InteractiveFly: GeneBrief

PDGF- and VEGF-receptor related : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Migration of border cells during Drosophila oogenesis is a simple and attractive model system in which to address the signaling pathways and mechanisms responsible for guiding cell migration in vivo. Pvr, a receptor tyrosine kinase related to mammalian PDGF and VEGF receptors, acts in border cells to guide them to the oocyte. The oocyte is the source of a ligand for Pvr, PDGF/VEGF factor 1 (Pvf1). Intriguingly, the guidance function of Pvr is largely redundant with that of Egfr. Rac and the Rac activator Myoblast city/DOCK180/CED-5 are implicated as mediators of the guidance signal (Duchek, 2001b).

Border cells are a cluster of 6-10 specialized somatic follicle cells which perform a stereotypic migration during oogenesis. At the beginning of stage 9 of oogenesis, border cells delaminate from the anterior follicular epithelium and initiate their migration between the germline derived nurse cells, toward the oocyte. About 6 hr later, at stage 10, the border cells reach the oocyte, and then migrate a short distance dorsally toward the germinal vesicle (GV). Thus, the migration occurs in two steps: an initial posteriorly directed (oocyte directed) migration, and subsequently, a shorter, dorsally directed migration. The migration of border cells is essential for female fertility (Duchek, 2001b and references).

EGF receptor (Egfr) signaling is responsible for guiding the second part of border cell migration, the dorsal migration (Duchek, 2001a). However, Egfr signaling is not essential for the first phase of migration of border cells toward the oocyte, indicating that an additional cue must direct this migration. A Drosophila ligand of the PDGF/VEGF family (Pvf1) and its receptor, PDGF/VEGF Receptor (Pvr), are required for the first phase of border cell migration. Pvr and Egfr act in a partially redundant manner to guide border cells to the oocyte. Pvr affects actin accumulation in follicle cells through Myoblast city (Mbc) and Rac. In addition, both Mbc and Rac are required for normal border cell migration. Together, these observations suggest that Pvr signaling controls actin accumulation via Mbc and Rac in migrating border cells (Duchek, 2001b).

Cell migration is guided by one or more spatial (guidance) cues. It was reasoned that uniform expression of a key guidance cue, or a rate-limiting component in its production, throughout the target tissue could be expected to confuse the migrating cells and thus cause inefficient migration. To identify candidate guidance molecules for border cell migration, a gain-of-function genetic screen was used. Controlled ectopic expression of random genes in the genome can be obtained using the modular misexpression, or EP element, system. In a screen of 8500 EP insertion lines, two lines were identified that cause inefficient border cell migration when overexpressed in the germline, consistent with the possibility that a guidance cue was being expressed. The first of these lines directed expression of the EGFR ligand Vein. For the second line, EPg11235, sequencing of flanking DNA showed that the EP element was positioned to drive expression of transcripts corresponding to the predicted gene CG7103. The gain-of-function phenotype was reproduced by expressing a corresponding cDNA. This cDNA was sequenced and shown to encode a protein with a signal sequence and a PDGF domain. It shows highest similarity to PDGF and VEGF ligands from vertebrates. It was therefore called PDGF/VEGF 1 (Pvf1). Pvf1 affects border cell migration whether overexpressed uniformly in the germline or in border cells themselves, consistent with production of a secreted molecule. From an existing collection of P element insertions, one line, EP1624, was found to have an insertion in the first intron of the Pvf1 gene. This insertion is a loss-of-function mutant of Pvf1 (Pvf1¹⁶²⁴) with no detectable transcript remaining in the ovary (Duchek, 2001b).

