InteractiveFly: GeneBrief

PDGF- and VEGF-related factor 1: Biological Overview | References

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. This study demonstrates that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands (Harris, 2007b).

Cell migration is an essential part of the development and function of many cell types in all multicellular organisms. Guidance by external spatial cues directs a migrating cell or tissue to maintain an appropriate migratory path within an organism and ultimately reach the correct target. There are many examples of this, including immune cells that receive chemical gradient cues throughout development, as well as during their lifetime as pathogen fighting cells, neurons that receive cues promoting axon guidance, the multistep migration of the primordial germ cells and migration of the border cells in the Drosophila ovary (Harris, 2007b).

The embryonic development of the Drosophila salivary glands provides a good system to study guided cell migration. The salivary glands consist of two cell types: gland cells and duct cells, which are specified on the ventral surface of parasegment 2. During stage 11, the circular salivary placodes form and are visible as two groups of cells on either side of the ventral midline. The placodes are separated ventrally by cells that will give rise to the salivary ducts. After specification, the salivary placodes begin to invaginate into the embryo. When the salivary glands reach the visceral mesoderm, the glands turn and begin posterior migration. The glands are completely internalized by stage 13 and lie parallel to the anteroposterior axis of the embryo. This posterior migration is a heavily regulated process involving attractive and repulsive cues and complex tissue-tissue interactions that are just beginning to be understood. Recent work on these cues has revealed startling similarities between salivary gland migration and axonal development (Harris, 2007b).

This study characterized the role in salivary gland development of Pvr, the gene coding for the single Drosophila homolog of the mammalian PDGF/VEGF receptors. Previous studies have shown that Pvr is needed for border cell migration, hemocyte migration and survival, thorax closure during metamorphosis, and the rotation and dorsal closure of the male terminalia. These processes involve concerted morphogenetic cell movements which are disrupted in Pvr mutants. This study reports that Pvr is expressed in Drosophila embryonic salivary glands and that mutations in Pvr disrupt the concerted migration of the salivary glands. Furthermore, at least two of the Pvf ligands, Pvf1 and Pvf2 are required for this migration (Harris, 2007b).

In Drosophila embryos the Pvr receptor is expressed in the hemocytes where it is necessary for cell survival and for migration of the hemocytes throughout the embryo. Another site of embryonic Pvr expression is the developing salivary gland. Salivary expression of Pvr mRNA is strongest at stage 11 of embryonic development, when salivary gland cells are still situated on the surface of the embryo as circular placodes. Transcript levels steadily decrease through stage 12, during which time the placode cells invaginate. At stage 13 Pvr transcripts are practically undetectable. PVR protein is detected in the gland beginning at stage 12 and is localized to the cell membrane (Harris, 2007b).

Three genes in the Drosophila genome code for Pvr ligands: Pvf1, Pvf2, and Pvf3. Both Pvf2 and Pvf3 are expressed in the ventral midline, where they are thought to act in a partially redundant manner as attractive cues for hemocytes migrating out of the head (Cho, 2002; Wood, 2006). Previous studies have shown that Pvf2 and Pvf3 share more than just a similar expression pattern. These genes are located only 16 kb apart and may have been generated by recent gene duplication. Sequence similarities indicate that they are likely to be functionally similar to each other as well (Cho, 2002). In contrast, Pvf1 contains unique, cysteine-rich CXCXC motifs not found in the other two ligands, and it has a distinct expression pattern. The developing salivary gland is the strongest site of Pvf1 expression, beginning at stage 12 and persisting through stage 17. Interestingly, Pvf1 protein is expressed at highest levels in the cells near the tip of the gland that are the most actively involved in migration. Pvf1 is not expressed in the ventral midline and has does not have a significant effect on embryonic hemocyte migration (Cho, 2002; Wood, 2006; Harris, 2007b and references therein).

The importance of Pvr and the Pvf ligands for salivary gland development was confirmed when Pvr mutant embryos were found to have salivary gland migration defects. Pvr null mutant embryos have salivary glands that curve abnormally toward the ventral surface of the embryo, instead of lying parallel to the A-P axis of the embryo as in wild-type embryos. In contrast to hemocytes, salivary gland survival, as tested by TUNEL staining, was not affected by Pvr mutations (Harris, 2007b).

Mutations in the ligand genes Pvf1 or Pvf2 caused ventral curving similar to that in Pvr mutant embryos. In contrast, Pvf3^EY09531, a P-element insertion located in the first intron of the Pvf3 gene, very infrequently affects embryonic gland positioning. This impenetrant phenotype may be due to the timing of Pvf3 expression, which occurs prior to salivary gland invagination and decreases during the time that the salivary gland migrates posteriorly. Alternatively, the EY09531 P-element insertion may be a weak, hypomorphic mutation that does not eliminate Pvf3 function (Harris, 2007b).

Several factors required for salivary gland guidance and migration have already been identified. After the salivary gland contacts the VM and begins to move posteriorly within the embryo, the attractant Netrin and two repellents, Slit and Wnt4, guide the salivary glands (Harris, 2007a; Kolesnikov, 2005). Netrin, which is expressed in the CNS and the visceral mesoderm, works as an attractant to maintain salivary gland positioning on the visceral mesoderm. At the same time, CNS expression of Slit and Wnt4 keeps the salivary glands away from the CNS and parallel to it. The early timing of the Pvr phenotype and its similarity to the slit and Wnt4 phenotypes suggest that PDGF/VEGF signaling is required at the same time as Netrin, Slit and Wnt4 signaling and might be required for the same process, salivary gland guidance. Near the end of salivary migration, a different signal-receptor pair, Wnt5 and Derailed, is required at the distal tip of the glands to mediate attachment to the longitudinal visceral mesoderm (Harris, 2007a; Harris, 2007b and references therein).

