InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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).

Drosophila PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity

PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine and endocrine mechanisms. This study investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). Genetic approaches and single-nuclei sequencing were combined to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. This study found that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. These findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells (Ghosh, 2020).

The presence in vertebrates of multiple PDGF/VEGF signaling ligands and cognate receptors makes it difficult to assess their roles in inter-organ communication. Additionally, understanding the tissue-specific roles of these molecules, while circumventing the critical role they play in regulating tissue vascularization, is equally challenging in vertebrate models. This study investigated the tissue-specific roles of the ancestral PDGF/VEGF-like factors and the single PDGF/VEGF-receptor in Drosophila in lipid homeostasis. The results demonstrate that in adult flies the PDGF/VEGF like factor, Pvf1, is a muscle-derived signaling molecule (myokine) that suppresses systemic lipid synthesis by signaling to the Drosophila hepatocyte-like cells/oenocytes (Ghosh, 2020).

The Drosophila larval and adult adipose tissues have distinct developmental origins. The larval adipose tissue undergo drastic morphological changes during metamorphosis and dissociate into individual large spherical cells. These free-floating adipose cells persist to the young adult stage where they play a crucial role in protecting the animal from starvation and desiccation. These larval adipose tissue cells are ultimately removed via cell death. Adult-specific adipose tissue cells develop during the pupal stage from yet unknown progenitor cells and have very little lipid stores in newly eclosed flies. Over the period of next 3-5 days the adult adipose tissue builds up its lipid reserves through feeding and de-novo lipid synthesis. However, at the end of the lipid build-up phase, the rate of lipid synthesis must be suppressed to avoid over-loading of the adipose tissue and prevent lipid toxicity. The data suggest that muscle Pvf1 signaling suppresses lipid synthesis at the end of the adult adipose tissue lipid build-up phase. Pvf1 production in the adult muscles peaks around the time when adult adipose tissue lipid stores reach steady state capacity. In turn, muscle-derived Pvf1 suppresses lipid synthesis and lipid incorporation by activating TOR signaling in the oenocytes (Ghosh, 2020).

This study reveals that Pvf1 is abundant in the tubular muscles of the Drosophila leg and abdomen. In these striated muscles, the protein localizes between individual myofibrils and is particularly enriched at the M and Z bands. Drosophila musculature can be broadly categorized into two groups, the fibrillar muscles and the tubular muscles, with distinct morphological and physiological characteristics. Drosophila IFMs of the thorax belong to the fibrillar muscle group and are stretch-activated, oxidative, slow twitch muscles that are similar to vertebrate cardiac muscles. By contrast, Drosophila leg muscles and abdominal muscles belong to the tubular muscle group. These muscles are striated, Ca2+ activated, and glycolytic in nature. The tubular muscles are structurally and functionally closer to vertebrate skeletal muscles. Expression of Pvf1 in the tubular muscles of the Drosophila leg may reflect a potentially conserved role of this molecule as a skeletal-muscle-derived myokine. The fact that most of the myokines in vertebrates were identified in striated skeletal muscles supports this possibility . Moreover, vertebrate VEGF ligands, VEGF-A and VEGF-B, have also been shown to be stored and released from skeletal muscles (Ghosh, 2020).

Interestingly, in vertebrates, the expression and release of VEGF ligands are regulated by muscle activity. In mice, expression of VEGF-B in the skeletal muscles is regulated by PGC1-α, one of the key downstream effectors of muscle activity. Additionally, expression of VEGF-B is upregulated in both mouse and human skeletal muscles in response to muscle activity. Similarly, expression of VEGF-A is induced by muscle contraction. No effect of muscle activity on the expression levels of pvf1 was observed in the Drosophila muscles. Whether muscle activity regulates release of Pvf1 primarily could not be demonstrated due to the difficulty in collecting adequate amounts of hemolymph from the adult males. However, the localization of Pvf1 to the M/Z bands suggests a potential role for muscle activity in Pvf1 release. The M and Z bands of skeletal muscles are important centers for sensing muscle stress and strain. These protein-dense regions of the muscle house a number of proteins that can act as mechano-sensors and mediate signaling events including translocation of selected transcription factors to the nucleus. Pvf1, therefore, is ideally located to be able to sense muscle contraction and be released in response to muscle activity. Further work, contingent on the development of new tools and techniques, will be necessary to measure Pvf1 release into the hemolymph and study the regulation of this release by exercise (Ghosh, 2020).

Previous work has shown that Pvf1 released from gut tumors generated by activation of the oncogene yorkie leads to wasting of Drosophila muscle and adipose tissue (Song, 2019). Adipose tissue wasting in these animals is characterized by increased lipolysis and release of free fatty acids (FFAs) in circulation. However, no role was observed of Pvf signaling in regulating lipolysis in the adipose tissue of healthy well-fed flies without tumors. Loss of PvR signaling in the adipose tissue did not have any effect on lipid content. Additionally, over-expressing Pvf1 in the muscle did not lead to the bloating phenotype commonly seen in cachectic animals with gut tumors. It is concluded that Pvf1 affects wasting of the adipose tissue only in the context of gut tumors and that the effect could involve the following mechanisms: (1) the gut tumor releases pathologically high levels of Pvf1 into circulation leading to ectopic activation of PvR signaling in the adipose tissue, and, that such high levels of Pvf1 are not released by the muscle (even when pvf1 is over-expressed in the muscle); (2) Pvf1 causes adipose tissue wasting in the context of other signals that emanate from the gut tumor that are not available in flies that do not have tumors (Ghosh, 2020).