To investigate whether Pvf1 could serve as a guidance cue for the migration of border cells to the oocyte, its expression was analyzed. The Pvf1 transcript was detected in the germline of the ovary at mid-oogenesis, more concentrated toward the oocyte. To look at the protein expression directly, anti-Pvf1 antisera was raised. The anti-Pvf1 sera shows specific staining in the ovary, which is absent from mutant egg chambers. Pvf1 is detected in the oocyte at stage 7 and at stage 8, filling the cytoplasm. At stage 9, when border cells have initiated migration, Pvf1 is still enriched in the oocyte, but now only in the subcortical area of the large oocyte. Thus, the oocyte appears to be the major site of Pvf1 protein production. Pvf1 is expressed both before and during border cells migration, consistent with the possibility that Pvf1 serves as an attractant for border cells (Duchek, 2001b).

The Pvf1¹⁶²⁴ mutant is homozygous viable, and analysis of egg chambers from mutant females reveals minor delays in border cell migration. However, two additional PDGF, VEGF-like ligands appear to exist in Drosophila. To overcome the potential redundancy between PVF ligands, a PVF receptor was sought in order to directly investigate its role in border cells (Duchek, 2001b).

Gene predictions indicate that the Drosophila genome contains a single gene encoding a protein related in sequence and structure to mammalian PDGF and VEGF receptors. The protein has been called Pvr for PDGF/VEGF receptor. It appears to be the only Drosophila member of this family of receptor tyrosine kinases, and thus could be the receptor for all three PVF ligands. Pvr transcripts are detected in mRNA from ovaries and from embryos. Pvr mRNA is detected in embryonic hemocytes and in Schneider cells, the related tissue culture cells. Using a specific antiserum directed against the C-terminal tail of Pvr, endogenous Pvr protein was detected in Schneider cell extracts as an approximately 180 kDa protein, corresponding well to the predicted molecular weight of 170 kDa (Duchek, 2001b).

Does Pvr mediate the effect of Pvf1 on border cell migration and is the effect direct? Immunofluorescence analysis of wild-type ovaries indicates that endogenous Pvr protein is present in all follicle cells and thus might respond to Pvf1. Pvf1 has been identified based on the ability of uniform expression to impede border cell migration. Direct uniform activation of the Pvf1 receptor in border cells should give the same effect or a stronger effect. To test whether Pvr would do this, an activated form of the receptor, lambda-Pvr, was made. This was done by exchanging the normal extracellular ligand binding domain for a constitutive dimerization domain, as has been done for other receptor tyrosine kinases. The Gal4-UAS system and the slboGal4 driver were used to drive expression of lambda-Pvr in border cells, centripetal cells, and a few other follicle cells. Lambda-Pvr is functional in vivo since it stimulates the MAP-kinase pathway (dpERK staining). Expression of lambda-Pvr in border cells also completely blocks their migration. In over 90% of control stage 10 egg chambers, border cells had reached the oocyte, and the rest were only slightly delayed. In contrast, almost none of the border cell clusters expressing lambda-Pvr had moved at all. Thus, uniform activation of Pvr in border cells blocks migration, as expected for a guidance receptor (Duchek, 2001b).

Ectopic expression of the ligand Pvf1 has a detectable but modest effect on migration: all border cell clusters have moved by stage 10, and one-third have arrived at the oocyte. Increased expression of the wild-type Pvr receptor in border cells has, on its own, a negligible effect on migration, but it sensitizes the cells to ectopic expression of Pvf1. Upon coexpression of Pvr and Pvf1, one-fourth of the stage 10 border cell clusters are at the oocyte, but another fourth have not moved at all. The effect is specific to Pvf1, since border cells are not sensitized to ectopic expression of the Egfr-ligand Vein. In fact, Pvr overexpression ameliorates the effect of ectopic Vein expression. The synergy between Pvf1 and Pvr expression supports a specific interaction between the two proteins on border cells (Duchek, 2001b).