In addition to its role in the salivary gland, Pvr is essential for hemocyte migration throughout the embryo (Cho, 2002; Wood, 2006). During their migration hemocytes lay down components of the extracellular matrix needed by other cells for movement. For example, the extracellular matrix is indispensable for the process of ventral nerve cord condensation. In Pvr mutants, this condensation fails due to defects in hemocyte migration and extracellular matrix deposition. This extracellular matrix might also be important for salivary gland migration. Therefore, whether the salivary gland defects caused by mutations in Pvr and its ligands were autonomous to the salivary gland was investigated. When expression of a dominant negative allele of Pvr is driven in the salivary glands by fkh-GAL4, the glands curve ventrally as they do in Pvr mutants. In contrast, expression with hemocyte specific driver pxn-GAL4 results in near background levels of ventral curving, despite the inhibition of hemocyte migration. Thus, Pvr activity is autonomously required in the salivary glands. Neither proper hemocyte migration nor the extracellular matrix that hemocytes deposit is required for salivary gland migration. Furthermore, the gland migration defects in Pvr mutants are rescued in embryos carrying fkh-GAL4 and either UAS-Pvr or the constitutively active UAS-λPvr construct (Harris, 2007b).

Previous studies in Drosophila have shown that the PVF ligands act as attractants. In the border cells, ectopic expression of PVF1 is sufficient to redirect border cells towards the site of expression (McDonald, 2003). Similarly, PVF2 and PVF3 in the ventral midline act as attractants for migrating hemocytes. Ectopic expression of PVF2 using breathless-GAL4 has been shown to induce a chemotactic response to the new source of PVF expression from the embryonic hemocytes (Cho, 2002). The ventral midline expression of PVF2, along with its ventral curving, mutant phenotype, suggests that PVF2 might be acting as a repellent for the salivary gland, despite the fact that there is no previous indication of PVF ligands acting as repellents. However, ectopic expression of PVF2 in the visceral mesoderm was not sufficient to redirect the salivary glands away from the visceral mesoderm (Harris, 2007b).

Overexpression of Pvf1 in the salivary glands results in ventrally curved glands similar to those seen in Pvr mutants. Similarly, misrouted glands result from salivary gland expression of Pvf2 and Pvf3. These results suggest that all three ligands may have the same or similar effects on the PVR receptor, despite their differing expression patterns and slightly different sequences. Interestingly, expression of the constitutively active receptor, λPVR in otherwise wild-type embryos results in salivary glands that are ventrally curved, similar to overexpression of the individual ligands in the gland. One possible explanation for these results is that the PVF ligands might be required for proper salivary gland positioning by regulating adhesion or another migratory event autonomous to the salivary gland (Harris, 2007b).

Alternatively, both the salivary gland expressed PVF1 and ventral midline expressed PVF2 may be required for a directional response to be received, perhaps forming a heterodimer to activate the PVR receptor. This possibility is attractive since vertebrate studies have shown that heterodimers are formed between PDGF-A and PDGF-B that have a unique binding affinity for PDGF receptor subtypes that differs from the affinity of either homodimer. Furthermore, the PDGF-AB heterodimer is capable of activating different signal transduction pathways thus eliciting a different response on proliferation as well as gene expression compared to the PDGF-AA or PDGF-BB monomers. It seems plausible that a similar situation may be occurring in the salivary glands, where both PVF1 and PVF2 are needed in order to activate PVR and direct the salivary glands (Harris, 2007b and references therein).

Developmental control of blood cell migration by the Drosophila VEGF pathway

A vascular endothelial growth factor (VEGF) pathway controls embryonic migrations of blood cells (hemocytes) in Drosophila. The VEGF receptor homolog is expressed in hemocytes, and three VEGF homologs are expressed along hemocyte migration routes. A receptor mutation arrests progression of blood cell movement. Mutations in Vegf17E (CG7103; also referred to as Pvf1) or Vegf27Cb (CG13780; also referred to as Pvf2) have no effect, but simultaneous inactivation of all three Vegf genes, including Vegf27Ca (CG13781/13782; also referred to as Pvf3) phenocopies the receptor mutant, and ectopic expression of Vegf27Cb redirects migration. Genetic experiments indicate that the VEGF pathway functions independently of pathways governing hemocyte homing on apoptotic cells. The results suggest that the Drosophila VEGF pathway guides developmental migrations of blood cells, and it is speculated that the ancestral function of VEGF pathways was to guide blood cell movement (Cho, 2002).

Blood cells (hemocytes) in Drosophila migrate extensively during development. They originate in the head mesoderm, and over a 7 hr period in midembryogenesis they migrate along specific pathways to disperse throughout the body, where they function as immune and interstitial cells. Like vertebrate monocytes and macrophages, insect hemocytes phagocytose or encapsulate foreign material and apoptotic cells. This is important during development because cell death is widespread, and as hemocytes disperse through the embryo they recognize and remove cell remnants. Hemocytes also produce many extracellular matrix molecules, including collagen IV and laminin, that compose the basement membrane surrounding internal organs. Although there has been progress in understanding the genetic control of blood cell differentiation in Drosophila and the ability of blood cells to recognize and engulf dying cells, little is known of the genetic and molecular mechanisms controlling their developmental migrations. It is also unclear how developmental migrations are coordinated with hemocyte homing toward dying cells along the migration pathway (Cho, 2002).