Only oenocyte-specific loss of PvR signaling phenocopies the obesity phenotype caused by muscle-specific loss of Pvf1, indicating that muscle-Pvf1 primarily signals to the oenocytes to regulate systemic lipid content. Additionally, muscle-specific loss of Pvf1, as well as oenocyte-specific loss of PvR and its downstream effector TOR, leads to an increase in the rate of lipid synthesis. These observations indicate a role for the Drosophila oenocytes in lipid synthesis and lipid accumulation in the adipose tissue. Oenocytes have been implicated in lipid metabolism previously and these cells are known to express a diverse set of lipid metabolizing genes including but not limited to fatty acid synthases, fatty acid desaturases, fatty acid elongases, fatty acid β-oxydation enzymes and lipophorin receptors. Functionally, the oenocytes are proposed to mediate a number of lipid metabolism processes. Oenocytes tend to accumulate lipids during starvation (presumably for the purpose of processing lipids for transport to other organs and generation of ketone bodies) and are necessary for starvation induced mobilization of lipids from the adipose tissue. This role is similar to the function of the liver in clearing FFAs from circulation during starvation for the purpose of ketone body generation, and redistribution of FFAs to other organs by converting them to TAG and packaging into very-low density lipoproteins. However, a [1-14C]-oleate chase assay did not show any effect of oenocyte-specific loss of PvR/TOR signaling on the rate of lipid utilization, indicating that this pathway does not affect oenocyte-dependent lipid mobilization (Ghosh, 2020).

Oenocytes also play a crucial role in the production of very-long-chain fatty acids (VLCFAs) needed for waterproofing of the cuticle (Storelli, 2019). Results of a starvation resistance assay indicate that loss of the muscle-to-oenocyte Pvf1 signaling axis does not affect waterproofing of the adult cuticle. Storelli (2019) recently showed that the lethality observed in traditionally used starvation assays is largely caused by desiccation unless the assay is performed under saturated humidity conditions. Since a starvation assay was performed under 60% relative humidity (i.e. non-saturated levels), it is likely that desiccation played a partial role in causing starvation-induced lethality. Any defects in waterproofing of the adult cuticle would have led to reduced starvation resistance. However, both muscle-specific loss of Pvf1 and oenocyte-specific loss of PvR led to increased starvation resistance suggesting normal waterproofing in these animals. The increased starvation resistance in these animals is likely the result of these animals having higher stored lipid content that helps them to survive longer without food (Ghosh, 2020).

Insect oenocytes were originally believed to be lipid synthesizing cells because they contain wax-like granules. These cells express a large number of lipid-synthesizing genes and the abundance of smooth endoplasmic reticulum further suggest a role for this organ in lipid synthesis and transport. However, evidence for potential involvement of the oenocytes in regulating lipid synthesis and lipid deposition in the adipose tissue has been lacking. The fact that two of the three fatty acid synthases (fasn2 and fasn3) encoded by the Drosophila genome are expressed specifically in adult oenocytes suggests a potential role for these cells in lipid synthesis. The observation that oenocyte-specific loss of PvR and its downstream effector TOR leads to increased lipid synthesis and increased lipid content of the adipose tissue strongly supports this possibility. The data further suggests that involvement of the oenocytes in mediating lipid synthesis is more pronounced in newly eclosed adults when the adipose tissue needs to actively build up its lipid stores. In later stages of life, the lipid synthetic role of the oenocytes is repressed by the muscle-to-oenocyte Pvf1 signaling axis. This observation also raises the question of whether FFAs made in the oenocytes can be transported to the adipose tissue for storage. This possibility was tested by over-expressing the lipogenic genes fasn1 and fasn3, which regulate the rate limiting steps of de-novo lipid synthesis, in the oenocytes. It was found that excess lipids made in the oenocytes do end up in the adipose tissue of the animal leading to increased lipid droplet size in the adipose tissue. Taken together, these results provide evidence for the role of Drosophila oenocytes in lipid synthesis and storage of neutral lipids in the adipose tissue of the animal. Interestingly, the vertebrate liver is also one of the primary sites for de-novo lipid synthesis and lipids synthesized in the liver can be transported to the adipose tissue for the purpose of storage. Hence, the fundamental role of the oenocytes and the mammalian liver converge with respect to their involvement in lipid synthesis (Ghosh, 2020).

Oenocyte-specific loss of the components of the Pi3K/Akt1/TOR signaling pathway was observed to lead to increased lipid synthesis. The increased rate of lipid synthesis in flies lacking TOR signaling in the oenocytes is paradoxical to current knowledge of how TOR signaling affects expression of lipid synthesis genes. In both vertebrates and flies, TOR signaling is known to facilitate lipid synthesis by inducing the expression of key lipid synthesis genes such as acetyl CoA-carboxylase and fatty acid synthase via activation of SREBP-1 proteins. Therefore this study checked how oenocyte-specific loss of TOR signaling affects expression of oenocyte-specific fatty acid synthases (fasn2 and fasn3) and oenocyte non-specific fatty acid synthesis genes (fasn1 and acc). Oenocyte-specific loss of TOR strongly down-regulated only fasn2 and fasn3, while the expression of adipose tissue specific fasn1 and acc did not change, indicating that TOR signaling is required for the expression of lipogenic genes in the oenocytes. An increase in lipid synthesis in response to loss of TOR in the oenocytes is quite intriguing and the mechanism remains to be addressed. The increase in lipid synthesis is thought not to happen in the oenocytes since loss of TOR signaling rather reduces expression of lipogenic genes in the oenocytes. The increase in lipid synthesis could happen either as a result of compensatory upregulation of lipid synthesis in the adipose tissue or due to disruption of an as yet unknown role of the oenocytes in lipid synthesis that hinges on TOR signaling. The fact that the expression levels of fasn1 and acc does not change significantly in animals lacking TOR signaling in the oenocytes indicates that compensatory upregulation of lipid synthesis, if present, does not happen through transcriptional upregulation of lipid synthesis genes in the adipose tissue. It is still possible, however, that the increase in lipid synthesis is caused by post-translational modifications of the enzymes. Alternatively, loss of TOR in the oenocyte could affect tissue distribution of lipids or impair clearing of dietary lipids via formation of cuticular hydrocarbons. Understanding the tissue specific alterations in gene expression and changes in the phosphorylation states of key lipogenic proteins in the adipose tissue of animals lacking TOR signaling in oenocytes could shed more light on the mechanisms involved (Ghosh, 2020).

