InteractiveFly: GeneBrief

Pvf2 and Pvf3: Biological Overview | References

BIOLOGICAL OVERVIEW

Drosophila hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. The migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and Pvf3; these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, PI3K is shown to be essential for hemocyte chemotaxis toward wounds and Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis (Wood, 2007).

BLAST searches identified three genes encoding proteins with sequence similarity to vertebrate VEGFs. Pvf3 and 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 were generated by a recent gene duplication. The VEGF homolog at cytological position 17E (Pvf1) produces two splice variants that differ by 11 N-terminal residues. 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. Pvf1 is slightly more similar to vertebrate VEGFs than to PDGF, whereas Pvf2 and Pvf3 are equally similar to both (Cho, 2002).

During Drosophila embryogenesis, hemocytes derive exclusively from head mesoderm at around 2 h after gastrulation. From this point of origin, these cells migrate along stereotypical routes to populate the whole embryo by stage 17. It has been shown that the developmental migration of these cells is dependent on the expression of the VEGF/PDGF ligands Pvf1, Pvf2, and Pvf3 (Cho, 2002). The PDGF/VEGF receptor (PVR) is expressed in hemocytes (Heino, 2001), and pvr mutant embryos fail to exhibit normal hemocyte migrations, resulting in an accumulation of these cells at their head end (Cho, 2002; Sears, 2003). A recent study has demonstrated a role of PVR in controlling anti-apoptotic cell survival of embryonic hemocytes (Bruckner, 2004) and suggests that the defect in hemocyte distribution observed in the mutant is largely due to high numbers of hemocytes undergoing apoptosis and becoming engulfed by their neighbors. However, this study also showed that Pvr expression within hemocytes is required for the directed migration of a subset of these cells that enter the extended germ during normal development (Bruckner, 2004), suggesting that this population of hemocytes may well be using Pvf signals as a chemoattractant to guide their migrations. Additionally, ectopic expression of Pvf2 within the embryo has been shown (Cho, 2002) to be sufficient to induce a chemotactic response from embryonic hemocytes (Wood, 2007).

In addition to migrating along developmental pathways, embryonic hemocytes have been shown to migrate toward a laser-induced wound in a process that resembles the vertebrate inflammatory response. For a hemocyte to chemotax toward a chemotactic source, be it a wound or a guidance cue expressed along developmental migration routes, it has to be able to sense a chemotactic gradient and polarize in alignment with that gradient. Studies using Dictyostelium discoideum and mammalian neutrophils have demonstrated that the phosphoinositides PtdIns(3,4,5)P₃ (PIP₃) and PtdIns(3,4)P₂ (PIP₂) are key signaling molecules that become rapidly and highly polarized in cells that are exposed to a gradient of chemoattractant (Stephens, 2002; Weiner, 2002; Merlot, 2003). In these actively chemotaxing cells, phosphoinositide 3-kinases (PI3Ks) rapidly translocate from the cytosol to the membrane at the leading edge of the cell, whereas phosphatase and tensin homologue (PTEN) dissociates from the leading edge and becomes restricted to the sides and the rear. The difference in localization of these two enzymes leads to localized PIP₃ production at the leading edge of the cell (Funamoto, 2002; Iijima, 2002). Down- or up-regulation of PIP₃ by deletion of PI3Ks or of PTEN, respectively, results in severely reduced efficiency of chemotaxis (Funamoto, 2002). Though PI3K has been shown to be important for cell motility using these model systems, its role for single-cell chemotaxis in vivo in a multicellular organism has yet to be clarified. D. melanogaster has one class I PI3K, Dp110, whose role in cell growth control and cell survival has been well characterized; however, no role in cell migration and chemotaxis in Drosophila for this protein has been shown (Wood, 2007).

This study analyzed the developmental migrations of hemocytes and characterized in detail their migration patterns along the ventral midline. Quantitative analysis shows that ventral midline hemocytes undergo a rapid lateral migration, during which they are highly polarized. Pvf2 and -3 expression in the central nervous system (CNS), and Pvf2 alone in the dorsal vessel, are essential for directing the migration of hemocytes along these structures, and a decrease in expression of these ligands in the CNS is essential for the normal lateral migration of hemocytes in this region. The function of PI3K was analyzed in hemocytes. Using both dominant-negative PI3K-expressing hemocytes and the specific PI3K inhibitory drug LY294002, PI3K is shown not to be required for the Pvf-dependent normal dispersal of hemocytes during development but is essential for chemotaxis toward wounds. Additionally, hemocyte chemotaxis toward wounds is shown to be dependent on actin polymerization but that PI3K is not required for lamellipodial formation and instead appears to be required to sense a chemotactic gradient from a wound and polarize the hemocyte accordingly. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the Pvf growth factor signals that coordinate their developmental migrations along the ventral midline and dorsal vessel during embryogenesis (Wood, 2007).