A large-scale mutagenesis was carried out by mobilizing a piggyBac[w⁺] transposable element. Inverse PCR and DNA sequencing of piggyBac[w⁺] insertion sites have identified three lines with insertions in Vegfr. Vegfr^c2195 is a homozygous lethal insertion located within the small (67 base) 11^th intron. RNA in situ hybridization has demonstrated that Vegfr transcript is undetectable in Vegfr^c2195 embryos, and genetic studies indicate it is an amorphic allele (Cho, 2002).

Vegfr^c2195 mutants display a striking defect in hemocyte migration. Formation of hemocytes and their initial migrations are normal, as judged by staining of Croquemort (CRQ) and Peroxidasin (PXN). By stage 11, posteriorly directed hemocytes reach the caudal margin normally. However, unlike Vegfr⁺ blood cells, which rapidly enter the tail, blood cells in mutant embryos never enter the region, instead accumulating at the caudal margin. By stage 13, wild-type hemocytes are dispersed throughout the embryo, whereas mutant hemocytes have clumped together in aggregates concentrated in the anterior. The mutant blood cells continue to express CRQ and PXN, suggesting that hemocyte differentiation is grossly intact. Interestingly, CRQ staining is stronger than in wild-type, even in isolated blood cells, implying their phagocytic function is activated. Aside from the hemocyte defects, the mutant embryos appear normal, and no defects are detected in the CNS, muscles, and tracheal system after staining with tissue-specific markers (Cho, 2002).

The severity of the hemocyte phenotype of Vegfr^c2195 homozygotes is the same as that of Vegfr^c2195 hemizygotes and homozygous deficiency embryos, implying that Vegfr^c2195 is an amorphic allele. Vegfr^c2859, a lethal piggyBac[w⁺] insertion in the first intron, and Vegfr^c3211, a homozygous viable insertion in the 3' noncoding region, do not exhibit the hemocyte phenotype. However, the disruption of hemocyte migration clearly reflects a requirement for Vegfr function, since the migration defect is also seen when endogenous Vegfr transcripts are depleted by RNAi. It is concluded that inactivation of the Vegfr gene blocks progression of blood cell movement; hence, the gene name stasis (stai, pronounced 'stay'), which means slowing or stopping, is proposed (Cho, 2002).

The RAS-MAPK pathway is activated by signaling through VEGFRs and other RTKs, so whether RAS-MAPK is involved in hemocyte migration was investigated. Immunostaining with a diphospho-MAPK antiserum shows that MAPK is activated in migrating hemocytes. MAPK activation is greatly reduced in Vegfr mutants, and it is increased by ectopic expression of a VEGF ligand. It was not possible to test the effect of complete loss of RAS-MAPK pathway activity in embryonic hemocytes, because pathway components are both maternally and zygotically required and expressed. Zygotic loss of function mutations in any of the three genes encoding adaptor proteins (drk, dshc) or a RAS exchange factor (sos) have no effect on hemocyte migration. However, expression of a dominant-negative RAS protein (DRAS1^N17) in hemocytes causes an early migration arrest similar to that seen in the Vegfr mutant, implicating RAS in the process (Cho, 2002).

BLAST searches have identified three genes encoding proteins with sequence similarity to vertebrate VEGFs. Vegf27Ca (CG13781/13782; also referred to as Pvf3) and Vegf27Cb (CG13780; also referred to as Pvf2) are adjacent genes at cytological position 27C. The genes are tandemly arrayed, separated by ~16 kb. Their close proximity, sequence similarity, and nearly identical expression patterns suggest they have been generated by a recent gene duplication. The VEGF homolog at cytological position 17E (Vegf17E; CG7103; also referred to as Pvf1) produces two splice variants (A and B) that differ by 11 N-terminal residues (Cho, 2002).

All the Drosophila VEGF proteins have a predicted signal peptide and central domain common to VEGF/PDGF superfamily members. All also have a cysteine-rich C-terminal domain, as do vertebrate VEGF-C and VEGF-D, but lack the C-terminal heparin binding domain found in human VEGF-A and VEGF-B. VEGF17E is slightly more similar to vertebrate VEGFs than to PDGF, whereas VEGF27Ca and VEGF27Cb are equally similar to both (Cho, 2002).

Embryonic expression patterns of Vegf genes were analyzed by RNA in situ hybridization. The genes are expressed in dynamic spatial and temporal patterns that line many of the migratory paths of developing blood cells. Vegf17E begins to be expressed at the end of stage 10 in an ectodermal patch at the caudal margin of the germband where blood cells enter the tail. It is also expressed in the developing trachea and salivary glands. General tracheal expression persists through stage 12, after which it restricts to the tips of growing ganglionic branches and more strongly in the visceral branches. The latter is the site where a novel population of Vegfr-positive cells cluster in the embryo. From stage 12 on, Vegf17E is expressed in Malpighian tubules, and beginning at stage 13, in a posterior ring of ectodermal cells (Cho, 2002).