Interestingly, the data suggests that the Drosophila InR does not play a role in activating TOR signaling in the oenocytes. While loss of TOR signaling in the oenocytes leads to obesity, loss of InR signaling does not. Additionally, loss of oenocyte specific InR signaling did not have any effect on p4EBP levels in oenocytes. Moreover, InR signaling and TOR signaling also diverge in their roles in regulating the size of oenocytes. While loss of InR signaling leads to a significant reduction in the size of oenocytes, loss of TOR does not. Further suggesting that TOR does not act downstream of InR in oenocytes. Rather, the data suggests that in wildtype well-fed flies TOR signaling in oenocytes is activated by the Pvf receptor. Interestingly, insulin dependent activation of TOR is not universal. For instance, in the specialized cells of non-obese mouse liver, InR does not play any role in activation of TOR and downstream activation of SREBP-1c (Ghosh, 2020).

Drosophila larval oenocytes are known to accumulate lipids in response to starvation. It has also been showed that starving adult females for 36 hr is capable of inducing lipid accumulation in the oenocytes and that this response is dependent of InR signaling. Since TOR signaling is a known metabolic regulator, one alternate hypothesis that could explain some of the data is that loss of PvR/TOR signaling leads to a starvation like response specifically in the oenocyte leading to InR-dependent accumulation of lipid droplets. To address this possibility, single nuclei sequencing of the adult male abdominal cuticle (and the tissues residing within) derived from oenots>tsc1,tsc2 flies. The animals were raised under identical experimental conditions as control animals. Then the two snRNA-seq data sets were re-analyzed after correcting for batch effects using harmony. The resulting UMAP plots for both genotypes look similar to the original UMAP plot for the control flies and identifies all the clusters reported (see Differential snRNA-seq of the abdominal cuticle upon oenocyte-specific loss of TOR). The percentage of nuclei that constitute each of the major clusters remained similar in both genotypes and the top marker genes for each of the clusters did not change. The oenocyte-specific gene expression profiles from both data sets were subsequently converted to pseudobulk expression for the genes that were detected. This allowed comparison of the expression profiles of the oenocytes from control animals and animals lacking TOR signaling in oenocytes. The effect of losing TOR on the expression of the 47 genes that had been reported to be up-regulated in oenocytes in response to starvation was specifically looked at. Thirty-six of these genes were detected by single nuclei sequencing analysis, however, none of them changed significantly. Based on this observation, it is concluded that loss of TOR signaling most likely does not mount a starvation like response in the oenocytes (Ghosh, 2020).

Serum levels of VEGF-A is high in obese individuals and drops rapidly in response to bariatric surgery, suggesting a role for VEGF-A in obesity. However, evidence on whether VEGF-A or other VEGFs are deleterious vs beneficial in the context of the pathophysiology of obesity is unclear. Adipose tissue-specific over-expression of both VEGF-B and VEGF-A has been shown to improve adipose tissue vascularization, reduce hypoxia, induce browning of fat, increase thermogenesis, and protect against obesity. At the same time, blocking VEGF-A signaling in the adipose tissue of genetically obese mice leads to reduction of body weight gain, improvement in insulin sensitivity, and a decrease in adipose tissue inflammation. Moreover, systemic inhibition of VEGF-A or VEGF-B signaling by injecting neutralizing monoclonal antibodies have also shown remarkable effects in improving insulin sensitivity in the muscle, adipose tissue, and the liver of high-fat diet-induced mouse models of obesity and diabetes. Although the evidence on the roles of VEGF/PDGF signaling ligands in obesity and insulin resistance is well established, the mechanisms clearly are quite complex and are often context dependent. Consequently, a wider look at various tissue specific roles of PDGF/VEGF signaling will be necessary to comprehensively understand the roles of PDGF/VEGF signaling in lipid metabolism. The current work demonstrates an evolutionarily conserved role for PDGF/VEGF signaling in lipid metabolism and a non-endothelial cell dependent role of the signaling pathway in lipid synthesis. Additionally, these findings suggest an atypical tissue-specific role of TOR signaling in suppressing lipid synthesis at the level of the whole organism. Further studies will be required to determine whether vertebrate VEGF/PDGF and TOR signaling exerts similar roles either in the vertebrate liver or in other specialized organ (Ghosh, 2020).

This study made use of snRNA-Seq technology to identify expression of Pvr precisely in certain tissues in the complex abdominal region, which harbors several metabolically active tissues including adipose tissues, oenocytes, and muscle in Drosophila. As yet, there is no systematic study of a complete transcriptomics resource of each of these tissues considering the difficulty in dissecting and segregating these tissues for downstream sequencing. Thus, this study also provides a rich resource of gene expression profiles, paving way for a systems-level understanding of each of these tissues in Drosophila (Ghosh, 2020).

