site of origin but also split into two groups. Taking advantage of high-resolution real-time imaging, this study shows that in Drosophila PGCs are polarized and migrate directionally toward the SGPs, avoiding the midline. Unexpectedly, neither PGC attractants synthesized in the SGPs nor known midline repellents for axon guidance are required to sort PGCs bilaterally. Repellent activity provided by wunen (wun) and wunen-2 (wun-2) expressed in the central nervous system, however, is essential in this migration process and controls PGC survival. These results suggest that expression of wun/wun-2 repellents along the migratory paths provides faithful control over the sorting of PGCs into two gonads and eliminates PGCs left in the middle of the embryo (Sano, 2006).

Using high-resolution in vivo imaging, Drosohila PGCs were followed as they emerged from the midgut and moved toward the SGPs. PGCs are polarized during migration and they move steadily toward the lateral mesoderm and SGPs. During this migration, PGCs sort into two bilateral groups, each group moving toward one set of SGP clusters. Genetic analysis of this process led to the following conclusions. First, Wun and Wun-2 LPP activity in the CNS acts as a long-range guidance factor during bilateral sorting and lateral migration of PGCs. Second, known axonal repellent guidance signals produced by ventral midline cells and PGC attractants produced by HMGCoAr-expressing cells in the lateral mesoderm and SGPs are not required for lateral migration. Finally, high levels of Wun and Wun-2 in the CNS eliminate PGCs that fail to sort properly from the middle. It is concluded that D. melanogaster LPPs play a major role in guiding Drosophila germ cells to the bilateral gonads and eliminating germ cells left at the midline. The data suggest repulsion and midline exclusion as an alternate mechanism to attraction and protection during the lateral sorting of germ cells (Sano, 2006).

Time-lapse analysis showed that PGCs start migrating laterally soon after they emerge from the PMG and, thus, before SGPs are specified. Consistent with this finding, PGCs sort bilaterally in abd-A mutants that lack SGPs. Because PGCs fail to leave the gut in mutants lacking lateral mesoderm, it was not possible to test whether lateral mesoderm, by itself, is required for the lateral migration of PGCs. Instead, the ability of PGCs to divide into two groups in hmgcr mutants was tested because hmgcr is broadly expressed in the lateral mesoderm and plays an important role in PGC attraction. hmgcr mutants did not show defects in lateral migration, suggesting that attraction by hmgcr is not critical for bilateral sorting. Finally, it is conceivable that as the PGCs are leaving the PMG, the movement of the developing PMG toward the mesoderm squeezes PGCs into two groups, thereby indirectly causing bilateral cluster formation. However, this is unlikely because in embryos doubly mutant for the integrin ßPS and integrin ßnu the morphological changes of the PMG do not occur but the two gonads form normally. Together, the fact that neither abd-A or hmgcr mutants nor embryos that fail to undergo normal midgut morphogenesis affect the bilateral movement of PGCs suggests that neither physical guidance nor attraction play a major role in lateral sorting of PGCs (Sano, 2006).

Instead, wun/wun-2 expression in the CNS is required to generate lateral clusters of PGCs by repulsion. High-resolution live imaging demonstrated that wun/wun-2 mutations affect the polarity but not the motility of PGCs. This change in morphology is particularly striking among cells that remained in the middle of the embryo, whereas more lateral cells continue to migrate toward the epidermis. Loss of polarity is accompanied by more frequent and less stable cytoplasmic extensions within a given time frame, slightly slower velocity, and overall shorter tracking distance. PGCs that exited the gut from a more lateral position in wun/wun-2 embryos have a more normal morphology and migrate into the epidermis. One possible interpretation of these results is that PGCs in more medial locations are in a generally attractive environment produced by hmgcr expression and possibly other attractants and that this environment may cause germ cells to stop migrating. PGCs in more lateral regions, on the other hand, may not experience sufficient levels of attractants and therefore continue to migrate. It remains open whether this migration is directed by specific somatic cues and if so what these cues are. Because these lateral PGCs in wun/wun-2 mutants often end up in the epidermis, it is likely that wun/wun-2 expression in the epidermis of wild-type embryos repels PGCs. wun and wun-2 expression in the gut, CNS, and epidermis flank the migratory route of PGCs. This and the striking defects in migration observed in wun/wun-2 mutants suggest that Wun/Wun-2 is a major long-range guidance factor that guides PGCs toward the two gonads. Based on these observations, it is proposed that dynamic expression of wun/wun-2 guides PGCs successively: first to reorient them dorsally on the midgut, then to move them from the gut into the mesoderm, and finally to steer them toward the lateral mesoderm and the SGPs away from the ventral midline, the CNS, and the epidermis (Sano, 2006).

wun/wun-2 expression in the CNS not only repels PGCs away from the midline but is also responsible for the elimination of ectopic PGCs that failed to sort bilaterally. These results demonstrate a role for somatic wun/wun-2 in normal PGC survival that had previously been revealed only after overexpression of either wun or wun-2 in the soma. In a rescue experiment, wun-2 expression driven by 9-201av-Gal4 rescued lateral migration but not death in the middle of the embryo. These results suggest that low levels of Wun/Wun-2 are sufficient to direct migration away from Wun/Wun-2–expressing cells, whereas continuous and possible high levels of exposure to Wun/Wun-2 leads to elimination of PGCs. Wun and Wun-2 are likely to regulate the distribution of a phospholipid substrate. PGCs may migrate along a gradient toward high levels of phospholipids, whereas low, evenly sustained levels of phospholipid may be sufficient for survival (Sano, 2006).

It is unclear why PGCs need to be removed from the middle region of Drosophila embryos because teratomas, similar to those observed in mouse and humans, do not seem to originate from lost PGCs in flies. However, it has been reported that PGCs are able to transdifferentiate into somatic cells when they lack the translational regulator Nanos. In this case, PGCs have to be prevented from apoptotic death. Interestingly, apoptotic death of Drosophila PGCs has so far only been observed in mutant backgrounds, such as nanos, which causes inappropriate somatic gene expression in PGCs. The data show that a nonapoptotic death pathway acts during PGC elimination in the middle of the embryo. There is no evidence that PGCs lose their germ cell character in wun/wun-2 mutants, and thus this pathway may be specific to germ cells. In mouse, ectopic PGCs are eliminated by apoptotic death. It will be interesting to see whether nonapoptotic pathways, mediated by LPPs similar to Wun/Wun-2, also play a role in the control of germ cell survival in vertebrates (Sano, 2006).