Vegf27Ca and Vegf27Cb are also expressed along blood cell migration routes. Both display the same expression pattern. Beginning at stage 9 and into stage 11, the genes are expressed in caudal ectoderm and developing hindgut, foregut, and ventral nerve cord. The hindgut (and subsequent Malpighian tubule) expression during stages 11-13 corresponds precisely to the position where blood cells cluster after entering the tail. There is also a striking correlation between the location of Vegf27Ca/27Cb expression and Vegfr-expressing blood cells at the ventral nerve cord, along which hemocytes move to reach the middle of the embryo. In late embryogenesis, Vegf27Ca/27Cb expression is detected in Malpighian tubules and a foregut derivative, both of which are also associated with migrating hemocytes (Cho, 2002).

In aggregate, the expression pattern of the three Vegf genes coincides with many blood cell migratory paths. This correlation is especially clear at the entry site into the tail and along the ventral midline, paths that Vegfr^- hemocytes fail to engage (Cho, 2002).

To determine if Vegf genes are required for hemocyte migration, Vegf17E and Vegf27Cb mutants were isolated. Three Vegf17E mutants were examined, including Vegf17E^ex3.6, a transcript null allele. No blood cell migration defects were detected in any of the mutants. A piggyBac[w⁺] insertion in Vegf27Cb, Vegf27Cb^c6947 was characterized. It is a homozygous viable insertion five nucleotides downstream of the 5' splice site of the fourth intron. This would likely disrupt exon 4-5 splicing and prevent inclusion of C-terminal coding sequences. No hemocyte migration defects were detected (Cho, 2002).

The above results suggested that if Vegf ligands are required for blood cell migration, they are likely to be redundant. To test for redundancy, RNAi was used to inactivate multiple Vegf genes simultaneously. As a control, it was first demonstrated that inactivation of Vegfr by RNAi could phenocopy the blood cell migration defects in Vegfr mutants. 59% of embryos showed mild (class I) to severe (class III) defects in hemocyte migration, with 24% of affected embryos showing a severe phenotype similar to that observed in null Vegfr mutants. RNAi of either Vegf27Ca or Vegf27Cb alone has little effect above background, and simultaneous RNAi of both Vegf27Ca and Vegf27Cb has only a moderate effect. Simultaneous inactivation of all three Vegf genes, however, results in a defect very similar to Vegfr inactivation: 71% of injected animals showed blood cell migration defects, with 14% of affected embryos showing the extreme phenotype. It is concluded that Vegf ligands are redundantly required for blood cell migration, and they are required for the same function as Vegfr (Cho, 2002).

The Gal4/UAS system was used to test if misexpression of a Vegf ligand could alter hemocyte migration. Misexpression of Vegf27Cb in the developing foregut, salivary duct, trachea, and midline glia using breathless-Gal4 driver (btl-Gal4) and UAS-Vegf27Cb (XP d2444) causes misrouting of hemocytes. In many embryos, most blood cells are redirected to anteroventral positions on and around the foregut, the site of ectopic expression closest to where they originate. Similar experiments using btl-Gal4 and XP d5686 to misexpress Vegf17E did not show an effect on hemocyte migration, nor did experiments using a UAS-Vegf17E transgenic line (UAS-Vegf17E-B). This suggests that the activity or diffusion properties of VEGF17E ligands may differ from those of VEGF27Cb (Cho, 2002).

The only gene previously known to alter the developmental migration of hemocytes is singleminded, a transcription factor that controls ventral midline development. In sim^- embryos, ventral midline cells do not develop normally, and hemocytes do not migrate along this tissue. Ventral midline expression of Vegf27Cb is selectively eliminated in sim^- embryos. Thus, sim functions upstream of Vegf27Cb in control of blood cell migration in the nervous system (Cho, 2002).

The simplest model of how VEGF signaling controls blood cell migration is that VEGFs serve as chemoattractants for blood cells expressing VEGFR. Vertebrate VEGFs function in vitro as chemoattractants for leukocytes and blood vessel enthothelial cells, and the ligand-expressing cells in Drosophila are located at the entry site to the tail and along most hemocyte migratory routes in the posterior. Thus, they are perfectly positioned to guide hemocytes along these routes. Not only are Vegf ligand genes required for migration, but ectopic expression of one of them (Vegf27Cb) in the foregut caused rerouting of blood cells to this tissue, demonstrating that localized expression of the ligand provides guidance information. Duchek (2001) proposes a similar role for VEGF17E in border cell migration in the egg (Cho, 2002).

In the chemoattraction model, VEGFs guide most blood cell migrations into and around the posterior. Because there are multiple sites of Vegf gene expression, the question arises as to how cells progress from one VEGF source to another. What causes them to leave the first source encountered? Perhaps ligand expression is highly dynamic, turning off transiently after a blood cell arrives, or perhaps a cell's arrival triggers mechanisms that selectively desensitize the cell to ligand produced from that source. The different ligand and receptor isoforms could also play a role if they have different functional properties, as suggested by misexpression studies with Vegf27Cb and Vegf17E. There also could be auxiliary factors that promote blood cell movement away from one VEGF source and on to the next (Cho, 2002).

VEGF pathway mutants divide blood cell migration into three phases. The Vegf expression patterns and loss of function phenotype suggest that VEGF signaling controls many instances of migration of blood cells, particularly those in and around the posterior. But the results also imply involvement of other signaling pathways before and after their arrival at the posterior. In Vegfr^-embryos, the initial migration of hemocytes to the caudal margin is unaffected. Anteriorly and ventrally directed migrations during these stages also appear grossly normal. This defines an early, Vegf-independent phase of migration (Phase I). Also, the late dispersal of hemocytes is not associated with Vegf expression, defining another Vegf-independent phase (Phase III). It will be important to identify the pathways that control these early and late migrations and learn how they are coordinated with the VEGF pathway. It will also be of interest to explore the function of VEGF signaling in the few domains of Vegf expression not obviously associated with blood cell migration (Cho, 2002).