Many obvious parallels exist between the migration of hemocytes along the ventral midline CNS and another developmentally regulated migration in Drosophila, that of border cell migration. Border cells take ~6 h to migrate a distance of 100 µm, a speed consistent with that describe for hemocyte migration along the CNS. Successful border cell migration, like hemocyte migration, requires the expression of the Pvr in the migrating cells and, just as is seem for hemocytes, the chemotactic signals detected by the PVR in the border cells are not transduced through PI3K. Successful migration of border cells does, however, require Rac signaling and the Rac activator myoblast city (mbc), the D. melanogaster homologue of Dock 180. It has been shown that hemocyte-specific expression of dominant-negative Rac^N17 disrupts all hemocyte developmental migrations, demonstrating that Rac is required for the successful migration of ventral midline hemocytes along the CNS. Given that Pvr couples to the Dock 180 signaling pathway during border cell migration and that Dock 180 has been shown to be involved in the migration of lymphocytes, Mbc/Dock 180 is a potentially important protein for hemocyte migration. Despite the fact that mbc mutant embryos display a grossly normal pattern of hemocyte dispersal, it would be interesting to look in detail at the migration of these mutant cells along the ventral nerve cord. More work is needed to investigate what other similarities may exist between border cell migration and ventral midline hemocyte migration. During development, only a subset of the hemocytes present in the embryo respond to the midline Pvf expression and migrate along the CNS accordingly. Other cells follow other migratory pathways. What specifies these cells to migrate along the midline? Important studies in border cell migration have shown that the JAK-Stat signaling pathway signaling through the Domeless receptor (Dome) is necessary and sufficient to transform nonmotile epithelial cells into invasive ones. Whether a similar signaling mechanism is operating to specify future ventral midline hemoctyes and initiate their migration remains to be seen (Wood, 2007).

From stage 14 onwards, once hemocytes occupy the entire ventral midline, individual cells begin to rapidly leave the midline and occupy more lateral positions. At this stage of development, hemocytes appear to be highly polarized, exhibiting large lamellipodia at their leading edges and migrating at a speed more than three times faster than their earlier midline migration. This lateral movement requires a down-regulation in the attractive signal provided by Pvf2 in the midline, but is this the only driving force for the lateral movement? One possibility is that a different source of chemoattractant exists in the more lateral positions and that once Pvf2 expression is sufficiently down-regulated, this chemoattractant source operates to pull hemocytes laterally. Alternatively, hemocytes may be actively repelled from the midline or from one another, and the lateral migration observed by a subset of these hemocytes is a consequence of these cells attempting to maximize the distance between one another while maintaining contact with the CNS. It remains to be seen which, if any, of these hypotheses is true, but what is certain is that the guidance of hemocytes along the ventral midline of the embryo is not as simple as was first thought, and more studies are required to determine the exact relationship between this subpopulation of hemocytes and the different structures within the CNS as well as the overlying ectodermal cells, any of which could provide either chemoattractants or repellents for the migrating hemocytes to respond to (Wood, 2007).

This study has demonstrated a requirement of PI3K for the polarization and active chemotaxis of hemocytes toward an epithelial wound. This is the first demonstration of the role of PI3K for single-cell chemotaxis in Drosophila and shows a striking correlation with the mechanism of cell chemotaxis used by D. discoideum and mammalian neutrophils. In these model systems, class I PI3Ks are activated upon stimulation of G protein-coupled chemoattractant receptors and, once activated, PI3Ks catalyze the production of the phosphoinositides PIP₃ and PIP₂ at the leading edge of the cell. The accumulation of PIP₃/PIP₂ leads to a rapid and transient recruitment of pleckstrin homology domain-containing proteins, including the serine/threonine kinase Akt/PKB. Akt/PKB itself becomes activated upon recruitment to the membrane and, in D. discoideum, activates the serine/threonine kinase p21-activated kinase a, which eventually leads to the phosphorylation of Myosin II and subsequent polarization of the cytoskeleton. Evidence also exists to support a role for the PI3K antagonist PTEN in helping to establish and maintain the intercellular PIP₃ gradient required for successful chemotaxis by down-regulating the PIP₃ pathway at the rear of the migrating cell. How much of this signaling pathway is operating in chemotaxing hemocytes remains to be seen. The current study demonstrates the involvement of PI3K, and previous work has shown that the small GTPase Rac is required for efficient hemocyte chemotaxis toward wounds. In neutrophils, PIP₃ production has been shown to be autocatalytic and to require Rac but not Cdc42. In the proposed positive feedback loop, it is thought that PIP₃ may stimulate Rac through activation of a specific Rac GEF, which in turn activates PI3K, as well as effectors that mediate lamellipodial protrusion. Because Rac is absolutely required for hemocyte chemotaxis and lamellipodia formation, it is tempting to speculate that a similar feedback loop may be operating in Drosophila hemocytes. Further work is required to determine the complex relationships operating among PI3K, Rho family small GTPases, and the actin cytoskeleton that coordinate chemotactic migration in these highly motile cells (Wood, 2007).