In most organisms germ cells originate at one location, whereas the somatic gonad forms bilaterally. Thus, sorting of germ cells along the midline is a conserved phenomenon. Furthermore, elimination of germ cells trapped at the midline is an important aspect of normal development, since germ cells trapped in the midline have been shown to give rise to germ line teratomas, one of the most frequent cancers among young adults. Like Drosophila PGCs, zebrafish and mouse PGCs migrate away from the dorsal midline toward the genital ridge. Repellents involved in this process have yet to be reported in zebrafish and mouse. It was shown that migration to the genital ridge is controlled by the G protein–coupled receptor CXCR4 and its ligand SDF-1 in both animals. CXCR4 is expressed in migrating PGCs, and SDF-1 is expressed in the somatic tissues and acts as an attractant. SDF-1 changes its expression pattern during embryonic development as it prefigures the route of PGC migration and, at least in zebrafish, seems to be the major guidance signal for PGCs. In CXCR4 mutant mice, germ cells that lack the receptor remain in the midline and die, presumably because of the lack of survival factors provided by the genital ridges. Thus, in mouse, attractants and survival factors seem to be the major determinants that sort PGCs into two clusters, whereas in Drosophila repellents and death dominate. Given the striking similarity in PGC behavior, it will be interesting to see if these disparities are because of differences in signaling mechanisms or reflect evolutionary changes in guidance strategy (Sano, 2006).

Quantifying the range of a lipid phosphate signal in vivo

Quantitative information about the range of influence of extracellular signalling molecules is critical for understanding their effects, but is difficult to determine in the complex and dynamic 3 dimensional environment of a living embryo. Drosophila germ cells migrate during embryogenesis and use spatial information provided by expression of lipid phosphate phosphatases called Wunens to reach the somatic gonad. However whether guidance requires cell contact or involves a diffusible signal is not known. This study substituted wild type Wunen expression for various segmentally repeated ectodermal and parasegmental patterns and used germ cell behavior to show that the signal is diffusible and to define its range. This was correlated back to the wild type scenario, and it was found that the germ cell migratory path can be primarily accounted for by Wunen expression. This approach provides the first quantitative information of the effective range of a lipid phosphate in vivo and has implications for the migration of other cell types that respond to lipid phosphates (Mukherjee, 2013).

This paper has explored the nature of the signal that regulates germ cell migration and survival in Drosophila embryos. Germ cells are excluded from Wunen expressing somatic domains in wild type embryos suggesting Wunen destroys an attractive signal. Ectopic mis-expression of Wunen in embryos otherwise somatically null for Wunens was used to show that Wunens are instructive for dictating the germ cell migration path. In particular Wunen expression in en or h ectodermal stripes causes germ cells to align parallel to and between the stripes. In such embryos, germ cells have an equidistance ratio closer to 1 as compared to control embryos indicating they are likely integrating a signal from both sides. By live imaging it was observed that germ cells reach and maintain these parallel positions without making any direct contacts with the stripes. Taken together these data strongly argue that the signal modulated by Wunen positive somatic cells is cell contact independent (Mukherjee, 2013).

The exclusion of germ cells from the entire ectoderm when Wun is expressed using the en or h drivers might result from germ cells being repelled by the ectodermal stripes or from death of germ cells that have entered the ectoderm. The former is favored because from live imaging germ cells were not seen to enter the ectoderm, and ectodermal Vasa positive cell remnants, which are indicative of germ cell death. were not seen. In both scenarios however, the conclusion that the mechanism is cell-contact independent remains valid (Mukherjee, 2013).

When presented with a single Wun stripe, using the NP5141 driver, the germ cells were repelled up to 33 μm. This represents an estimate of the maximal effective range of the Wun signal. This distance is comparable to assessments of the effective range of Wingless (Wg) and Hedgehog (Hh), which can form gradients over at least 50 μm in wing imaginal discs. When germ cells are faced with multiple wunen domains, such as wun expressing en stripes, then germ cells can tolerate being much closer, but make avoidance turns when 15 μm away. In wild-type 99% of germ cells are located up to 33 &mu:m from a wun2 expressing domain. Therefore although germ cells are likely influenced by Wunen expression for the entire duration of their migration, 59% of them are closer than 15 μm. It is postulated that the small patches of wild type wun2 positive cells in the lateral mesoderm and ectoderm might not repel as far as the much larger ectopic wunen expressing en stripes (Mukherjee, 2013).

Membrane bound ligands can also effect long-range signals via their presence on long actin rich cytoplasmic extensions. For example, lateral inhibition of Drosophila sensory organ precursor (SOP) fate is mediated by the transmembrane Notch ligand Delta which can signal 3 to 5 cell diameters away via cytoplasmic extensions of up to 20 μm. Therefore it was considered whether germ cells could use cytoplasmic projections to make direct contact with Wun expressing stripes. Germ cell filopodia at their leading edge and often a longer lagging tail (or uropod) are seen by both live and fixed tissue analysis. The filopodia are generally no longer than 2 μm whilst the later can be up to 8 μm. Therefore these projections are not sufficiently long to make contact with the stripes. Furthermore a constitutively active form of Moesin, MoeT559D, was used that disrupts the actin cytoskeleton in Drosophila photoreceptors and suppresses filopodia from the leading edge during dorsal closure. Expression of this construct in germ cells caused only minor defects in germ cell survival indicating that germ cells do not rely on Moesin- dependent filopodia for migration in wild-type embryos (Mukherjee, 2013).

wun2 over-expression in its endogenous pattern was used to evaluate the relative importance of somatic Wun expression levels versus their spatial distribution in permitting germ cell survival. The data show that the level of Wunen protein in endogenously Wunen expressing cells is not critical to regulate germ cell survival. Ectopic expression can be tolerated if it is spatially separated from the germ cells. Therefore it is the location of the Wunen expression, and not its overall level, which is critical for germ cells. This is similar for other signaling systems, for example mis-expression of Hh ubiquitously leads to patterning defects but over-expression in its normal locations has little effect on segmentation. Insensitivity to over-expression in endogenous domains may well be a common feature of signaling molecules (Mukherjee, 2013).

This data is consistent with a model in which the somatic Wunen expression sets up relatively short range repulsion zones within the embryo. These result were envisioned from a local depletion of a lipid phosphate substrate. This could take the form of a discrete change in levels ('all or nothing') or a gradient. The ability of cells to find equidistant positions between Wunen stripes and to be repelled over many cell diameters when faced with a single Wunen expressing stripe strongly favors a gradient (Mukherjee, 2013).

The canonical model of morphogen gradient formation is that the morphogen is secreted locally and diffuses to create a gradient. However morphogens may also be restricted to a narrower area by localized depletion accomplished by receptor-mediated endocytosis or sequestration. Whilst the source of the ligand in the case of germ cells is not known, the data is consistent with gradient formation based on dephosphorylation of an extracellular lipid phosphate by a LPP (Mukherjee, 2013).

What would be the nature of the lipid gradient? S1P is present in human plasma and serum bound to low- and high-density lipoprotein and albumin. LPA is also found in human serum bound to albumin. It is possible that a similar protein binding partner occurs in Drosophila embryos. The extracellular movement of morphogens, such as Hh and Wg, has been proposed to occur on membranous vesicles (also called argosomes or exosomes), or lipoprotein particles. It is possible that similar particles are important for the formation of a lipid gradient that affects germ cell migration (Mukherjee, 2013).