PVF1 is sufficient to guide border cells during oogenesis

The border cells of the Drosophila ovary undergo a well-defined and developmentally regulated cell migration. Two signals control where and when the cells migrate. The steroid hormone ecdysone, acting through its receptor and a coactivator known as Taiman , contributes to regulating the timing of border cell migration. PVF1, a growth factor related to platelet-derived growth factor and vascular-endothelial growth factor, contributes to guiding the border cells to the oocyte. To probe the mechanisms controlling border cell migration, a screen was performed for genes that exhibit dominant genetic interactions with taiman. Fourteen genomic regions were identified that interact with taiman. Within one region, Pvf1 was identified as the gene responsible for the interaction. Signaling by PVF1 has been proposed to guide the border cells to their proper target, but ectopic PVF1 has not been tested for its ability to redirect the border cells. The ability of PVF1 (as well as other factors such as Gurken) to guide the border cells to new targets was tested. Ectopic expression of PVF1 is sufficient to redirect border cells in some egg chambers but the other factors tested are not. These data suggest that the guidance of border cell migration is robust and that there are likely to be additional factors that contribute to long-range guidance of these cells. In addition, taiman and Pvf1 regulate the dynamic localization of E-cadherin in the border cells, possibly accounting for the interaction between these two pathways (McDonald, 2003).

The interaction of tai with Pvf1 appears to be specific because tai does not interact with either loss-of-function mutations or deficiencies that remove other genes known to regulate border cell migration, such as slbo or shotgun/DE-cadherin. Mutations in slbo or shotgun reduce DE-cadherin levels in the border cells, so tai does not interact with every gene that regulates DE-cadherin, possibly because tai regulates the distribution rather than the levels of DE-cadherin in the border cells. Identification of Pvf1 indicates that this screen provides a useful approach for identifying additional loci that affect border cell migration in general and regulate turnover of adhesion in particular (McDonald, 2003).

The genetic interaction between Pvf1 and tai indicates that the regulation of border cell migration timing and guidance might be linked. What is the nature of the interaction between tai and Pvf1 during border cell migration? Ecdysone signaling does not regulate PVF1 or PVR expression nor does Pvf1 regulate TAI expression, but the ecdysone and Pvf1 pathways both affect the distribution of DE-cadherin and Arm. A model is favored whereby tai and Pvf1 interact because they both regulate adhesion complex localization or turnover. The tai and Pvf1 genes could act independently to regulate cadherin-dynamics. Alternatively, tai and Pvf1 might function in a common pathway. TAI and PVR both function autonomously in the border cells, although they are unlikely to bind directly to each other because TAI localizes to the nucleus and PVR is a receptor tyrosine kinase localized to the membrane. One possibility is that PVR activates (or represses) the function of a protein whose expression is dependent on TAI, and that this protein in turn regulates cadherin dynamics in the border cells. Tyrosine phosphorylation of ß-catenin, the Arm homolog, causes destabilization of adhesion complexes in other cell types, so perhaps PVR activity destabilizes E-cadherin/Armadillo complexes specifically in the border cells. Identification of additional genes identified in this screen, in particular those that affect adhesion turnover in border cells, should help clarify the biochemical relationship between TAI and PVF1 (McDonald, 2003).

The results reported here demonstrate that ectopic expression of PVF1 is sufficient to redirect border cells even though, in Pvf1 null mutants, border cell clusters migrate normally in the majority of egg chambers. When PVF1 is ectopically expressed in random follicle cells, the border cells are attracted to these sources of PVF1. The border cells are attracted more efficiently to sources of PVF1 signal close to the anterior pole, indicating that they respond better to high concentrations of the ligand. The finding that doubling the dose of ectopically expressed PVF1 dramatically increases the frequency with which the cells respond to the ectopic signal confirms the idea of a concentration dependent effect (McDonald, 2003).

The concentration of ectopic PVF1 at the anterior end of the egg chamber appears to exceed the concentration of endogenous PVF1 at that position, even when only a single UAS-Pvf1 transgene is included in the experiment. Consistent with that idea, elimination of endogenous PVF does not significantly alter the response of the border cells to ectopic ligand. The border cells still migrate normally in many cases, apparently ignoring ectopically expressed PVF1. The most likely explanation for this is that there are additional germ-line-derived attractive cues that instruct the cells to migrate correctly in the absence of endogenous PVF1 and in the presence of ectopic PVF1 (McDonald, 2003).

PVF2 does not seem to be a good candidate for a redundant guidance cue because loss of function of the PVR receptor produces a phenotype that is indistinguishable from loss of PVF1 alone. Moreover UAS-Pvf2 was not able to redirect the border cells. This finding is surprising because PVF2 is thought to bind and activate the same receptor as PVF1. It is especially surprising because only PVF2 (expressed from the same UASPVF2 transgene) and not PVF1 is effective at misguiding hemocytes in the embryo. Together, these findings suggest a striking, and as-yet inexplicable, specificity of ligand action that will be interesting to study further (McDonald, 2003).