The PI3K-dependent mechanism of polarization required for hemocyte chemotaxis toward a wound is extremely fast and perfectly suited for mature, highly motile hemocytes that need to rapidly react to a source of attractive signal, be it a wound, an invading organism, or an apoptotic cell. In contrast, the mechanics to developmentally disperse need not be so rapid, since the aim during development is simply to ensure that hemocytes migrate toward and arrive at their target tissue in a given amount of time and does not require the rapid response to constantly changing environments required for mature hemocytes. The mechanism controlling the developmental migration of hemocytes along the ventral midline is consequently much slower and is dependent on slow-diffusing growth factors of the Pvf family providing short-range guidance information signaling through the receptor tyrosine kinase PVR. These two mechanisms may not be the only ways in which hemocytes are able to chemotax toward an attractive source; indeed, the observation that hemocytes travel different migratory routes in the embryo suggests that they may not all be using the same machinery to polarize and migrate. What does seem to be consistent for both chemotaxis toward developmental signals and toward wounds, like motility in many cell types, is a requirement for Rac signaling and the formation of actin protrusions (Wood, 2007).

The fact that hemocyte migrations within the embryo are strictly regulated and adhere to a stereotyped pattern is important in a developmental context. Throughout embryogenesis, hemocytes carry out important developmental functions within the embryo, such as the engulfment and removal of apoptotic cells and the laying down of many extracellular matrix molecules, including collagen IV and laminin, that compose the basement membrane surrounding internal organs. The failure of hemocytes to travel along their normal migratory routes therefore has serious consequences. Such defects have been described in pvr mutants, where a lack of hemocyte migration along the ventral nerve cord results in a failure in CNS condensation (Olofsson and Page, 2005), as well as a disruption in axon patterning (Sears, 2003). It is therefore vital for the embryo to ensure that hemocytes arrive at their correct target tissues during development. For this to occur, it is not sufficient to allow these cells to passively disperse throughout the embryo by random migrations; instead, a directed and tightly controlled migration is required (Wood, 2007). In this study, drugs were directly applied to Drosophila embryos using bead implantation. The application of drugs has been a powerful tool in cell culture and in vitro cell motility studies but remains largely unused in Drosophila. Using a bead assay, it will be possible to take advantage of the many useful drugs available to block both specific signaling pathways as well as important cytoskeletal processes. Combined with the powerful genetics available in Drosophila and the relative ease of live imaging in this system, the study of Drosophila hemocytes provides a powerful model to address the process of cell motility and chemotaxis and will undoubtedly provide a clearer understanding of the regulation and mechanics of single-cell migration in the complex setting of a multicellular organism (Wood, 2007).

Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007 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, 2007).

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, 2007).

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. 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, 2007).

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, 2007).

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, 2007).

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, 2007).

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, 2007 and references therein).

Tsuzuki, S., Matsumoto, H., Furihata, S., Ryuda, M., Tanaka, H., Jae Sung, E., Bird, G. S., Zhou, Y., Shears, S. B. and Hayakawa, Y. (2014). Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP. Nat Commun 5: 4628. PubMed ID: 25130174

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. This study used insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. A Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade (see Small wing) to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, this study describes mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection (Tsuzuki, 2014).

Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor

Age-associated changes in stem cell populations have been implicated in age-related diseases, including cancer. However, little is known about the underlying molecular mechanisms that link aging to the modulation of adult stem cell populations. Drosophila midgut is an excellent model system for the study of stem cell renewal and aging. This study describes an age-related increase in the number and activity of intestinal stem cells (ISCs) and progenitor cells in Drosophila midgut. Oxidative stress, induced by paraquat treatment or loss of catalase function, mimics the changes associated with aging in the midgut. Furthermore, an age-related increase in the expression of PVF2, a Drosophila homologue of human PDGF/VEGF, is associated with and required for the age-related changes in midgut ISCs and progenitor cell populations. Taken together, these findings suggest that PDGF/VEGF may play a central role in age-related changes in ISCs and progenitor cell populations, which may contribute to aging and the development of cancer stem cells (Choi, 2008).

Autocrine platelet-derived growth factor-vascular endothelial growth factor receptor-related (Pvr) pathway activity controls intestinal stem cell proliferation in the adult Drosophila midgut

A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. This study uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. Pvr is coexpressed with its ligand Pvf2 in ISCs, and hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, it was determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. These findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states (Bond, 2012).

The metazoan gut is under constant bombardment from environmental pressures that damage exposed epithelial cells and corrupt intestinal tissue integrity. The human intestinal tract alone is home to over 10 trillion bacteria, which equals approximately 10 fold more bacterial cells than human somatic and germ cells combined. As a result, the intestinal microbiome may contain greater than 100 times more unique genetic sequences than are present in the entire human genome. This highlights the remarkably complex relationship between metazoans and their intestinal environment, and the requirement for sophisticated intercellular communication networks that coordinate homeostatic responses to protect organ function from enteropathogenic challenges (Bond, 2012).