Dictyostelium cells can respond at a distance of approximately 70 μm away from a micropipette containing LPA. S1P is important for the movement of heart progenitor cells from bilateral positions to the midline in zebrafish which involves a distance of approximately 100 μm. S1P also regulates the circulation of T-lymphocytes in mouse, in particular allowing T-cells to exit from lymph nodes which are several millimeters in length. In spite of these essential roles it is not always clear whether absolute levels or gradients of S1P are required. Drug treatments that increase the S1P levels of the lymphoid organs but not the circulatory system (effectively reversing the normal difference in S1P levels between these locations) are sufficient to block T-cell exit, is suggestive that a gradient is required in this circumstance however its contour is not known. This work has shown that lipid gradients in a Drosophila embryo can exist over distances comparable to their protein counterparts. Whether such distances are scaled up in the larger embryos and tissues of other species remains an open question (Mukherjee, 2013).

EFFECTS OF MUTATION

A novel genetic locus, wunen, is required for guidance of germ cell migration in early Drosophila development. Loss of wun function does not abolish movement but disrupts the orientation of the motion causing the germ cells to disperse even though their normal target, the somatic gonad, is well formed. The product of this gene enables a signal to pass from the soma to the germ line. It is proposed that the function of this signal is to selectively stabilize certain cytoplasmic extensions resulting in oriented movement. To characterize this guidance factor, wun was mapped to within 100 kb of cloned DNA (Zhang, 1996).

In wild type as gastrulation proceeds (from 5:00 to 5:30 hr) most germ cells are found at the end of the PMGP. After this they pass across the gut epithelium and enter the embryo. Any difference between the wild-type and wun mutant embryos before exit are too subtle to be seen, and as in wild type, wun embryos show germ cells clustered inside the distal part of the PMGP in a 5:00-5:30 hour collection. After exit the wild-type and wun mutants begin to show differences. Normally the germ cells cluster on the dorsal posterior surface of the PMGP proximal to the mesoderm and then separate into two groups in the mesoderm. In wun mutants these processes fail, and germ cells do not cluster dorsally and can be found as far as the primordia of the Malpighian tubules. Examination of the embryonic gut primordium using an enhancer trap and forkhead expression did not reveal significant molecular or morphological changes in the PMGP (Zhang, 1996).

Examination of the development of the somatic gonad in these mutants using expression of the 412 transposon did not show detectable changes in this tissue. Even at this relatively late stage in development, the germ cells continue to display ameboid morphologies both as individual cells and in clusters (Zhang, 1996).

All three wun alleles show identical and temperature-independent migration phenotypes. Furthermore, the phenotype of Df(ZRjNp5, which removes the flanking lethals l(Zj06736 and 1(2)k16806, is very similar to that of these alleles. It is concluded that the migration phenotype of these alleles show the consequences of loss of wun function (Zhang, 1996).

To address the question of the site of action of wun, mosaic animals were generated where the germ cells were wild type and the soma either wun mutant (experimental samples) or wild type (control samples). The results of this experiment show that wun function is required in the somatic tissue of the embryo. The reciprocal experiment of placing wun mutant germ cells in a wild-type somatic environment and scoring for migration in the embryo is technically challenging since individual donor embryos need to be genotyped and the results of transplantation from each donor to be followed. This experiment was not attempted (Zhang, 1996).

Germ cell-autonomous Wunen2 is required for germline development in Drosophila embryos

To identify new maternally acting genes required for pole cell development, a genetic screen was performed for maternal mutations causing defects in this process. A screen was carried out for EMS-induced mutations on the chromosome arm 2R using the FLP/FRT/DFS system, which produces homozygous germline clones in heterozygous mothers. Embryos derived from homozygous germline clones were stained for Vasa, a marker protein for the germline. From the screening of 1156 independent lines, one line, which was named N14, was recovered that exhibits developmental defects in pole cells during their migration (Hanyu-Nakamura, 2004).

Deficiency mapping revealed that N14 was uncovered by Df(2R)w45-19g. In embryos derived from N14/Df(2R)w45-19g mothers (hereafter referred to as N14^m- embryos), normal numbers of pole cells were formed (stage 5). These pole cells were carried into the embryo along with the invagination of the posterior midgut; they passed through the midgut epithelium, and migrated dorsally along the surface of the midgut. However, at stage 11, when pole cells normally associate with the mesoderm, the number of Vasa-positive pole cells was dramatically reduced in N14^m- embryos. In these embryos, the remaining pole cells, if any, associated with the surface of the midgut, and in subsequent development, few or no pole cells were incorporated into the gonads. N14^m- embryos showed no discernible morphological defects in somatic tissues and developed into adults. However, consistent with the loss of pole cells during embryogenesis, over 80% of the adult females developed from N14^m- embryos had agametic ovaries. This defect in pole cell development was not rescued by a paternally supplied wild-type copy of the N14 gene, and zygotic N14 mutation did not affect the maternal N14 mutant phenotype. These results indicate that maternal N14 function is required for the maintenance of pole cells during their migration to the gonads (Hanyu-Nakamura, 2004).

The above observations allow two possible interpretations: that the pole cells are eliminated after exiting the midgut in N14^m- embryos, or that they survive but lose pole cell-specific markers, such as Vasa. To discriminate these alternative possibilities, the fate of pole cells was examined in N14^m- embryos by using a photoactivatable lineage tracer, caged fluorescein. Caged fluorescein was injected into cleavage-stage embryos and photoactivated in pole cells at the cellular blastoderm stage. The majority of the pole cells was successfully marked, and the marked cells were traced until they formed the embryonic gonads in wild-type embryos (Hanyu-Nakamura, 2004).

In N14^m- embryos, pole cells, which were marked with both fluorescein and anti-Vasa antibody, were observed on the surface of the midgut at stage 10. However, at stage 11, the fluorescein-marked cells rapidly disappeared. Fluorescein-marked pole cells were occasionally observed with no or very faint Vasa signals on the surface of the midgut. At stage 11, the pole cells appeared to have experienced a significant reduction in size accompanied by an increase in the intensity of fluorescein signal, and few fluorescein-marked pole cells were detectable by stage 12. From these observations, it is concluded that pole cells in N14^m- embryos die after they pass through the midgut epithelium. Additional experiments have suggested that pole cell death in N14^m- embryos occurs via a caspase 3-independent pathway, and that the N14 mutation does not affect early events in pole cell development (Hanyu-Nakamura, 2004).

Through complementation tests, five overlapping deficiencies were identified that uncovered the N14 mutant. The breakpoints of these deficiencies defined the N14 locus within a ~100 kb genomic region containing nine identified or predicted genes. To identify the gene responsible for the N14 mutant phenotype, a series of transgenes were generated containing genomic DNA fragments in this region. Of these, a ~17 kb genomic DNA fragment, which contained two genes, wun2 and CG13955, completely rescued the N14 mutant. To determine which gene was responsible for the N14 mutant phenotype, constructs were made in which only one of the genes was intact. Only the 8 kb HincII fragment (Pwun2-8k), containing the entire wun2 locus, rescued the N14 mutant. Furthermore, a genomic fragment that had a partial deletion in the wun2 RNA-coding region (Pwun2-8kDelta) failed to rescue the N14 mutant. Finally, it was found that the N14 mutant chromosome had a nonsense mutation in the wun2 gene at the 111th Trp codon. Thus, the N14 mutant is referred to as the wun2^N14 allele (Hanyu-Nakamura, 2004).