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).

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 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 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 (Macías, 2004).

Mondal, B. C., et al. (2011). Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance. Cell 147(7): 1589-600. PubMed ID: 22196733

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche (see Hematopoetic progenitor maintenance in the Drosophila blood system). In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. This study shows that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. These findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche (Mondal, 2011).

The mammalian hematopoietic niche displays complex interactions between populations of HSCs and progenitors to maintain their numbers. The relative in vivo contributions of cues emanating from the microenvironment in regulating stem cell versus progenitor maintenance remains unclear. Several stem cell and progenitor populations demonstrate slow cell cycling and this property of 'quiescence' is critical for maintaining their integrity over a period of time (Mondal, 2011).

In vivo genetic analysis in Drosophila allows for the study of stem cell properties in their endogenous microenvironment (Losick, 2011). Drosophila blood cells, or hemocytes, develop within an organ called the lymph gland, where differentiating hemocytes, their progenitors, and the cells of the signaling microenvironment or niche, are found. Differentiated blood cells in Drosophila are all myeloid in nature and are located along the outer edge of the lymph gland, in a region termed the cortical zone (CZ. These arise from a group of progenitors located within an inner core of cells termed the medullary zone (MZ). The MZ cells are akin to the common myeloid progenitors (CMP) of the vertebrate hematopoietic system. They quiesce, lack differentiation markers, are multipotent, and give rise to all Drosophila blood lineage. MZ progenitors are maintained by a small group of cells, collectively termed the posterior signaling center (PSC), that function as a hematopoietic niche. Clonal analysis has suggested the existence of a niche-bound population of hematopoietic stem cells, although such cells have not yet been directly identified (Mondal, 2011).

The PSC cells express Hedgehog (Hh), which is required for the maintenance of the MZ progenitors. Cubitus interruptus (Ci) is a downstream effector of Hh signaling similar to vertebrate Gli proteins; it is maintained in its active Ci¹⁵⁵ form in the presence of Hh and degraded to the repressor Ci⁷⁵ form in the absence of Hh. PSC-derived Hh signaling causes MZ cells to exhibit high Ci¹⁵⁵ (Mondal, 2011).

Proliferation of circulating larval hemocytes is also regulated by Adenosine Deaminase Growth Factor-A (Adgf-A), which is similar to vertebrate adenosine deaminases (ADAs). Adgf-A is a secreted enzyme that converts extracellular adenosine into inosine by deamination. Two distinct adenosine deaminases, ADA1 and ADA2/CECR1, are found in humans. CECR1 is secreted by monocytes as they differentiate into macrophages. In Drosophila, mutation of Adgf-A causes increased adenosine levels and increase in circulating blood cells (Mondal, 2011 and references therein).

Extracellular adenosine is sensed by the single Drosophila adenosine receptor (AdoR) that generates a mitogenic signal through the G protein/adenylate cyclase/cAMP-dependent Protein Kinase A (PKA) pathway (Dolezelova, 2007). A target of PKA is the transcription factor Ci, which also transduces the Hedgehog signal. This study explored the potential link between adenosine and Hedgehog signaling, both through PKA mediated regulation of Ci, and a model was proposed that the niche signal and the CZ signal interact to maintain the progenitor population in a quiescent and undifferentiated state within the MZ of the lymph gland (Mondal, 2011).

The first cells that express differentiation markers appear stereotypically at the peripheral edge of the lymph gland. These differentiating cells will eventually populate an entire peripheral compartment that will comprise the CZ. The timing of the first signs of differentiation matches closely with the onset of quiescence among the precursor population, eventually giving rise to the medullary zone (MZ) (Mondal, 2011).

The close temporal synchronization of CZ formation and the quiescence of MZ progenitors raised the intriguing possibility that the onset of differentiation might regulate the proliferation profile of the progenitors. To test this hypothesis, cell death was induced by expressing the pro-apoptotic proteins Hid and Reaper in the differentiating hemocytes, and the effect of their loss was assayed in the progenitor population. Loss of CZ cells was found to induce proliferation of the adjacent progenitor cells, which are normally quiescent at this stage (Mondal, 2011).

Candidate ligands in the lymph gland were knocked down by RNA interference (RNAi) and monitored for a loss of progenitor quiescence. This survey identified Pvf1 as a signaling molecule that is required for the maintenance of quiescence within the lymph gland. Expressing Pvf1^RNAi using Gal4 drivers specific to either niche (PSC) cells using Antp-gal4, progenitor cells using dome-gal4, or differentiating cells using Hml-gal4 showed that PSC-specific knockdown is sufficient to induce progenitor proliferation, whereas Pvf1 knockdown in progenitors or differentiating cells has no effect on the lymph gland. These results indicate that Pvf1 synthesized in the PSC is required for progenitor quiescence (Mondal, 2011).

To determine the site of Pvf1 function, its receptor Pvr was knocked down in the lymph gland using a similar approach. Interestingly, it was found that Pvr^RNAi expressed under the control of drivers specific to differentiating cells (Hml-gal4 and pxn-gal4) causes a loss of progenitor quiescence. The BrdU incorporating cells do not express differentiation markers. Thus, differentiation follows the proliferative event. Lymph glands are not similarly affected when Pvr function is downregulated in the progenitors themselves. These results indicate that Pvf1 originates in the niche and activates Pvr in maturing hemocytes, and that this signaling system is important for the quiescence of MZ progenitors. These results did not explain, though, how maturing cells might signal back to the progenitors causing them to maintain quiescence (Mondal, 2011).