Studies of the Drosophila midgut model revealed that ISC homeostasis is maintained through an elaborate balance of multiple pathways that respond to extrinsic insults and intrinsic requirements for the orderly development of mature epithelial cell types. ISCs proliferate and differentiate rapidly in response to stress-signals. However in the absence of these signals, intrinsic cues guide low level ISC division to ensure a stable population of progenitor cells. Previous studies highlighted the overlapping contributions of Jak/ Stat, EGFR, InR, Hippo/Wrts, and JNK pathways to meet intestinal tissue requirements. The Jak/Stat pathway is a major regulator of intestinal homeostasis in response to injury or stress with additional contributions to stem cell differentiation under unstressed conditions. The EGFR pathway amalgamates paracrine stress responsive signals with autocrine signals to regulate ISC growth and proliferation. The InR pathway is a general regulator of homeostatic proliferative controls in posterior midgut ISCs and responds to nutritional requirements and epithelial damage. Along with the strong non-cell-autonomous requirement for the Wrt/Hippo pathway in the generation of stress-signals, there is also evidence that Wrt/Hippo plays a role in the regulation of ISC-autonomous homeostatic signals. Finally, oxidative stress activates the dJNK pathway to guide the production of mitogenic signals that drive the rapid proliferation (Bond, 2012).

In the current studies, a novel requirement was uncovered for the Pvr/Ras signal transduction pathway in the regulation of ISC homeostatic controls in the posterior midgut. Loss of the Pvr receptor in ISCs completely blocks the ISC/EB/EC developmental program. Instead, mutant cells fail to proliferate and retain their identity as Dl positive ISCs. As the simultaneous deletion of pvf2 and pvf3 exclusively from ISCs in an otherwise heterozygous background phenocopies the pvr mutant phenotype, it is concluded that Pvf2 and Pvf3 are ISC-autonomous regulators of ISC proliferation. Furthermore, these observations indicate that autocrine Pvf/Pvr signals guide ISC homeostasis. This hypothesis is entirely consistent with the observed ISC expression patterns for Pvr and Pvf2, where both ligand and receptor are restricted to ISCs. These findings also highlight a noteworthy distinction between Pvr and previously described intrinsic regulators, as extrinsic stress cues are epistatic to Pvr in relation to proliferation. This is in contrast to the findings of EGFR and InR pathway mutants that display proliferative defects under unstressed conditions and upon enteropathegenic infection. Thus, these studies suggest that Pvr is an ISC-autonomous homeostatic regulator (Bond, 2012).

Age-associated decline in stem cell activity has been implicated in the development of several disease conditions such as progressive organ failure and cancer. As intrinsic signals are responsible for the maintenance of ISC pools over the lifetime of the animal, the loss or disruption of these pathways significantly affect age-related disease progression. In aged Drosophila posterior midguts, ISCs hyperproliferate and the resultant pool of daughter cells fail to differentiate correctly causing dysplasia and gradual degeneration of the intestinal epithelium. In agreement with a connection between aging and deregulated ISC homeostasis, genetic manipulation of factors that suppress ISC proliferation are associated with reduced age-related intestinal dysplasia and prolonged longevity. Pvf/Pvr hyperactivity in ISCs drives intestinal dysplasia and previous studies found that production of Pvf2 by ISCs engages the Pvr pathway to activate p38 and contributes to age-related changes in the Drosophila posterior midgut. These observations support the model of Pvr as an intrinsic regulator of ISC homeostasis (Bond, 2012).

The Drosophila Pvr protein shares significant sequence and structural similarity with the human VEGF- and PDGF-families of RTKs. In mammals, the VEGF- and PDGF-receptors function in multiple cellular processes that include growth, proliferation, migration and differentiation. For example, studies of mice mutant in PDGF-A and PDGFR-α showed a spectrum of development defects in organogenesis. Of particular relevance to these studies is the finding that PDGF-A and PDGFR-α mutant mice display severe defects in gastrointestinal tract architecture predominantly in the upper small intestine. During organogenesis the paracrine expression of PDGF-A by epithelial cells engages PDGFR-α in underlying mesenchymal cells to cause mesenchymal cell proliferation. A breakdown of epithelial-mesenchymal PDGF-signals results in disrupted intestinal morphogenesis and epithelial differentiation defects. It is currently unclear if the differentiation defects are secondary to the morphogenetic requirements for PDGF or if they reflect direct contributions of PDGFR positive mesenchymal cells to epithelial differentiation (Bond, 2012).