To generate new wun2 alleles, a P element insertion, EP2650, which locates ~30 bp distal to the 5' side of the wun2 locus, was mobilized. As a result, a deletion of wun2 (wun2^Delta) was isolated in which one-third of the wun2 locus was deleted. The wun2^Delta mutant was homozygous viable, and lacked wun2 RNA expression. In embryos derived from wun2^Delta/Df(2R)w45-19g and wun2^Delta/wun2^N14 mothers, pole cells formed normally, but most of them died after exiting the midgut; phenotypes essentially the same as those of the N14^m- embryos. The maternal wun2^Delta mutant phenotype was rescued by introducing the Pwun2-8k transgene. These data demonstrate that maternal wun2 is essential for pole cell survival (Hanyu-Nakamura, 2004).

wun2 encodes an LPP that dephosphorylates a number of phospholipids in vitro (Burnett, 2003; Starz-Gaiano, 2001). It was next asked whether the phosphatase activity of Wun2 is responsible for maternal Wun2 function. It has been shown that the putative catalytic residues of Wun2, His274 or His326, are essential for its activity (Starz-Gaiano, 2001). His274 or His326 were mutated into Lys (H274K or H326K) and the ability of such mutant transgenes to rescue the wun2^N14 phenotype was examined. The two mutant Pwun2-8k transgenes with H247K or H326K failed to rescue the wun2^N14 phenotype, indicating that the effect of Wun2 on pole cell survival is dependent on its phosphatase activity (Hanyu-Nakamura, 2004).

The distribution of wun2 RNA during early embryogenesis was examined. wun2 RNA is detected ubiquitously in cleavage-stage embryos (stage 2). Although the signal in the somatic cell region becomes undetectable by the cellular blastoderm stage, it remains at high levels in pole cells (stage 4). At stage 5, wun2 signal is also detected in the somatic region in a posterior stripe pattern. These observations were consistent with a previous report (Renault, 2002). In embryos from wun2^Delta/Df(2R)w45-19g females crossed to wild-type males, wun2 RNA is undetectable in pole cells, but is expressed in the posterior stripe. These results indicate that wun2 RNA in pole cells is supplied maternally, while it is expressed zygotically in the posterior stripe (Hanyu-Nakamura, 2004).

To examine whether wun2 activity is required in pole cells for their survival, whether the pole cell-specific zygotic expression of wun2 rescues the maternal wun2 mutant phenotype was examined. For this purpose, wun2^N14 mutant females carrying the nanos-Gal4-VP16 transgene were crossed with males possessing EP2650, which expresses wun2 under UAS control (Starz-Gaiano, 2001). In embryos resulting from this cross, pole cells escaped from cell death. Although these pole cells were moderately dispersed, as in wun2⁺ embryos that overexpressed wun2 in pole cells, they were incorporated into the gonads. These results indicate that the expression of wun2 in pole cells is sufficient to rescue pole cell death in wun2^m- embryos, and that wun2 is required in pole cells for their survival (Hanyu-Nakamura, 2004).

It has been shown that zygotic wun2 acts redundantly with wun in somatic tissues to guide pole cell migration. In embryos that are double mutants for wun and wun2, pole cells are dispersed on the midgut at stage 10, and fail to migrate to the mesoderm (Starz-Gaiano, 2001; Zhang, 1996; Zhang, 1997). Interestingly, somatic Wun and Wun2 are also capable of affecting pole cell survival. Overexpression of either of them in somatic tissues causes dramatic pole cell loss after stage 11 (Burnett, 2003; Starz-Gaiano, 2001). Since this latter phenotype is very similar to that observed in wun2^m- embryos, it was asked whether there are any functional links between maternal wun2 and zygotic wun and wun2 in regard to pole cell survival (Hanyu-Nakamura, 2004).

Initially, the effect was examined of the loss of both zygotic wun and wun2 on pole cell death caused by a maternal wun2 mutation. Embryos lacking both maternal wun2 and zygotic wun and wun2 (referred to as wun^m+z- wun2^m-z- embryos) showed a partial rescue of the pole cell death phenotype of the maternal wun2 mutation). In wun^m+z- wun2^m-z- embryos, an average of 10.5±3.5 pole cells survived at stage 14-15, although they failed to migrate toward the gonads as in the wun^m+z- wun2^m+z- embryos. This result revealed that maternal wun2 interacts genetically with zygotic wun and wun2 in regulating pole cell survival (Hanyu-Nakamura, 2004).

Next to be examined was the overexpression of wun2 in pole cells affects the pole cell loss phenotype caused by the overexpression of wun2 in somatic cells. Females carrying both the nanos-Gal4-VP16 transgene and a mesoderm driver twist-Gal4 were crossed with males possessing EP2650 to overexpress wun2 both in pole cells and the mesoderm. The embryos that overexpressed wun2 both in pole cells and the mesoderm had an average of 10.4±4.0 pole cells, while embryos overexpressing wun2 in the mesoderm alone had an average of 4.4±3.6 pole cells per embryo at stage 14-15. This result revealed that the overexpression of wun2 in pole cells suppresses the pole cell loss caused by the overexpression of wun2 in somatic cells. These observations show that pole cell survival requires a balance between LPP activities in pole cells and somatic cells. This further suggests that Wun2 activity in pole cells competes with the Wun and Wun2 activities in somatic cells to promote pole cell survival (Hanyu-Nakamura, 2004).

Earlier work has shown that the two related LPPs, Wun and Wun2, act redundantly in somatic cells to provide a repulsive environment to steer pole cell migration (Starz-Gaiano, 2001; Zhang, 1996; Zhang, 1997). The directional migration of pole cells is disrupted only when the expression of both wun and wun2 is lost in somatic cells. Wun and Wun2 also influence pole cell viability, because the overexpression of either of them in somatic cells causes a severe loss of pole cells (Burnett and Howard, 2003; Starz-Gaiano, 2001). These observations had led to the proposal that Wun and Wun2 were indistinguishable in their somatic function, which is required for pole cell migration and survival. However, the current study has shown that Wun and Wun2 have separable functions, and that Wun2 plays a novel cell-autonomous role in pole cell survival. Furthermore, a balance between the LPP activities in pole cells and the surrounding somatic cells is crucial for pole cell survival. It is proposed that this balance is also involved in the directional migration of pole cells (Hanyu-Nakamura, 2004).

Several maternal mutations that affect pole cell survival have been isolated. These include nanos, pum and pgc. However, the current results indicate that the phenotypes of maternal wun2 mutant embryos are different from those of nanos, pum or pgc mutant embryos. For example, although nanos, pum and pgc are required for transcriptional repression in early pole cells, maternal wun2 plays no role in either transcriptional repression or in the onset of zygotic gene expression in pole cells. Thus, it is likely that maternal wun2 is required for pole cell survival at a different developmental step from nanos, pum and pgc (Hanyu-Nakamura, 2004 and references therein).