Given the previously known role of Adgf-A in the control of hemocyte number in circulation (Dolezal, 2005), whether this protein plays a similar role in the lymph gland was investigated. Remarkably, downregulation of the secreted Adgf-A protein in the differentiating hemocytes of the CZ, achieved by expressing Adgf-A^RNAi under Hml-gal4 control, induces loss of quiescence of MZ progenitors, similar to that seen with loss of Pvr in the CZ. This suggests that Adgf-A may act as a signal originating from differentiating hemocytes that is required for maintaining progenitor quiescence. In support of this idea, while overexpression of Adgf-A in differentiating hemocytes alone does not affect normal zonation, it suppresses the induced progenitor proliferation caused by downregulation of Pvr. For loss of signaling molecules, it is the break in the signaling network necessary for reducing adenosine that causes continued proliferation and eventual differentiation. For rpr/hid the signaling cell itself has been removed, thereby causing a lack in a backward signal. Quantitative analysis of the data is consistent with a role for Adgf-A downstream of Pvr (Mondal, 2011).

The role of a niche signal is well established in many developmental systems that involve stem cell/progenitor populations. In the Drosophila lymph gland the niche expresses Hh and maintains a group of progenitor cells (Mandal, 2007). This current study establishes an additional mechanism, parallel to the niche signal that originates from differentiating cells, which also regulates quiescence of hematopoietic progenitors (Mondal, 2011).

The cells of the lymph gland proliferate at early stages, from embryo to mid second instar. At this stage, cells farthest from the PSC initiate differentiation and the rest enter a quiescent phase defining a MZ. In wild-type, the cells of the MZ remain quiescent and in progenitor form throughout the third instar, and this process requires a combination of the PSC and CZ signals. If either signal is removed, the progenitor population will eventually be lost due to differentiation. In many different genetic backgrounds, if quiescence is lost, the progenitor population initially continues to incorporate BrdU during the second instar without expressing any maturation markers. The differentiation phenotype, characterized by the expression of such markers, follows this abnormal proliferation. The net result is that whenever the progenitors accumulate BrdU (but not express any markers of differentiation) in the second instar, all cells of the lymph gland are differentiated and no MZ remains in the third instar. While the nature of the signal that triggers hemocyte differentiation is not known, withdrawal of Wingless may play a role in this process (Mondal, 2011).

Experimental analysis has demonstrated a novel role for Pvr in maturing hemocytes and its ligand, Pvf1, in the cells of the PSC. Pvf1 expression increases at a stage when the lymph gland is highly proliferative. At this critical window in development, Pvf1 originating from the PSC is transported to the differentiating hemocytes, binds to its receptor Pvr, and activates a STAT-dependent signaling cascade. At this stage, Pvf1 is sensed by all cells but it is only in the differentiating hemocytes that it activates Adgf-A in an AdoR/Pvr-dependent manner. This secreted factor Adgf-A is required for regulating extracellular adenosine levels. High adenosine would signal through AdoR and PKA to inactivate Ci and reduce the effects of the niche-derived Hedgehog signal leading to differentiation of the progenitor cells. The function of the Adgf-A signal is to reduce this adenosine signal and therefore reinforce the maintenance of progenitors by the Hedgehog signal. Thus, the Adgf-A and Hh signals work in the same direction but Adgf-A does so by negating a proliferative signal due to adenosine. In wild-type, equilibrium is reached through a signal that does not originate from the niche that opposes this proliferative process. The attractive step in this model is that the CZ and niche (in this case Hh-dependent) signals both impinge on common downstream elements allowing for control of the progenitor population relative to the niche and the differentiated cells. Most importantly, this is a mechanism for maintaining quiescence within a moderately large population of cells that is not in direct contact with a niche. By the time the three zone PSC/MZ/CZ system is set up in the late second instar all the cells of the MZ express high levels of E-cadherin, become quiescent and are maintained as progenitors and are capable of giving rise to all blood cell lineages. Under such circumstances, the interaction between a niche-derived signal and an equilibrium signal originating from differentiating cells can maintain homeostatic control of the progenitor population. Several vertebrate stem cell/progenitor scenarios such as during bone morphogenesis and hematopoiesis or in the Drosophila intestine have progenitors and differentiating cells in close proximity that could pose an opportunity for a similar niche and differentiating cell-derived signal interaction. In fact, evidence for such interactions have recently been provided for vertebrate skin cells (Mondal, 2011).

The role of small molecules such as adenosine has not yet been adequately addressed in vertebrate progenitor maintenance. A small molecule such as extracellular adenosine is unlikely to form a gradient over the population of cells and maintain such a gradient over a developmental time scale. It is much more likely that this system operates similar to the 'quorum sensing' mechanisms described for prokaryotes. A critical level of adenosine is required for proliferation and by expressing the Adgf-A signal this threshold amount is lowered, causing quiescence in the entire population (Mondal, 2011).

This study describes a developmental mechanism that is relevant to the generation of an optimal number of blood cells in the absence of any overt injury or infection. However, a system that utilizes such a mechanism to maintain a progenitor population could potentially sense a disruption upon induction of various metabolic stresses to cause differentiation of myeloid cells. Various mitochondrial and cellular stresses can cause an increase in extracellular adenosine (Fredholm, 2007), but whether they are relevant to this system remains to be studied. In the past, dual use has been observed of reactive oxygen species (ROS) as well as Hypoxia Inducible Factor-a (HIF-a) in both development and stress response of the Drosophila hematopoietic. Responses to injury have been described in the Drosophila intestine, and in satellite cells that respond during injury, a stress related signal could be the initiating factor that overrides a maintenance signal. Thus, the equilibrium generated through developmental interactions is disrupted to promote a cellular response to stress signals (Mondal, 2011).