Although this study found that autocrine signals guide Pvr activity, it was also found that loss of Pvr results in profound defects in the differentiation program of the intestinal epithelium. Therefore, further studies of the morphogenetic requirements for Pvr signals in ISC differentiation within the Drosophila posterior midgut model may illuminate specific requirements for PDGF- and VEGF-pathway signals in epithelial cell development in mammals. In addition to developmental roles, deregulation of VEGF- and PDGF-receptor signals contributes significantly to the generation and progression of numerous cancer types. One important hallmark of cancer is growth factor independence. In this regard, PDGF has long been recognized as an important autocrine growth factor in the stimulation of neoplastic transformation. PDGF/PDGFR proliferative signals promote tumorigenesis in preneoplastic or genetically unstable cells that accumulate genetic changes and become malignant. For example, nearly all glioblastomas express a multitude of PDGFs and PDGFRs that establish an autocrine PDGF/PDGFR signal loop. More recently, autocrine VEGF/ VGFR signals have been directly implicated in cancer progression through the increased renewal of cancer stem cells. Given the similarities between Pvr and the established roles of autocrine feedback loop activation of VEGF- and PDGF families in cancer progression, it is felt that further studies in the genetic regulation of Pvr signals in posterior midgut ISCs provides a fruitful model to study how these pathways promote disease (Bond, 2012).

PVF2, a PDGF/VEGF-like growth factor, induces hemocyte proliferation in Drosophila larvae

Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, antibodies were raised against a tumorous blood cell line, and an antigenic determinant was identified that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, a dramatic increase was indidued in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis (Munier, 2002; full text of article).

Since Drosophila blood cells are difficult to obtain in sufficient numbers for immunization of mice, antibodies were raised against the cells of the tumorous blood cell line, mbn-2, which are functionally close to plasmatocytes. These cells phagocytose microorganisms and also synthesize and secrete antimicrobial peptides when exposed to bacteria or bacterial cell components. Live mbn-2 cells were injected into BALB/c mice and hybridomas were selected for their capacity to produce antibodies that recognize cell surface antigens on live mbn-2 cells. This recognition was monitored by flow cytometric analysis, and selected antibodies were tested for their abilities to affect the mitotic rate of mbn-2 cells. In these series, one antibody, 18G, strongly inhibited the proliferation of mbn-2 cells when added to the culture medium. The antiproliferative capacity of 18G was further ascertained by measuring its dose-dependent effect. Western blots performed with the 18G antibody on extracts of mbn-2 cells (under non-reducing conditions) revealed several protein bands of high molecular weight (>75 kDa;). Stained high-molecular-weight proteins were also detected in extracts of larval blood cells, as well as in Drosophila Schneider 2 (S2) cells (Munier, 2002).

The distribution of the 18G determinant on the different Drosophila hemocyte types was examined by immunocytochemistry. Wild-type and hop^Tum-l mutants were used for this purpose. hop^Tum-l is a Janus kinase (JAK) gain-of-function mutation resulting in an overproliferation of circulating blood cells, of which a large number are lamellocytes. Plasmatocytes were stained and lamellocytes were not. In hop^Tum-l mutants, the most strongly reacting cells were small rounded cells that correspond to circulating progenitor blood cells (prohemocytes). In wild-type larvae, the presence of prohemocytes is mostly restricted to lymph glands. Strong staining was observed on the prohemocytes of wild-type lymph glands. In hop-^Tum-l lymph glands, the small rounded prohemocytes were also strongly stained, but other cells that have been described as lamellocytes but did not react with the antibody. Stained circulating crystal cells could not be observed due to their fragility. No staining was ever observed on larval tissues other than hemocytes, namely on fat body, muscles, imaginal discs, epidermal cells, brain or trachea (Munier, 2002).

Three protein bands positive for 18G staining in mbn-2 cell extracts were purified by immunoaffinity chromatography and subjected to Edman degradation. The N-terminal sequences obtained with each of the three protein bands were identical: VPLQQFSPDP. The Drosophila genome contains a single match to this sequence, namely in a gene encoding a homolog of mammalian receptors for PDGF and VEGF. Independent studies have identified expression of this gene in ovarian border cells and embryonic hemocytes, and the receptor is now referred to as PVR (PDGF/VEGF receptor). Pvr RNA interference experiments in S2 cells confirmed that 18G recognizes this receptor (Munier, 2002).

Attempts were made to study the role of PVR in Drosophila blood cell proliferation in vivo, using mutants or transgenes of Pvr. A transposon insertion (line PBc2195) in the 11th intron of the Pvr gene generates a null mutant that leads to embryonic lethality. Larvae heterozygous for this mutation (Pvr^c2195/CyO) did not show defects in hemocyte counts. Overexpression of the Pvr cDNA by using a UAS-Pvr transgenic line crossed with either a daughterless-GAL4 line (for ubiquitous expression) or e33c-GAL4 line (for preferential lymph gland expression) is lethal at the embryonic stage. As an alternative, to establish the role of this receptor in hemocyte proliferation in larvae, the effect of ectopic expression of two of its putative ligands: [Pvf1 (CG7103) and Pvf2 (CG13780)] was analyzed (Munier, 2002).