Surprisingly, dying pole cells in wun2^m- embryos were negative for cleaved caspase 3. It was also found that the overexpression of the caspase inhibitor p35 in pole cells did not rescue the pole cell death phenotype in wun2^m- embryos. Furthermore, TUNEL-positive pole cells have never been detected in wun2^m- embryos. Thus, the pole cell death in wun2^m- embryos seems to occur via a mechanism different from typical caspase-dependent apoptosis. It has become evident that caspase-independent cell death pathways do exist, and it is supposed that such caspase-independent cell death might be occurring in these pole cells. Further morphological study may reveal how pole cell death occurs in wun2^m- embryos (Hanyu-Nakamura, 2004).

Wun2 belongs to a conserved family of LPPs (Burnett, 2003; Starz-Gaiano, 2001). LPPs are integral membrane proteins that dephosphorylate a number of bioactive lipid phosphates in vitro, such as lysophosphatidic acid, phosphatidic acid, sphingosine-1-phosphate and ceramide-1-phosphate. These lipid phosphates act as extracellular signaling molecules and/or intracellular second messengers and affect a variety of cellular processes, including cell survival and motility. LPPs can attenuate cell activation by dephosphorylating bioactive lipid phosphates and/or they can generate alternative signals from dephosphorylated lipids, such as diacylglycerol, sphingosine and ceramide. The active sites of LPPs are exposed either on the outer surface of the plasma membrane or on the luminal surface of intracellular organelles, depending on their subcellular localization. It has been proposed that LPPs promote the incorporation of lipid phosphate substrates into the outer leaflet of the membrane before their dephosphorylation. Although the exact subcellular distribution of the endogenous Wun2 protein remains elusive, it localizes to the plasma membrane in Drosophila embryos when overexpressed (Starz-Gaiano, 2001). Therefore, Wun2 is likely to be an ecto-enzyme that promotes the uptake and the dephosphorylation of extracellular lipid phosphate substrates (Hanyu-Nakamura, 2004 and references therein).

The data indicate that maternally supplied Wun2 acts in a cell-autonomous manner to promote the survival of pole cells. By contrast, zygotically expressed Wun and Wun2 act in somatic cells to direct pole cell migration (Starz-Gaiano, 2001; Zhang, 1996; Zhang, 1997). Furthermore, pole cell survival requires a balance between LPP activities in pole cells and somatic cells. Considering that Wun and Wun2 are likely to function as ecto-enzymes (Burnett, 2003; Starz-Gaiano, 2001), the same extracellular lipid phosphate could be degraded by both Wun2 in pole cells, and Wun and Wun2 in somatic cells. Thus, it is propose that Wun2 activity in pole cells competes with somatic Wun and Wun2 activities for the uptake and the dephosphorylation of a common substrate. When Wun or Wun2 is overexpressed in somatic cells, the extracellular substrate would be depleted in the hemocoel surrounding the pole cells, so that Wun2 in pole cells is unable to produce the survival signal any longer. In embryos overexpressing Wun2 in both somatic cells and pole cells, increased Wun2 activity in pole cells leads to the increased incorporation of the substrate, promoting pole cell survival. In wun^m+z- wun2^m-z- embryos, pole cells escaped from cell death, suggesting that pole cells are capable of producing the survival signal even in the absence of pole cell-autonomous Wun2 activity. There are eight LPP genes, including wun and wun2, in the Drosophila genome; wun mRNA is also maternally supplied in cleavage-stage embryos (Renault, 2002). Although wun mRNA does not become concentrated in pole cells, it is conceivable that trace amounts of Wun and/or other LPP become partitioned into pole cells, promoting their survival in the wun^m+z- wun2^m-z- embryo. However, in normal embryos, such activity would have only a subtle effect on pole cell survival, because it would promote the survival of only a small number of pole cells (Hanyu-Nakamura, 2004).

Somatic LPP activity also functions to repel pole cells. An extracellular substrate might direct somatic cells to produce a repellant molecule, while directing pole cells to produce a distinct survival signal. However, based on the finding that LPP activity in somatic cells competes with that in pole cells for an extracellular substrate, the idea is favored that LPP activity in somatic cells provides a repulsive environment that directs pole cell migration by depleting a substrate that is required by pole cells for their survival. It is expected that similar LPP-mediated mechanisms of cell migration and survival may be widely used, since LPPs play important roles in various developmental processes such as axon growth, axis patterning and extra-embryonic vasculogenesis in mammals (Bräuer, 2003; Escalante-Alcalde, 2003). Future work will focus on identifying the endogenous substrate for Wun2 and resolving the mechanism by which Wun2 exerts its effects on pole cell survival (Hanyu-Nakamura, 2004).

Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. Wunens mediate germ cell-germ cell repulsion and this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. It is proposed that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, it was found that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. It is suggested that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells (Renault, 2010).

These data show that Wun and Wun2 act redundantly, not only in the soma, but also in germ cells. Analysis of loss-of-function double mutants reveals that wunens are required earlier than previously described, during transepithlelial migration, the first active migratory step of germ cells. Wunens act specifically and cell-autonomously in germ cells and this function is needed to maintain cellular integrity necessary for germ cell survival and migration. Wunen-mediated germ cell-germ cell repulsion provides a mechanism to disperse and distribute the germ cells evenly between the embryonic gonads, thereby maximizing adult fecundity (Renault, 2010).

The early transcriptional quiescence of Drosophila germ cells requires that many germ cell components be maternally supplied. Loss of maternal wun2 leads to germ cell death. Mutations in wun, although having no maternal phenotype by themselves, substantially enhance the phenotype caused by loss of maternal wun2. Therefore, similar to the situation that occurs in somatic cells, wun2 is redundant with wun in germ cells. Expression of Wun or Wun2 individually is able to fully substitute for the loss of both genes in germ cells. Overexpression of either gene in somatic cells causes identical amounts of germ cell death. It is concluded that Wun and Wun2 are fully functionally redundant, which supports the notion there is a single in vivo substrate that both proteins can dephosphorylate with equal efficiency. The germ cell death caused by lack of Wun2 in germ cells, which does not occur upon removal of just Wun, is probably caused by differences in the expression levels of the two genes in germ cells (Renault, 2010).

The requirement of germ cells for wunens for their survival can be abrogated if the migration of the germ cells is blocked. Although the possibility cannot be ruled out that germ cells become reliant on wunens owing to a signalling event that occurs concomitant with migration or that the midgut provides a protective environment, the idea is favored that the migration process itself creates physical stress that, without wunens, leads to cell disintegration and death (Renault, 2010).

Transepithelial migration involves the rapid dispersal of the germ cells, which move as individually migrating cells across the midgut epithelium. Wunens play a role in this process based upon germ cell behaviour in two genetic backgrounds. First, in embryos lacking germ cell and somatic wunens (wun wun2 M^-Z^- embryos), the majority of germ cells remain inside of the midgut. Second, in embryos lacking just germ cell wunens (wun wun2 M^-Z⁺ embryos), the germ cells fail to individualize but do cross the midgut epithelium. The germ cells never appear as individual cells but remain in a tight cluster. Their migration is delayed compared with wild type and is concurrent with the epithelial-to-mesenchymal transition of the midgut cells, which might be necessary to permit this movement of clustered cells (Renault, 2010).

