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).
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).
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).
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 N14m- 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 N14m- 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. N14m- 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 N14m- 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 N14m- 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 N14m- 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 N14m- 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 N14m- embryos die after they pass through the midgut epithelium. Additional experiments have suggested that pole cell death in N14m- 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 wun2N14 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 (wun2Delta) was isolated in which one-third of the wun2 locus was deleted. The wun2Delta mutant was homozygous viable, and lacked wun2 RNA expression. In embryos derived from wun2Delta/Df(2R)w45-19g and wun2Delta/wun2N14 mothers, pole cells formed normally, but most of them died after exiting the midgut; phenotypes essentially the same as those of the N14m- embryos. The maternal wun2Delta 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 wun2N14 phenotype was examined. The two mutant Pwun2-8k transgenes with H247K or H326K failed to rescue the wun2N14 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 wun2Delta/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, wun2N14 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 wun2m- 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 wun2m- 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 wunm+z- wun2m-z- embryos) showed a partial rescue of the pole cell death phenotype of the maternal wun2 mutation). In wunm+z- wun2m-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 wunm+z- wun2m+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 wun2m- 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 wun2m- embryos. Furthermore, TUNEL-positive pole cells have never been detected in wun2m- embryos. Thus, the pole cell death in wun2m- 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 wun2m- 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 wunm+z- wun2m-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 wunm+z- wun2m-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).
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).
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date revised: 15 February 2011
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