UAS-Pvf1 and UAS-Pvf2 transgenic fly lines were generated and PVF1 and PVF2 expression was directed using daughterless-GAL4 and e33C-GAL4 drivers, and larval hemocytes were examined. PVF2 expression in both cases results in a dramatic increase (up to 300-fold) in the number of blood cells in third instar larvae and leads to pupal lethality. Apart from hemocytes and lymph glands, no proliferation was observed in other tissues. In contrast, overexpression of PVF1, using the same GAL4 drivers, results in a mild and variable effect on blood cell counts that does not exceed a 2-fold increase compared with controls. Overexpression of PVF1 also results in pupal lethality (Munier, 2002).

On average, the hemocytes in PVF2-overexpressing flies were noticeably smaller than in the wild type. All cells were reactive to the 18G antibody, indicating that they are prohemocytes or plasmatocytes. However, only a small percentage was able to phagocytose injected India ink, thus qualifying as fully mature plasmatocytes. Staining with anti-phosphohistone H3 antibody shows that a large number of circulating hemocytes were in the process of division, indicating that PVF2 stimulates proliferation rather than promotes cell survival. The vast majority of the PVF2-induced hemocytes are therefore to be considered as prohemocytes. Crystal cells were noticeably absent in w; UAS-Pvf2/+; e33C-GAL4/+ larvae (Munier, 2002).

Blood cells were counted in two lines with a transposon inserted in the Pvf2 gene, XPd2444 and PBc6947. These lines are homozygous viable and showed no obvious defects in blood cell counts. A Pvf1 loss-of-function mutant, Pvf1¹⁶²⁴, similarly showed no defect in larval blood cell counts. However, in this line, pupal lethality was observed (Munier, 2002).

Therefore, by using an antibody screening approach against a Drosophila blood cell line, the receptor tyrosine kinase PVR was identified as a marker of larval Drosophila hemocytes. This receptor is found on lymph gland prohemocytes and on the surface of mature circulating plasmatocytes/macrophages. The anti-PVR antibody inhibits thymidine incorporation in blood cell lines, whereas overexpression of one of its ligands, PVF2, induces the proliferation of hemocytes in vivo. These results strongly suggest that the PVF2/PVR couple is involved in hemocyte proliferation. PVR belongs to the PDGF/VEGF subfamily of RTKs. These receptors are characterized by an extracellular sequence composed of either five Ig domains (in c-Kit, Flt-3, c-Fms, PDGFRalpha and ß) or seven Ig domains (in VEGFRS: Flt1, KDR, Flt4) and a cytoplasmic split tyrosine kinase domain. In vertebrates, receptors of this RTK family function in both cell proliferation and cell migration (Munier, 2002).

PVR has recently been identified as a chemotactic receptor guiding cells to a source of PVF ligand, both in the context of ovarian border cell migration and in embryonic macrophage migration. The three PVFs (PVF1, PVF2 and PVF3) encoded by the Drosophila genome are thought to function redundantly during migration. The misexpression of a PVR ligand can disrupt the normal migration of border cells (i.e., PVF1 misexpression) or embryonic hemocytes (i.e., PVF2), but the removal of a single ligand is insufficient to block the migration process. A similar misexpression strategy was used to study larval hemocyte proliferation, with markedly different outcomes depending on the misexpressed ligand. This study clearly establishes that PVF2, and not PVF1, promotes hemocyte proliferation. More complex to explain is the complete absence of crystal cells in larvae overexpressing PVF2. This phenotype could result from the persistence of PVR-positive prohemocytes in a constant mitotic state by PVF2 stimulation, preventing their differentiation into crystal cells. Finally, the absence of any abnormal blood cell phenotype in transposon insertion lines of the Pvf2 gene implies that PVF2 is sufficient, but not absolutely required, in hemocyte proliferation (Munier, 2002).

The overexpression of PVF1 or PVF2 results in lethality during pupal development. In the case of PVF2, this phenotype is attributed to the enormous amount of blood cells that could disturb overall physiology of the larvae instead of metamorphosis per se. However, PVF1 misexpression reveals a more complex role for the PVR pathway in metamorphosis, possibly by disrupting the homing of hemocytes to, and/or engulfment of, larval apoptotic tissue. PVF1 seems important in metamorphosis, because a loss-of-function allele in the gene (Pvf1¹⁶²⁴) results in 40%-60% pupal lethality but has wild-type hemocyte numbers (Munier, 2002).

In vertebrates, hematopoietic stem cells are defined by their ability to self-renew and contribute to all lineages of mature blood cells. This self-renewal and differentiation are driven by numerous receptors that co-exist on the surface membrane of hematopoietic cells, among which are receptor tyrosine kinases of the PDGFR family (c-Kit, Flt-3, c-Fms, PDGFR). These factors act in combination with intracellular signal transducers. For example, it has been shown that JAK2 activation triggers extensive self-renewal of stem cells only if it is complemented by a second signal from c-Kit or Flt-3. Each of these proteins, JAK2, Flt-3 or c-Kit, alone is unable to sustain this activity. (Munier, 2002).