Two interpretations of these data are possible. First, that germ cell wunens act to reduce adhesion between the germ cells, allowing them to disperse. However, reducing DE-cadherin in germ cells, sufficient to get germ cell dispersal, is not sufficient to cause the germ cells to migrate through the midgut in wun wun2 M^-Z^- embryos. The alternative explanation is that wunen-expressing germ cells also repel each other, given that wunen-expressing somatic cells repel germ cells. Wunen-mediated germ cell-germ cell repulsion would thereby provide a mechanism to explain the rapid movement of these cells away from each other that occurs during transepithelial migration (Renault, 2010).

It was asked whether germ cell-germ cell repulsion might confer any advantages to the organism. It was found that germ cell-germ cell repulsion serves to distribute the germ cells evenly between the two embryonic gonads, which are located on either side of the midline. Full fertility of the adult can be achieved even if only a few germ cells reach the embryonic gonad. This is because germ cell division during larval stages can compensate for deficits in the number of germ cells reaching the gonad. If, however, no germ cells reach a gonad, compensatory proliferation is not possible and the adult ovary or testis will be devoid of eggs or sperm, respectively. Therefore, distribution of germ cells between the gonads becomes crucial when there are few germ cells. For germ cells to reach both gonads, they must sort bilaterally to either side of the midline. Wunens are expressed in the central nervous system in the middle of the embryo and this tissue repels germ cells from the midline toward the lateral sides. This mechanism might be sufficient to get germ cells to the lateral sides but could actually hinder the equal distribution of germ cells because it also prevents germ cells from crossing the midline from one side of the embryo to the other. Therefore, it is important that germ cells are already evenly spread laterally when they exit the midgut. Wunen-mediated germ cell-germ cell repulsion inside the midgut is well placed to ensure that germ cells exit the midgut in all directions and therefore are likely to sort equally to lateral sides and hence to both gonads (Renault, 2010).

If germ cells repel each other, then how do they come together to cluster at the embryonic gonad? Although it is possible that attractive cues from the gonad act to overcome repulsive effects of the germ cells, it is noted that the coalescence of the germ cells into the embryonic gonad is driven by the somatic cells of the gonad and not by the germ cells themselves. Wun2 expression in germ cells remains constant throughout embryogenesis; therefore, it is not believed that regulation of Wun2 levels plays a role in germ cell coalescence (Renault, 2010).

These findings have provided new insight into how wunens act at the molecular level in transepithelial migration (see Model showing how wunen activity leads to germ cell-germ cell repulsion), and germ cell migration in general, and extend the previous model. Wunens dephosphorylate a lipid phosphate, which is required for germ cell survival and attraction. Expression of wunens on somatic cells leads to spatial differences in lipid phosphate levels. Germ cells move out of the gut owing to expression of wunens on midgut cells, which depletes lipid phosphate levels. Expression of wunens on germ cells causes local depletions in lipid phosphate levels and hence germ cells migrate away to avoid contact with each other (Renault, 2010).

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. This study now shows that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. The integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs it was shown that wun function in the trachea is distinct from its role in germ cell migration (Ile, 2012).

This study demonstrates a role for an LPP in development of the trachea. In the absence of wun function, the trachea suffers from breaks in the dorsal trunk (DT), non-uniform lumen diameter, and loss of luminal components resulting from ineffective paracellular barrier function. wun functions tissue autonomously and Wun activity can be replaced by that of the close paralog Wun2 and two mouse homologs, but not by a catalytically dead LPP (Ile, 2012).

Defects are only seen in the trachea when wun is removed both maternally and zygotically and this is likely to explain why wun has not been previously uncovered in screens performed to identify genes required for tracheal development. The genetic data suggest that maternally provided Wun protein or the product of the reaction it catalyses lasts at least until the start of tracheal system formation and that zygotically expressed Wun in tracheal cells is sufficient to provide this activity. (Ile, 2012).

In germ cells the Wunens function redundantly: the germ cell death caused by loss of both proteins from germ cells can be rescued by expression of either protein alone, indicating that the two proteins have overlapping substrate specificities. In the trachea the situation is similar in that overexpression of wun2 in the trachea is able to substitute for loss of wun. To determine whether the roles of Wunens in germ cell migration and tracheal development are identical two mammalian LPPs with different activities were used. mLPP3 is able to substitute for Wunens in germ cell migration and survival assays, whereas mLPP2 is not, in spite of both proteins being highly expressed and localizing to the cell surface. As expected, it was found that mLPP3-GFP is able to rescue the tracheal phenotypes of wun wun2 M-Z− embryos; however, mLPP2-GFP is also able to do so. Thus, mLPP2 lacks an activity required for germ cell migration but possesses activity sufficient for tracheal development. It is concluded that the crucial LPP substrate or substrates for germ cell and tracheal development are different. Wunens and mLPP3 show relatively little substrate specificity and can dephosphorylate the lipid essential for both germ cell and tracheal development. mLPP2, by contrast, shows more restrictive specificity and can only dephosphorylate the lipid that is crucial for tracheal development. (Ile, 2012).

The localization of Wun-GFP to particular regions of the tracheal cell plasma membrane is intriguing. Mammalian LPPs have been demonstrated to localize to specific plasma membrane domains. Human (h) LPP1 (PPAP2A -- Human Genome Nomenclature Committee) sorts apically, whereas hLPP3 colocalizes with E-cadherin in Madin-Darby canine kidney (MDCK) cells and the C-terminal domain of hLPP3 has been shown to bind the AJ protein p120 catenin (catenin δ1). However, it remains to be confirmed whether specific Wun-GFP localization is crucial for activity. Wun2-myc can also rescue and, although the protein shows no specific localization, there might be sufficient present at SJs or apical membranes to fulfill the requirement for LPP activity. What is clear is that the LPPs, just as in germ cell migration, are playing more than a structural role because Wun2-H326K showed no rescue ability (Ile, 2012).

How does loss of Wun affect the tracheal epithelial cells? Overall, the polarity of these cells is unaffected, but AJ proteins are weaker at the apical surface. In this respect the tracheal phenotype is reminiscent of weak alleles of shotgun, which encodes Drosophila E-cadherin. Such mutants exhibit incomplete fusion of the DT and uneven luminal diameter. However, no genetic interaction between wun wun2 and shotgun is seen, suggesting that reduced DE-cadherin is not the critical factor in causing the tracheal defects in wun wun2 mutants. (Ile, 2012).

SJs were also affected in the wun wun2 M-Z− mutants: SJ components were not confined to the subapical region, as in wild type, and paracellular barrier function was lost. However, mutants for all essential SJ components reported to date display an abnormally elongated and convoluted DT. This phenotype is not seen in wun wun2 M-Z− mutants, indicating that although barrier activity may be lost, SJs must still be present and indeed they are seen by EM. This situation is similar to that of yrt M-Z− embryos, which also show compromised paracellular barrier function despite a normal complement of septa when examined ultrastructurally (Ile, 2012).