Gurken, the major Egfr ligand in the ovary, is not an effective guidance cue for the border cells, either when expressed alone or in combination with PVF1. The inability of Grk to affect border cells is striking because even class II and III phenotypes are absent, even though these are not uncommon following PVF misexpression. This is consistent with the observation that migration of the border cells to the oocyte is completely normal in grk mutant egg chambers and in mosaic egg chambers in which border cells lack EGF receptor function. Grk does, however, have a role in the dorsal migration of the border cells after they reach the oocyte. Currently, the evidence supporting a role for Grk in migration of the border cells to the oocyte is the combined effect of dominant-negative PVR and dominant-negative Egfr. Taken together with the results supplied in this study, the evidence in favor of a role for Egfr is somewhat better than the evidence in favor of a role for Grk, possibly suggesting the involvement of other Egfr ligands (McDonald, 2003 and references therein).

In addition to ligands for Pvr and Egfr, this study might imply the existence of other, as-yet-unidentified cues, that participate in the long-range guidance of the border cells. It is proposed that PVF1, and possibly additional unknown ligands, guide the border cells to the oocyte. Similarly, in the Drosophila central nervous system, multiple short-range and long-range cues are required to guide motor axons properly to their appropriate muscle targets. Perhaps even a simple migration, such as that of the border cells, uses multiple cues, each of which might only have a small contribution. Screens such as the one reported here might help identify the full set of border cell migration cues as well as additional genes that function in adhesion complex turnover (McDonald, 2003).

Polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton

Epithelial tissue functions depend largely on a polarized organization of the individual cells. The roles of the Drosophila PDGF/VEGF receptor (PVR) in polarized epithelial cells were examined, with specific emphasis on the wing disc epithelium. Although the receptor is broadly distributed in this tissue, two of its ligands, PVF1 and PVF3 are specifically deposited within the apical extracellular space, implying that polarized apical activation of the receptor takes place. The apical localization of the ligands involves a specialized secretion pathway. Clones for null alleles of Pvr or expression of RNAi constructs showed no phenotypes in the wing disc or pupal wing, suggesting that Pvr plays a redundant role in this tissue. However, when uniform expression of a constitutively dimerizing receptor was induced, loss of epithelial polarity, formation of multiple adherens and septate junctions, and tumorous growth were observed in the wing disc. Elevation of the level of full-length PVR also gave rise to prominent phenotypes, characterized by higher levels of actin microfilaments at the basolateral areas of the cells and irregular folding of the tissue. Together, these results suggest that polarized PVR activation is necessary for the proper organization of the wing disc epithelium, by regulating the apical assembly of the actin cytoskeleton (Rosin, 2004).

Examination of PVR protein has revealed a broad expression in epithelial tissues in the embryo from stage 14, and in the imaginal discs. PVR expression is not confined along the apicobasal axis of the cells. In contrast to the uniform distribution of the receptor, there is restricted apical localization of the ligands PVF1 and PVF3 within the wing disc epithelium (Rosin, 2004).

The mechanism responsible for the apical accumulation of PVF1 and PVF3 in the wing disc is intriguing. Since cell junctions are likely to form barriers that can not be bypassed by exogenous ligand, apical accumulation may imply preferential secretion of PVR ligands at the apical compartment. It is possible that PVF1 and PVF3 are targeted to vesicles that are specifically marked for secretion at the apical surface. The observation that PVF3 overexpression compromises the secretion of PVF1 but not that of sGFP, supports such a possibility. The presence of distinct secretory vesicles that are targeted to apical versus basolateral compartments has been previously established (Rosin, 2004).

What further interactions do PVF1 and PVF3 undergo, once secreted to the apical extracellular compartment? One possible interaction involves binding to heparan-sulfate proteoglycans on the cell surface. The vertebrate VEGF proteins have a defined heparin-binding domain at the C terminus that is distinct from the receptor-binding moiety. The equivalent C-terminal domain of PVF1 does not show a distinct homology to the heparin-binding domain of VEGF. However this study shows that PVF1 secreted by S2 cells can bind heparin beads. To examine if PVF1 is trapped on the cell surface following secretion, marked clones of cells overexpressing PVF1 were created. The ligand is uniformly redistributed along the entire apical surface, including the surface of cells not secreting the ligand. Although the ligand is capable of spreading readily within the apical plane, it is incapable of crossing the cell junctions, and is thus excluded from the basolateral extracellular compartment (Rosin, 2004).

Accumulation of PVF1 and PVF3 at the extracellular apical compartment implies that the PVR receptor is activated in a polarized fashion. Does such an apically polarized pattern of activation play a role in shaping the wing disc epithelium? The most direct way to examine PVR function in the wing disc is to generate clones for null Pvr alleles, and follow their phenotype. The Pvr-mutant clones are similar in size to their wild-type twins, and within the clones no aberrant morphology or misorganization of actin was detected. It is thus concluded that PVR has a redundant role in the wing. Nevertheless, a series of dramatic wing phenotypes is induced following expression of various PVR constructs. This analysis leads to a proposal that these phenotypes represent gain-of-function circumstances following inappropriate activation of PVR on the basolateral side of the wing disc epithelium (Rosin, 2004).

This interpretation was suggested by the dramatic effects of non-restricted and constitutive receptor activation, achieved by expression of lambdaPVR in the wing disc epithelium. The epithelium lost its polarity, multiple cell layers were generated, and giant tumorous discs were formed. Ectopic accumulation of F-actin around the circumference of the cells was observed, and corroborated by the identification of multiple adherens junctions in EM images (Rosin, 2004).