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeded, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that form a ring shape and surround the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011).

To visualize the genitalia rotation in living animals, the His2Av-mRFP Drosophila line was used whose nuclei are ubiquitously marked by a fluorescent protein. The genital disc is a compound disc comprised of cells from three different embryonic segments: A8 (male eighth tergite), A9 (male primordium) and A10 (anal). During metamorphosis, the genital disc is partially everted, exposing its apical surface, and adopts a circular shape. The results captured the male genitalia undergoing a 360° clockwise rotation. Inhibiting apoptosis by expressing the baculovirus pan-caspase inhibitor p35 driven by engrailed-GAL4 (en-GAL4), which is expressed in the posterior compartment of each segment, results in genital mis-orientation at the adult stage (Kuranaga, 2011).

In flies expressing nuclear fluorescent protein driven by en-GAL4, it was observed that the posterior part of the A8 segment (A8p) formed a ring of cells surrounding the A9-A10 part of the disc. First, the images were recorded at a low resolution (10× objective lens) to measure the rotation speed accurately in control and p35-expressing flies, because long-term time-lapse imaging at a high resolution can cause photodamage, and thus alter pupal development. Most of the cells in the A8p that seem to disappear actually moved out of the plane of focus. The imaging results, the rotation started around 24 hours APF (after puparium formation) and stopped about 12 hours later. To confirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, the rotation was observed in flies expressing p35 under the en-GAL4 driver. In the p35-expressing flies, the rotation began, but it stopped before it was complete, after about 12 hours, i.e. with the same timing as in control flies. This suggested that the reduced caspase activation in A8p prevented the genitalia from completing the rotation, resulting in mis-oriented adult genitalia (Kuranaga, 2011).

To compare complete rotation with incomplete rotation, the rotation speed was calculated by measuring the angle (theta_control and theta_p35) of the A9 genitalia every 30 minutes on time-lapse images. The normal rotation was composed of at least four steps: initiation, acceleration, deceleration and stopping. The velocity of rotation V=dtheta/dt was calculated by measuring theta as a function of time t. At first, the genitalia rotated at an average velocity (V_control) of 7.67±3.72°/hour by 1 hour after initiation, then the rotation accelerated, with V_control gradually increasing to 53.83±7.11°/hour by 7 hours after initiation. Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, and the average velocity (V_p35) from the initial rotation to 1 hour later was 7.45± 2.98°/hour, which was not significantly different from the normal rotation. However, the acceleration of the rotation in the p35-expressing flies was lower than normal, with V_p35 gradually increasing to 21.35±7.45°/hour at 5.5 hours after initiation. The first peak of the acceleration rate, which was defined as the initiation of rotation, was observed in the p35-expressing flies (a_p35) and was the same as in the control flies (a_control). However, the duration of the acceleration period was shorter in the p35-expressing flies. These data suggest a relationship between apoptosis and the acceleration of genitalia rotation (Kuranaga, 2011).

Next, the signaling mechanism(s) involved in the acceleration of genitalia rotation wee examined. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling in male flies has been shown to result in mis-oriented adult male terminalia, and it has been hypothesized that the PVF/PVR (PVF receptor) may affect the genitalia rotation via JNK-mediated apoptosis (see Benitez, 2010). Consistent with previous reports, the acceleration of genitalia rotation was significantly impaired in flies expressing dominant-negative forms of JNK (JNK-DN) and PVR (PVR-DN). These data implied that caspase activation and JNK signaling contribute to driving the acceleration of genitalia rotation (Kuranaga, 2011).

To analyze how the genitalia accelerate their rotation, the movement of A8p was traced at the single-cell level. For this experiment, live imaging was performed at a high resolution (20× objective lens), which enabled the cells in A8p to be tracked at single-cell resolution. Cells that were neighbors of A9 rotated with A9, whereas cells located in the anterior half of A8p rotated later than A9. Based on this imaging, A8p was divided into two sheets, named A8pa (anterior of A8p) and A8pp (posterior of A8p). It was found that a part of the cells in A8p underwent apoptosis (Kuranaga, 2011).

To observe caspase activation in living animals, a FRET (fluorescence resonance energy transfer)-based indicator, SCAT3 (sensor for activated caspases based on FRET) was generated. To precisely evaluate apoptosis, a nuclear localization signal-tagged SCAT3 (nls-SCAT3; UAS-nls-ECFP-venus) was used. The nls-SCAT3 signal was clearly observed in A8p. Cells exhibiting high caspase activity were extruded into the body cavity and disappeared, consistent with their apoptotic death and engulfment by circulating hemocytes. Each cell was tracked in the A8p region during the first half of the rotation, and it was found that at least three types of cellular behavior were observed: (1) cells located in A8pp moved with A9, (2) cells underwent apoptosis and (3) cells located in A8pa rotated later (Kuranaga, 2011).