The lack of luminal accumulation of Serp and Verm in the wun wun2 M-Z animals is striking. Embryos mutant for serp and verm also display trachea with an abnormally elongated and convoluted DT. As this is not seen in wun wun2 M-Z animals, it is suspected that the loss of luminal Serp and Verm is not absolute. Indeed, extremely weak luminal staining id occasionally seen, but mostly Serp and Verm are detected in the hemolymph of late embryos. Although the possibility cannot be excluded that Serp and Verm are incorrectly secreted at the basolateral surface, the possibility is favored that Serp and Verm are apically secreted but owing to defects in the SJ-mediated paracellular barrier they diffuse from the lumen into the hemolymph. (Ile, 2012).

The differential accumulation of Serp and Verm versus the 2A12 antigen in the tracheal lumen in various mutant backgrounds has been interpreted to suggest that multiple secretory pathways exist. The first is actin dependent and is based on the observation that in dia mutants the 2A12 antigen is not present in the tracheal lumen whereas Verm is. The second is SJ dependent and is based on the fact that in mutants for the α subunit of the Na/K-ATPase, the 2A12 antigen accumulates in the lumen but Verm, which at stage 15 can been seen both in the lumen and in the tracheal cells, is undetectable in the lumen and tracheal cells by stage 16. Similar results have been obtained with mutants for other SJ components, including those encoded by sinuous, Lachesin, varicose, coracle and kune-kune. Based on these data, it is likely that the failure in Serp and Verm accumulation results, at least in part, from their diffusion out of the trachea. The difference in behavior between Serp, Verm and ANF-GFP versus the 2A12 antigen might depend more on the strength of their interaction with luminal components than on differences in their secretion. (Ile, 2012).

A model is proposed in which Wun expression at the cell surface leads to changes in intracellular lipid levels, which affects both AJs and SJs. These changes result in paracellular barrier defects and prevent particular luminal components from accumulating. Although the identification of which lipid or lipids are being affected and how changes in their levels and/or localization result in defects in specific tissues is ongoing, one potential Wun substrate, S1P, is known to increase barrier function in HUVEC cells via an S1P₁-dependent pathway (Ile, 2012).

What is particularly striking is that the role of Wun in barrier function for the trachea and ventral nerve cord in Drosophila appears to be representative of a more conserved aspect of LPP function. mLPP3 has an essential embryonic role in establishing vascular endothelial cell interactions during early development. In addition, mice with postnatal inactivation of Lpp3 specifically in the vascular endothelium are viable but have impaired vascular endothelial barrier function leading to vascular leakage, particularly in the lungs . Thus, it appears that mLPP3 is required in both the establishment and maintenance of vascular integrity in a tissue-autonomous fashion (Ile, 2012).

Recent studies have demonstrated crucial roles for lipids in establishing or maintaining epithelial cell plasma membrane identity. For example, phosphoinositides are central to establishing the apical surface during lumen formation in MDCK cells and glycosphingolipids are needed to maintain apicobasal domain identity in C. elegans intestinal cells. Modulation of lipid levels coupled with cell biological analyses in a developmental context will be invaluable in exploring this fascinating field further (Ile, 2012).

Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure

The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure this study shows that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. It was also shown that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype (Haack, 2014).

The movement of the heart progenitor cells to the midline has long been established to be dependent on dorsal closure. The cardioblasts were noted as being several cell diameters away from the dorsal edge of the epidermal primordium and moving relatively little compared to the ectoderm. Heart cell movement to the midline in Drosophila occurs through additional autonomous heart cell migration. Several lines of evidence support this conclusion. Firstly, live imaging of heart cells and ectodermal cells during dorsal closure shows uncoupling of the movement of heart and ectodermal cells. Secondly, cardioblasts make extensive protrusions during dorsal closure. It is speculated that these protrusions are required for motility, but they could be used for attachment to contralateral cardioblasts. In support of the former hypothesis, the protrusions occur from the onset of dorsal closure several hours before heart cells meet and are not suppressed through genetic mechanisms that are sufficient to suppress protrusions of ectodermal leading edge cells. The latter are generally longer (up to 10μm) than cardioblast protrusions (below 2μm) and are required for attachment to contralateral leading edges cells. Thirdly the strength of adhesion of heart cells to the ectoderm, as judged by the ablation experiments, is reduced as the heart cells approach the midline. It is speculated this is because the heart cells are moving partly autonomously at this time. Finally, when dorsal closure is delayed, as occurs in wun wun2 mutants, the heart cells migrate up to the amnioserosa before dorsal closure has completed. This phenotype appears to be a general feature when dorsal closure is delayed as it is reported to occur in other mutants (Haack, 2014).

Several molecular players are implicated in linking heart cells to the ectoderm: Spot adherens junctions (AJ) have been reported between cardioblasts and ectodermal cells. The AJ component DE-Cadherin (encoded by shotgun, shg) is highly expressed in heart cells and shg mutants display defects in cardioblasts reaching the midline as well as lumen formation. The extracellular collagen-like protein Pericardin (Prc) is expressed by pericardial cells and surrounds both them and cardioblasts Reduction in Prc levels causes interruptions in the cardioblasts lines, which appears to result from a loss of interaction with the ectoderm. Finally, disruption of integrin complexes, which are receptors for extracellular matrix proteins, using scab or mys mutants (encoding integrin α and β subunits respectively) causes mislocalisation of pericardial cells. If and how and these complexes and proteins are regulated to allow the heart cells to dynamically attach to the ectoderm remains an open question (Haack, 2014).

How might Wun and Wun2 be working mechanistically to promote heart formation and dorsal closure? The forces for dorsal closure arise from three sources. Firstly, actin rich filopodia from leading edge cells make contact with contralateral partners at the anterior and posterior most ends (canthi) and act in a zippering fashion. These filopodia are also important for correct alignment of the ectoderm with respect to parasegmental boundaries. Secondly, an actin-myosin-rich cable at the leading edge acts as a supracellular purse-string. Finally contractility and coordinated internalization of amnioserosa cells pulls the leading edges towards the midline (Haack, 2014).

This study found in wun wun2 mutants that the actin cable and leading edge filopodia are present, and internalization of amnioserosa cells is seen. Amnioserosa cells have highly wavy edges, normally only observed in much earlier embryos, during germ band retraction. Therefore the idea is favored that there are defects in tension in the ectoderm in wun wun2 mutants. wun and wun2 are expressed in the ectoderm and ectodermal wun2 expression is needed (along with heart cell expression) to rescue the heart defects of wun wun2 mutants. This loss of tension would also explain why the pericardial cells often lie away from the cardioblasts in wun wun2 mutants. In wild type the pericardial cells are strongly associated with the cardioblasts during dorsal closure. However, by the time the embryo is ready to hatch these two cell types are not tightly attached as can be seen during a heartbeat when the pericardial cells are thrust laterally and normally immediately rebound. It is speculated that if tension is lost then this rebound is weak leading to displacement of the pericardial cells (Haack, 2014).

EVOLUTIONARY HOMOLOGS

Cloning and characterization of lipid phosphate phosphohydrolases

A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42, polyclonal antibodies were raised in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. Hydrophobic segments H1, H3, and H5 had a signal/anchor function, and membrane insertion of Dri 42 is achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed (Barila, 1996).