The phenotype created by expressing lambdaPVR in the wing disc is reminiscent of the phenotype described for loss of the septate junction proteins DLG and Scribbled, as well the LGL protein. It is believed that the alterations in cell polarity following lambdaPVR expression are less severe than the dlg, scribble or lgl mutant phenotypes. Although excess adherens junctions are established and the septate junctions are mislocalized, the LGL protein, which requires intact septate junctions for its insertion into the membrane, is found associated with the membrane in wing discs expressing lambdaPVR. The tumorous growth of the cells is believed to be a secondary consequence of the loss of polarity, which may lead to impairment of cell-cell communication (Rosin, 2004).

The consequences of misexpressing full-length PVR were examined. Significantly higher levels of F-actin were noticed in the basolateral area of the cells expressing PVR, while the level of actin monomers was lower. This indicates that PVR has a localized effect on actin polymerization, rather than a general role in actin monomer synthesis. Elevation in Profilin (Chickadee) protein levels was also noticed. Profilin binds actin monomers in a way that inhibits nucleation and elongation of pointed ends but promotes rapid elongation of uncapped barbed ends, leading to depletion of the actin monomer pool (Rosin, 2004).

Overexpression of the ligands alone did not lead to any phenotype, while even mild overexpression of the receptor resulted in pronounced phenotypes. Moreover, these phenotypes were strongly enhanced by elevating the levels of PVF1 in the apical domain. There are two possible explanations for the overexpression phenotype. The overall levels of receptor activation may be important. The receptor could be present in limited amounts, so that increasing its levels allows more ligand at the apical side to bind and activate receptors. Alternatively, polarized activation of the receptor that normally takes place is disturbed, because of redistribution of the ligand. This may happen by recycling of the ligand-bound receptor inside the cells. The fact that elevation in PVR levels resulted in basolateral polymerization of actin, while the ligand is normally found on the apical side supports this possibility. Mislocalized activation of the receptor may also take place because of spontaneous dimerization caused by the higher levels of the receptor (Rosin, 2004).

It is interesting to note that while lambdaPVR, a constitutively dimerized form of PVR, gave rise to a dramatic phenotype when expressed in the wing disc or the follicular epithelium, no apparent phenotypes were observed following expression in the embryonic ectoderm or the eye disc. Some of the intracellular elements that may be essential for relaying the signals resulting from PVR activation could thus be expressed or active only in a restricted set of tissues (Rosin, 2004).

In the embryonic ectoderm and eye disc where lambdaPVR was inactive, apical accumulation of PVF1 was not seen. The correlation between the capacity of the wing epithelium to localize the ligands apically, on the one hand, and to respond to uniform PVR activation, on the other, strengthens the notion that apical activation of PVR is instructive in this tissue (Rosin, 2004).

What can the ectopic phenotypes teach us with regard to the normal downstream responses to PVR activation in the wing epithelium? The primary defect upon overexpression of PVR is misorganization of the actin cytoskeleton at the basolateral side. In addition, expression of the constitutively active receptor results in multiple adherens junctions. It is thus suggested that apically restricted PVR activation provides signals that facilitate the formation of F-actin at the adherens junctions. This role is reminiscent of the activity of PVR in the border cells of the ovary, where polarized activation by PVF1, expressed in the oocyte, participates in guiding the migration of the border cells. It is tempting to suggest that polarized PVR activation regulates migration or cell polarity, using a common set of intracellular responses leading to localized actin polymerization (Rosin, 2004).

PVF1/PVR signaling and apoptosis promotes the rotation and dorsal closure of the Drosophila male terminalia

The Drosophila adult male terminalia originate from the genital disc. During the pupal stages, the external parts of terminalia evert from two ventral stalks; the everted left and right dorsal halves fuse at the dorsal midline. At the same time the male terminalia perform a 360o clockwise rotation. Several mutations are known to affect the rotation of the male terminalia, while none is known to affect dorsal closure. This study shows that the Pvf1 gene, encoding one of the three Drosophila homologues of the mammalian VEGF/PDGF growth factors, is required for both processes. Males either mutant for Pvf1 or bearing a dominant negative form of Pvr, the PVF receptor, do not complete either rotation or dorsal closure. Pvf1 expression in the genital disc is restricted to the A8 cells. However, PVF1/PVR signaling influences A8, A9 and A10 cells, suggesting that the PVF1 protein diffuses from its source. Flies hemizygous for the apoptotic genes hid, reaper and grim, or mutant for puckered which encodes a phosphatase that down-regulates the n-Jun-N terminal kinase pathway, lead to the same phenotypes as mutations in PVF1/PVR. These results indicate that PVF1/PVR signaling functions not only in apoptotic phenomena but are also required during rotation and dorsal closure of the Drosophila male genital disc (Mac̀as, 2004).