Thus, to observe the behavior of the cells in A8pa, Abdominal B (AbdB) was used as an A8 marker. AbdB is a homeotic gene that is required for the correct development of the genital disc, and AbdB-GAL4^LDN is expressed in the segment A8 (in A8a and A8p) of the genital disc during the 3rd instar larval stage. At 24 hours APF, AbdB was expressed in A8 and formed a ring. Time-lapse images were taken, and unexpectedly it was found that most of the cells in the AbdB-expressing region underwent a 180° clockwise movement, suggesting that AbdB was not expressed in the A8pp region that moved 360° with A9. To determine the speed of the AbdB-expressing cells, three individual cells were traced in each fly, and the value of the turning angle of the cells (theta_AbdB) was calculated. The findings confirmed that the AbdB-expressing region moved halfway around. Although cells in the AbdB-expressing region moved only 180°, the A8pp (inner ring), which was encircled by the AbdB-expressing region (outer ring), still moved 360°. Furthermore, the imaging data indicated that the movement of the outer ring started 1-2 hours later than that of the A9 region, when the acceleration of the genitalia rotation occurred. These observations raise the possibility that the outer ring movement is related to the acceleration of the genitalia rotation (Kuranaga, 2011).

It was therefore considered that the outer ring movement was restricted in the p35-expressing flies, resulting in an incomplete genitalia rotation of about 180°, which mimics the movement of only the inner ring. To verify this possibility, the movement of the outer ring was examined in the p35-expressing flies (en-GAL4+UAS-p35). Although the inner ring rotated normally, the rotation of the outer ring was impaired in the p35-expressing flies. The turning angles were determined by tracing cells in the p35-expressing flies and it was found that theta_{p35 _inner} increased, while the increase of theta_{p35 _outer} was impaired. These data suggest that the A8 segment is composed of two independently regulated rings, and when apoptosis is inhibited, the inner ring can move only 180° with no outer ring movement, resulting in incomplete genitalia rotation (Kuranaga, 2011).

Thus, to determine whether apoptosis correlates with the outer ring movement, the apoptosis was quantified in A8pa every 10 minutes from 0-8 hours after the start of genitalia rotation. The frequency of apoptosis (R_apoptosis) was normalized to the total number of apoptotic cells in each individual. Pulsatile increases in R_apoptosis were observed, with peaks at 1, 2.5 and 4 hours after the start of genitalia rotation. To verify the participation of R_apoptosis in the initiation of outer ring movement, the acceleration rate of theta_AbdB (a_AbdB) was calculated by measuring V_AbdB as a function of time t, and R_apoptosis was compared with a_AbdB. The starting time of outer ring movement was characterized by the early peaks of a_AbdB. The analysis suggested that the a_AbdB was related to the R_apoptosis, because a_AbdB showed its first two peaks at about 1 and 2.5 hours after genitalia rotation started. To quantify these observations, the correlation was calculated between R_apoptosis and a_AbdB. This analysis confirmed that there was a strong correlation between these parameters, because the correlation between a_AbdB and R_apoptosis is approximately linear during this time. Therefore, these data implied a possible mechanism of apoptosis that facilitates the outer ring movement (Kuranaga, 2011).

To verify this possibility, whether the upregulation of apoptotic signals induces an increase in genitalia rotation speed was meastured. Because the expression of apoptotic genes using an en-GAL4 driver, which is expressed at the embryonic stage, is lethal, the TARGET system was used to control gene expression temporally. Flies were allowed to develop at 18°C until the head of the pupae had just everted, to inhibit gene expression. The pupae were then heat-shocked at 29°C for 12 hours to induce gene expression. Live imaging was performed at 22°C, after the heat shock. At this temperature, the genitalia rotation in the control flies was slower than in control flies bred at 25°C, because a low breeding temperature affects the rate of fly development, including genitalia rotation. Therefore, it was necessary in this experiment to compare the rotation speeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that the upregulation of apoptotic signaling significantly increased the timing of acceleration and speed of genitalia rotation. These observations led to the proposal that the outer ring functions like a 'moving walkway' to accelerate the speed of the inner part of the structure, including the A9 genitalia, enabling genitalia to complete rotation within the appropriate developmental time window (Kuranaga, 2011).

According to these observations, it was found that apoptosis drives the movement of cell sheets during the morphogenesis of male terminalia. Further questions remain with regard to how apoptosis contributes to the cell sheet movement. A recent study indicated the possibility that local apoptosis acts as a brake release to regulate genitalia rotation, coupled with left-right determination (Suzanne, 2010). However, it has been reported that the cell shape change by apoptosis enables not only the extrusion of dying cells, but also the reorganization of the actin cytoskeleton in neighboring cells. Therefore, apoptosis could affect the behavior of neighboring cells, to act as a main driving force of the cell-sheet movement. Taken together, apoptosis may generally participate in the morphogenetic process of cell-sheet movement during morphogenesis (Kuranaga, 2011).

The forming limb skeleton serves as a signaling center for mammalian limb vasculature patterning via regulation of Vegf

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, evidence is provided for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, evidence is provided for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature (Eshkar-Oren, 2009).