A Mg2+-independent phosphatidate phosphohydrolase was purified from rat liver plasma membranes in two distinct forms, an anionic protein and a cationic protein. Both forms of the enzyme are able to dephosphorylate phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. When assayed at a constant molar ratio of lipid to Triton X-100 of 1:500, the apparent Km values of the anionic phosphohydrolase for the lipid substrates were 3.5, 1.9, 0.4, and 4.0 microM, respectively. The relative catalytic efficiency of the enzyme for phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate was 0.16, 0.14, 0.48, and 0.04 liter (min x mg)-1, respectively. The hydrolysis of phosphatidate is inhibited competitively by ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. The Ki(app) values were 5.5, 5.9, and 4.0 microM, respectively. The hydrolysis of phosphatidate by the phosphohydrolase conforms to a surface dilution kinetic model. It is concluded that the enzyme is a lipid phosphomonoesterase that can modify the balance of phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate relative to diacylglycerol, ceramide, monoacylglycerol, and sphingosine, respectively. The enzyme could thus play an important role in regulating cell activation and signal transduction (Waggoner, 1996).

Two human cDNA clones were obtained encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,158) and PAP-2b (Mr = 35,119), both of which contain six putative transmembrane domains. Both enzymes are glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b share 47% identical sequence and are the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34% to 39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings suggest critical roles for PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyze lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b is expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a is relatively specific, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation (Kai, 1997).

Phosphatidic acid (PA) and diacylglycerol (DG) are lipids involved in signal transduction and in structural membrane-lipid biosynthesis in cells. Phosphatidic acid phosphatase (PAP) catalyzes the conversion of PA to DG. This enzyme exists in at least two isoforms, one of which (PAP1) is presumed to be cytosolic and membrane associated and the other (PAP2) to be an integral membrane protein. Homology search of the GenBank database using a murine sequence probe enabled the cloning of several putative human isoenzymes. Two isoforms, presumed to be alternative splice variants from a single gene, designated as PAP2-alpha1 and PAP2-alpha2, have been cloned and expressed. The PAP2-alpha1 and PAP2-alpha2 have a 84% and a 72% overall match, respectively, with the published mouse PAP amino acid sequence. The area of alternative exon usage was confined to the coding region at amino acids 20 to 70. Ectopic expression of PAP2-alpha1 and PAP2-alpha2 cDNAs in ECV304 endothelial cells led to a 6- to 8-fold and a 2-fold increase in PAP activity, respectively, in cell-free extracts using an in vitro assay that measured the conversion of [14C]PA to [14C]DG. The increase in PAP activity in PAP2-alpha-transfected cells correlated with a >50% decrease in the steady-state PA level. Northern analysis showed that PAP2-alpha mRNA expression was suppressed in several tumor tissues, notably those derived from the lower alimentary tract. Subsequent analysis of colon tumor tissue derived from four donors confirmed lower expression of PAP2-alpha than in matching normal colon tissue. Considering these data and previous demonstrations that certain transformed cell lines have lower PAP activity, it is suggested that human PAP cDNAs may be candidates for gene therapy for certain tumors (Leung, 1998).

Phosphatidic acid (PA), lysophosphatidic acid, ceramide 1-phosphate (C1P), and sphingosine 1-phosphate (S1P) are lipid mediators generated by phospholipases, sphingomyelinases, and lipid kinases. The major pathway for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types 1 and 2) of phosphohydrolase activities (PAPs). cDNAs encoding two type 2 PAPs, PAP-2a and PAP-2b, have been expressed by transient transfection and shown to catalyze hydrolysis of PA, C1P, and S1P. A third type 2 PAP enzyme, PAP-2c, has been identified that exhibits 54% and 43% sequence homology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low endogenous PAP activity. Recombinant expression of the three PAP enzymes produces dramatic increases in membrane-associated PAP activity. Expression of PAP-2a but not PAP-2b or -2c resulted in high levels of cell surface PAP activity in intact insect cells. Kinetic analysis of PAP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C1P, and S1P revealed differences in substrate specificity and susceptibility to inhibition by sphingosine, Zn2+, and propranol (Roberts, 1998).

Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with LBP1, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, this study reports the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP1 can partially substitute for the function of LBP1. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate. Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, these results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate (Mandala, 2000).

The wunen gene of Drosophila encodes a multipass membrane-spanning protein that negatively regulates primordial germ cell migration. A mouse gene has been cloned that encodes a protein homologous to wunen and to the Type 2 phosphatidic acid phosphatases. This gene encodes a 251-amino-acid protein that most closely resembles the human Type 2 phosphatidic acid phosphatase PAP-2c. Northern blot analysis reveals the presence of a single 1.9-kb Ppap2c transcript. The Ppap2c gene was localized to the central portion of mouse Chromosome 10 by interspecific backcross analysis (Zhang, 2000a).

Characterization of enzymatic activity of lipid phosphate phosphohydrolases

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87% and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate when compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation is inhibited by extracellular Ca2+ and this inhibition is diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations does not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts show relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreases the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling (Jasinska, 1999).

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. The Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzes the dephosphorylation of DGPP. This reaction is Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and is inhibited by Mn2+ ions. PAP2 exhibits a high affinity for DGPP. DGPP inhibits the ability of PAP2 to dephosphorylate PA, and PA inhibits the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae is inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 shows homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, this work indicates that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. A model is proposed in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol (Dillon, 2003).

Lipid phosphate phosphohydrolase mutation

Phosphatidic acid phosphatases (PAPs) catalyze the conversion of phosphatidic acid to diacylglycerol and inorganic phosphate and have been postulated to function both in lipid biosynthesis and in cellular signal transduction. In Drosophila melanogaster, the Type 2 phosphatidic acid phosphatase protein encoded by the wunen gene, negatively regulates primordial germ cell migration. A mouse Ppap2c gene encodes the Type 2 phosphatidic acid phosphatase Pap2c. To analyze the in vivo role of the Ppap2c gene, a null mutation was constructed by gene targeting. Ppap2c(-/-) homozygous mutant mice are viable, fertile, and exhibit no obvious phenotypic defects. These data demonstrate that the Ppap2c gene is not essential for embryonic development or fertility in mice (Zhang, 2000b).

Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in beta-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit beta-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, are able to inhibit beta-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on beta-catenin-mediated TCF transcription remain to be determined (Escalante-Alcalde, 2003).

Lipid phosphate phosphohydrolases and axon guidance

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. A membrane-associated lipid phosphate phosphatase is described that was named plasticity-related gene 1 (PRG-1). PRG-1 facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, a novel mechanism is proposed by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space (Bräuer, 2003).

Lysophosphatidic acid-induced mitogenesis is regulated by lipid phosphate phosphatases and is Edg-receptor independent

Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors it has been demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7 (Hooks, 2001).

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. This study introduce a new assay for self-generated chemotaxis, and shows that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not of its homologues LPP1 or LPP2 - diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. These results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering. (Susanto, 2017).

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Search PubMed for articles about Drosophila wunen and wunen-2

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date revised: 5 December 2023

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