This demonstrates that either mutations at Pvf1 or the expression of a dominant negative form of its receptor, Pvr^DN, result in various degrees of male rotated terminalia and failure of dorsal closure. These observations indicate that the PVF1/PVR pathway is relevant in these morphogenetic processes. Although the Pvf1 gene is only expressed in a subset of cells from the segment A8, reduction or abolition of PVF1/PVR signaling affects the normal development of all terminalia precursors (A8, A9 and A10). Interestingly, mutations in the Abd-B m function, which affect only the A8 segment have a phenotype of rotated terminalia. Thus, these results highlight the importance of the A8 segment in this process. It is proposed that A8 cells affect the development of structures originated from A9 and A10 through the activity of the PVF1 protein diffusing from A8. Although the data concern transcript expression, Rosin (2004), demonstrated that PVF1 is capable of extensive lateral diffusion, so it has the properties of a long range signaling molecule. PVFs could form homo and heterodimers, what opens the possibility of different effects in the binding responses of the receptor. McDonald (2003), observed that homodimers are not equivalent, because PVF1 seems to be the relevant signal for the migration of border cells and Bruncker (2004), describe two function for PVR in the embryonic hemocytes, suggesting a diversity of functions. Other PVFs have not been examined, and although some partial redundancy was observed, it will be necessary to separate PVF individual or associated functions (Mac̀as, 2004).

Indirect evidence was obtained about where the PVR receptor is activated or expressed. First, evidence was obtained by recognition of factors that mediate the activity of the PVF1/PVR signaling mechanism (i.e. dpERK), whose expression is located at the periphery of the group of cells expressing Pvf1. Second, it was shown that blocking PVF1 activity using Pvr^DN and overexpressing Pvf1 results in stronger effects in the engrailed domain where Pvf1 is not expressed. These findings provide additional evidence that there are specific domains for ligand expression and for responsive cells. In the ovary Pvf1 is expressed in the ovule while Pvr is expressed in the follicle cells, the importance of this non-overlapping domains is reflected by the fact that overexpression of a constitutive active form of the receptor (lambda Pvr) produces the same phenotype of its lack of function. In the wing disc Rosin (2004) observed that the restrictions in the activity are regulated by a polarized secretion of the ligand in the apical membrane (Mac̀as, 2004).

Mutations in the pro-apoptotic gene hid have been shown to affect male terminalia rotation, although this phenotype was observed in trans heterozygotes for Df(3L)H99, which includes the three pro-apoptotic genes hid, rpr and grim. Trans heterozygotes for hid mutations are of wildtype phenotype, indicating that the rotated phenotype over deficiency is not only due to hid but to the haploinsufficiency of one or the two other genes. The result that preventing cell death with p35 leads to miss rotation and split dorsal is also consistent with an involvement of apoptosis in these processes (Mac̀as, 2004).

Additionally, it was shown that overexpressing puc results in the same phenotypes as PVF1/PVR and reduction of apoptosis; lowering puc rescues the rotated terminalia defects observed in DfH99/+ males. The level of puc is considered as indicative of the JNK pathway activity, so these experiments suggest that JNK promotes apoptosis, probably by upregulating hid (Mac̀as, 2004).

The fact that alterations in the PVF1/PVR pathway and in JNK/ apoptosis give rise to similar phenotypes suggests a functional link between these two pathways. The penetrance of the phenotypes of Pvf1 mutations in the terminalia increases when they are additionally heterozygous for Df(3L)H99. This increase is nonadditive, suggesting PVF1/PVR and the apoptotic machinery affect the same aspect of the process. The overexpression of Pvf1 ectopically activates puc and impedes the normal rotation and closure. This activation would down-regulate the JNK apoptotic pathway, thus reducing apoptosis and giving rise to the terminalia phenotype, but since the JNK pathway is a transcriptional activator of puc, this result opens up the possibility that Pvf1 ectopically activates JNK rather than puc (Mac̀as, 2004).

Apoptosis is necessary for terminalia rotation and dorsal closure and these results indicate that it is mediated by JNK activity. PVF1/PVR is also affecting these processes and the data suggest that PVF1/PVR may also affect JNK-mediated apoptosis. It is not clear however, whether all these elements act on the same developmental cascade (Mac̀as, 2004).

Search PubMed for articles about Drosophila Pvf1

Brückner, K., et al. (2004). The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev. Cell 7: 73-84. 15239955

Cho, N. K., et al. (2002). Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell 108: 865-876. PubMed citation; Online text

Duchek, P., et al. (2001). Guidance of cell migration by the Drosophila PDGF/VEGF Receptor. Cell 107: 17-26. 11595182

Harris, K. E. and Beckendorf, S. K. (2007a). Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration. Development 134(11): 2017-25. PubMed citation: 17507403

Harris, K. E., Schnittke, N. and Beckendorf, S. K. (2007b). Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning. Mech. Dev. 124(6): 441-8. PubMed citation: 17462868

Kolesnikov, T. and Beckendorf, S. K. (2005). NETRIN and SLIT guide salivary gland migration, Dev. Biol. 284: 102-111. PubMed citation: 15950216

Mac̀as, A., et al. (2004). PVF1/PVR signaling and apoptosis promotes the rotation and dorsal closure of the Drosophila male terminalia. Int. J. Dev. Biol. 48(10): 1087-94. PubMed citation: 15602694

McDonald, J. A., Pinheiro, E. M. and Montell, D. J. (2003). PVF1, a PDGF/VEGF homolog, is sufficient to guide border cells and interacts genetically with Taiman. Development 130(15): 3469-78. PubMed citation; Online text

Rosin, D., Schejter, E., Volk, T. and Shilo, B.-Z. (2004). Apical accumulation of the Drosophila PDGF/VEGF receptor ligands provides a mechanism for triggering localized actin polymerization. Development 131: 1939-1948. 15056618

Wood, W., Faria, C. and Jacinto, A. (2006). Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster. J Cell Biol. 173(3): 405-16 . PubMed citation; Online text

date revised: 28 February 2008

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