In Drosophila gain-of-function alleles in the JAK Hopscotch (e.g. hop^Tum-l) cause overproliferation of hemocytes. This report raises the obvious question as to what kind of interconnection exists between pathway(s) activated by PVR and the JAK/STAT pathway itself. Some cross-talk could occur, such as phosphorylation of STAT by PVF2-induced PVR activation. In mammals, for instance, PDGFR can directly activate some STATs. Conversely, evidence exists that JAK can activate the D-raf/D-MEK/MAP kinase pathway, one that is frequently activated by receptor tyrosine kinases. As is the case for PVF2, however, neither JAK nor STAT seem absolutely required for blood cell proliferation. Indeed, in loss-of-function mutants of hop or stat that permit larval development, blood cell counts are normal. This leaves open the possibility that upstream components of the JAK/STAT pathway, e.g., the receptor Domeless (DOME) and its ligand Unpaired, could act in synergy with the PVF2/PVR pathway. Both DOME and Upd are implicated in embryonic pair-rule gene expression, but their role in hematopoeisis awaits investigation (Munier, 2002).

In summary, the data indicate that PVR integrates two functions shared by mammalian receptors of the same subfamily. Like its mammalian VEGFR homologs (Flt1, KDR and Flt4), it regulates cell migration; and like c-Kit, Flt-3, c-Fms and most PDGFRs, it is implicated in the control of blood cell proliferation. In the light of the importance of hemocytes in development and in the innate immune response, it would be highly relevant to investigate further the interaction between PVFs, PVR, the JAK/STAT pathway and the downstream mitogenic factors that they induce (Munier, 2002).

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 (Pvf1) 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).

Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila

The easy accessibility of energy-rich palatable food makes it difficult to resist food temptation. Drosophila larvae are surrounded by sugar-rich food most of their lives, raising the question of how these animals modulate food-seeking behaviors in tune with physiological needs. This study describes a circuit mechanism defined by neurons expressing tdc2-Gal4 (a tyrosine decarboxylase 2 promoter-directed driver) that selectively drives a distinct foraging strategy in food-deprived larvae. Stimulation of this otherwise functionally latent circuit in tdc2-Gal4 neurons was sufficient to induce exuberant feeding of liquid food in fed animals, whereas targeted lesions in a small subset of tdc2-Gal4 neurons in the subesophageal ganglion blocked hunger-driven increases in the feeding response. Furthermore, regulation of feeding rate enhancement by tdc2-Gal4 neurons requires a novel signaling mechanism involving the VEGF2-like receptor, octopamine, and its receptor. These findings provide fresh insight for the neurobiology and evolution of appetitive motivation (Zhang, 2013).

Modulation of feeding responses to food sources is heavily influenced by nutritional quality, taste, and the energy costs of foraging. The current findings suggest that Drosophila larvae have evolved a complex neural network to regulate appetitive motivations. In hungry fly larvae, OA neurons seem to mediate a specialized circuit that selectively promotes persistent feeding of readily ingestible sugar food. This OA circuit functions in parallel to the previously characterized mechanism coregulated by the fly insulin and NPY-like systems that drives feeding response to non-preferred solid food. Because food deprivation triggers simultaneous activation of both circuits, hungry larvae become capable of adaptively responding to diverse energy sources of high or low quality. It remains to be determined how OA signaling promotes persistent feeding response to liquid sugar food in hungry larvae. One possible scenario is that OA neurons in the SOG may be conditionally activated by gustatory cues associated with rich palatable food to promote appetitive motivation (Zhang, 2013).

This study has has provided evidence, at both molecular and neuronal levels, that the OA-mediated feeding circuit has two opposing effects on food motivation. When surrounded by liquid sugar media, the OA circuit is essential to prevent fed animals from excessive feeding. Because targeted lesions in VUM1 neurons caused excessive feeding response, these neurons may define an inhibitory subprogram within the OA feeding circuit. However, targeted lesions in VUM2 neurons attenuated hunger-induced increases of feeding response, suggesting that VUM2 neurons, along with the OA receptor Octβ3R, may define a subprogram that enhances feeding in fasted larvae. Several lines of evidence suggest that the VUM2 neuron-mediated subprogram may be suppressed by the VUM1 neuron-mediated subprogram. First, fed larvae with double lesions in both VUM1 and VUM2 neurons failed to display excessive feeding, suggesting that increased feeding response of fed larvae deficient for VUM1 neuronal signaling requires VUM2 neurons. Second, targeted lesions in VUM2 neurons of fed tdc2-Gal4/UAS- dTrpA1 larvae completely blocked the increased feeding response induced by genetic activation of tdc2-Gal4 neurons. Finally, the anatomical data also show that VUM1 and VUM2 neurons project to many common regions of the larval brain implicated in the control of feeding. Future work will be needed to determine whether VUM1 neurons inhibit directly or indirectly the activity of VUM2 neurons (Zhang, 2013).