VEGF is required for dendritogenesis of newly born olfactory bulb interneurons

The angiogenic factor vascular endothelial growth factor A (VEGF) has been shown to have a role in neurogenesis, but how it affects adult neurogenesis is not fully understood. To delineate a role for VEGF in successive stages of olfactory bulb (OB) neurogenesis, a conditional transgenic system was used to suppress VEGF signaling at the adult mouse sub-ventricular zone (SVZ), rostral migratory stream (RMS) and OB, which constitute the respective sites of birth, the migration route, and sites where newly born interneurons mature and integrate within the existing OB circuitry. Following the development of fluorescently tagged adult-born neurons, it was shown that sequestration of VEGF that is constitutively expressed by distinct types of resident OB neurons greatly impaired dendrite development in incoming SVZ-born neurons. This was evidenced by reduced dendritic spine density of granule cells and significantly shorter and less branched dendrites in periglomerular neurons. Notably, the vasculature and perfusion of the SVZ, RMS and OB were not adversely affected when VEGF suppression was delayed until after birth, thus uncoupling the effect of VEGF on dendritogenesis from its known role in vascular maintenance. Furthermore, a requirement for VEGF was specific to newly born neurons, as already established OB neurons were not damaged by VEGF inhibition. This study thus uncovered a surprising perfusion-independent role of VEGF in the adult brain, namely, an essential role in the maturation of adult-born neurons (Licht, 2010).

Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling

The regulation of stem cell ontogeny is poorly understood. The leukemia-associated Ets transcription factor, Tel1/ETV6, specifies the first hematopoietic stem cells (HSCs) in the dorsal aorta (DA). In contrast, Tel1/ETV6 has little effect on embryonic blood formation, further distinguishing the programming of the long- and short-term blood populations. Consistent with the notion of concordance of arterial and HSC programs, it was shown that Tel1/ETV6 is also required for the specification of the DA as an artery. Tel1/ETV6 acts by regulating the transcription of VegfA in both the lateral plate mesoderm and also in the somites. Exogenous VEGFA rescues Tel1/ETV6 morphants, and depletion of VEGFA or its receptor, Flk1, largely phenocopies Tel1/ETV6 depletion. Few such links between intrinsic and extrinsic programming of stem cells have been reported previously. These data place Tel1/ETV6 at the apex of the genetic regulatory cascade leading to HSC production (Ciau-Uitz, 2010).

PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration

A fundamental property of neural crest (NC) migration is Contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibian and zebrafish by controlling cell polarity in a cell contact dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin (see Drosophila CadN), which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial to mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. This study shows that PDGFRα (see Drosophila Pvr) and its ligand PDGF-A (see Drosophila Pvf1) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently impairing CIL. Moreover, PI3K/AKT (see Drosophila Akt) was found to be a downstream effector of the PDGFRα cellular response during CIL. These results lead to a proposal that PDGF-A/PDGFRα signalling is a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, it was shown that once NC have undergone EMT, the same PDGF-A/PDGFRα works as NC chemoattractant guiding their directional migration (Bahm, 2017).

Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

The transcriptional pathways activated downstream of Vascular Endothelial Growth Factor (VEGF; see Drosophila Pvf1) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand, Delta-Like 4 (DLL4; see Drosophila Delta) in endothelial cells this study found that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG (see Drosophila Pointed), a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300 (see Drosophila Nejire). Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG ChIP-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG, and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (a transcription factor implicated in sprouting angiogenesis; see Drosophila Homeodomain protein 2.0) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis (Fish, 2017).

Search PubMed for articles about Drosophila Pvf1

Bahm, I., Barriga, E. H., Frolov, A., Theveneau, E., Frankel, P. and Mayor, R. (2017). PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration. Development [Epub ahead of print]. PubMed ID: 28526750

Benitez S., et al. (2010). Both JNK and apoptosis pathways regulate growth and terminalia rotation during Drosophila genital disc development. Int. J. Dev. Biol. 54: 643-653. PubMed ID: 20209437

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: PubMed ID; Online text

Ciau-Uitz, A., Pinheiro, P., Gupta, R., Enver, T. and Patient, R. (2010). Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling. Dev. Cell 18(4): 569-78. PubMed ID: 20412772

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

Eshkar-Oren, I., et al. (2009). The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development 136(8): 1263-72. PubMed ID: 19261698

Fish, J. E., Gutierrez, M. C., Dang, L. T., Khyzha, N., Chen, Z., Veitch, S., Cheng, H. S., Khor, M., Antounians, L., Njock, M. S., Boudreau, E., Herman, A. M., Rhyner, A. M., Ruiz, O. E., Eisenhoffer, G. T., Medina-Rivera, A., Wilson, M. D. and Wythe, J. D. (2017). Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network. Development. PubMed ID: 28536097

Ghosh, A. C., Tattikota, S. G., Liu, Y., Comjean, A., Hu, Y., Barrera, V., Ho Sui, S. J. and Perrimon, N. (2020). Drosophila PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity. Elife 9. PubMed ID: 33107824

Kuranaga, E., et al. (2011). Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia. Development 138(8): 1493-9. PubMed ID: 21389055

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 ID: 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 ID: 17462868

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

Licht, T., et al. (2010). VEGF is required for dendritogenesis of newly born olfactory bulb interneurons. Development 137(2): 261-71. PubMed ID: 20040492

Macías, 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 ID: 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 ID: PubMed ID; 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. PubMed ID: 15056618

Song, W., Kir, S., Hong, S., Hu, Y., Wang, X., Binari, R., Tang, H. W., Chung, V., Banks, A. S., Spiegelman, B. and Perrimon, N. (2019). Tumor-derived ligands trigger tumor growth and host wasting via differential MEK activation. Dev Cell 48(2): 277-286 e276. PubMed ID: 30639055

Storelli, G., Nam, H. J., Simcox, J., Villanueva, C. J. and Thummel, C. S. (2019). Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood. Dev Cell 48(2): 200-214 e206. PubMed ID: 30554999

Suzanne M., et al. (2010). Coupling of apoptosis and L/R patterning controls stepwise organ looping. Curr. Biol. 20: 1773-1778. PubMed ID: 20832313

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

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

date revised: 22 February 2022

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