Genetic and pharmacological evidence has been obtained for the critical role of OA in the regulation of acquiring readily accessible sugar media. OA has been reported to mediate diverse neurobiological functions including appetitive memory formation and modulation of the dance of honey bee foragers to communicate floral or sucrose rewards. It is postulated that the different OA receptors may mediate diverse OA-dependent behavioral responses to high-quality foods (Zhang, 2013).

Norepinephrine (NE), the vertebrate counterpart of OA, has been shown to promote ingestion of carbohydrate-rich food at the beginning of a natural feeding cycle. This feeding activity of NE resides in the paraventricular nucleus (PVN) of the feeding control center. In the PVN, α1 and α2 adrenergic receptors are organized in an antagonistic pattern. Activation of α1 receptor inhibits food intake, whereas activation of the α2 receptor stimulates food intake. The current results suggest that the insect OA system, like the NE system in mammals, exerts both positive and negative effects on the intake of preferred food. The activity of NE in PVN has been shown to antagonize that of 5-HT, which suppresses intake of carbohydrate- rich food. In Drosophila, 5-HT is also known to suppress feeding response. These findings suggest that the homeostatic control of intake of preferred food is likely mediated by a conserved neural network in flies and mammals (Zhang, 2013).

This study has identified a unique role of Pvr in physiological regulation of hunger-motivated feeding of preferred food (liquid sugar media). The feeding-related activity of the Pvr pathway involves two regulatory proteins, Drk and Ras, and oral introduction of OA restores the hunger-driven feeding response in tdc2-Gal4/ UAS-drkdsRNA larvae. Together, these results suggest that the Pvr pathway positively regulates OA release by tdc2-Gal4 neurons. Among the three identified ligands of Pvr, Pvf2 is enriched in the larval CNS. The current finding suggests that Pvf2 regulates the feeding-related activity of the Pvr pathway. It is possible that Pvf2 may transduce a metabolic stimulus to Pvr/tdc2-Gal4 neurons that signals the energy state of larvae. In the honey bee brain, OA neurons from the SOG have been reported to respond to sugar stimulation. Therefore, it would be interesting to test whether the Pvf2/ Pvr pathway is responsive to sugar stimuli (Zhang, 2013).

Previous studies have shown that the fly insulin and NPY-like systems coregulate hunger-elicited motivation to acquire solid sugar media. This study has now provided evidence that the fly VEGFR2- and NE-like systems control larval motivation to acquire liquid sugar media. These findings strongly suggest that the neural activities of different RTK systems play critical roles in different aspects of adaptive feeding decisions under various food and metabolic conditions. Therefore, further investigation of the mechanistic details of the food-related functions of RTK systems in the Drosophila model may provide novel insights into the neurobiology and evolution of appetitive control as well as pathophysiology of eating-related disorders (Zhang, 2013).

Search PubMed for articles about Drosophila Pvf2 and Pvf3

Bond, D. and Foley, E. (2012). Autocrine platelet-derived growth factor-vascular endothelial growth factor receptor-related (Pvr) pathway activity controls intestinal stem cell proliferation in the adult Drosophila midgut. J Biol Chem 287: 27359-27370. PubMed ID: 22722927

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

Cho, N. K., et al. (2002). Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell 108: 865-876. PubMed ID: 11955438

Choi, N. H., et al. (2008). Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor. Aging Cell. [Epub ahead of print]. PubMed ID: 18284659

Duchek, P., Somogyi, K., Jekely, G., Beccari, S., Rorth, P. (2001). Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107(1): 17-26. PubMed ID: 11595182

Funamoto, S., Milan, K., Meili, R. and Firtel, R. A. (2001). Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium. J. Cell Biol. 153: 795-810. PubMed ID: 11352940

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

Heino, T. I., et al. (2001). The Drosophila VEGF receptor homolog is expressed in hemocytes. Mech. Dev. 109: 69-77. PubMed ID: 11677054

Iijima, M. and Devreotes, P. (2002). Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109: 599-610. PubMed ID: 12062103

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 ID: PubMed ID; Online text

Merlot, S., and Firtel, R. A. (2003). Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J. Cell Sci. 116: 3471-3478. PubMed ID: 12893811

Munier, A. I., et al. (2002). PVF2, a PDGF/VEGF-like growth factor, induces hemocyte proliferation in Drosophila larvae. EMBO Rep. 3: 1195-200. PubMed ID: 12446570

Olofsson, B. and Page, D. T. (2005). Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity. Dev. Biol. 279(1): 233-43. PubMed ID: 15708571

Sears, H. C., Kennedy, C. J. and Garrity, P. A. (2003). Macrophage-mediated corpse engulfment is required for normal Drosophila CNS morphogenesis. Development 130: 3557-3565. PubMed ID: 12810602

Stephens, L., Ellson, C. and Hawkins, P. (2002). Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr. Opin. Cell Biol. 14: 203-213. PubMed ID: 11891120

Weiner, O. D. (2002). Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr. Opin. Cell Biol. 14: 196-202. PubMed ID: 11891119

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 ID: PubMed ID; Online text

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date revised: 25 April 2024

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