To investigate whether Pvr is required for guiding border cell migration to the oocyte, a dominant negative form of the receptor, DN-Pvr, was generated. DN-Pvr was made in the same way as the highly specific dominant negative Egfr. DN-Pvr contains only the extracellular and transmembrane domains of the receptor, allowing it to sequester ligand as well as to form inactive dimers with the endogenous receptor, and thus specifically attenuate signaling from this receptor. When expressed in border cells, DN-Pvr causes some delay of posterior migration. This result was confirmed by quantification of migration at stage 10. Upon expression of DN-Pvr, less than 60% of border cell clusters had reached the oocyte. This phenotype is similar to that seen in Pvf1 homozygous mutant females, indicating that Pvf1 is the major endogenous ligand for Pvr in this context. Thus, Pvr signaling, and lack thereof, affects the efficiency of border cell migration, but it is not essential for the process (Duchek, 2001b).

In addition to Pvr, Egfr also has properties consistent with a role in guiding border cells to the oocyte: both receptor tyrosine kinases are expressed in border cells, and their ligands are found in key locations in the germline. Both give similar gain-of-function effects, and both dominant negative receptors give subtle effects with respect to migration to the oocyte. One possible explanation for the subtle dominant negative effects is that the receptor/ligand pairs are partially redundant. This possibility was first addressed by coexpressing both dominant negative receptors in border cells. This gave a very dramatic effect. Border cells expressing both dominant negative receptors migrate very inefficiently. When quantified at stage 10, 90% of border cell clusters expressing both dominant negative receptors had migrated less than halfway to the oocyte. In 5% of egg chambers, border cell clusters were found off the direct track to the oocyte. This suggests that the cells are motile but poorly guided. This 'off track' phenotype is not observed in wild-type egg chambers or in egg chambers where border cell migration is impaired for another reason (slbo mutant) (Duchek, 2001b).

The effect of expressing dominant negative receptors in Pvf1¹⁶²⁴ mutant egg chambers was also tested. As expected, the Pvf1¹⁶²⁴ mutant phenotype is not made worse by removing activity of its cognate receptor, Pvr. However, reducing activity of the other pathway by expression of dominant negative Egfr has a strong effect. Border cells are not able to reach the oocyte by stage 10, and they also show a low level of 'off track' migration. This confirms the redundancy of function for the two receptors, as well as their ligand specificity. Thus, if either Egfr or Pvr (and corresponding ligand) are left intact, border cells can find the oocyte, but if both receptor functions are severely affected, they cannot. That Egfr is uniquely required for dorsal migration of border cells is explained by the ligand distribution. Only Egfr ligands are expressed differentially on the dorsal side. Gurken is expressed by the dorsally located germinal vesicle, and the protein is found in a gradient originating from there. Spitz and Vein are expressed in dorsal follicle cells (Duchek, 2001b).

These results indicate that Pvr and Egfr are guidance receptors for border cell migration toward the oocyte. A guidance function implies that the critical parameter for proper migration is the differential distribution of signal (ligand) rather than absolute level of signaling. This is supported by the observation that increased expression of Pvr in border cells suppresses the effect of the ectopically expressed Egfr ligand, Vein. The level of Pvr+Egfr signaling in border cells is likely higher upon coexpression, but the signal distribution might be more normal due to increased sensitivity to the spatially graded Pvr ligand relative to the ectopically expressed Egfr ligand. To test the importance of signal distribution versus level more directly, signaling from one receptor was reduced by expression of its dominant negative form and it was asked whether the deleterious effect of ectopic ligand for the other receptor would be enhanced or suppressed. For guidance signaling, the expectation is that cells which can only respond to one type of ligand will require this ligand to be properly distributed and thus be very sensitive to its misexpression. If just the correct level of signal is required, then simultaneously increasing and decreasing signaling should give a less severe phenotype than either alone. The experiment was done for both receptors, and in both cases, a strong enhancement of the migration defect was seen. Ectopic expression of one ligand and the dominant negative form of the other receptor causes a phenotype similar to one expressing both dominant negative receptors: border cells do not reach the oocyte at stage 10. They usually had migrated less than halfway, and sometimes were found off track. As expected, coexpression of a ligand with a dominant negative version of its cognate receptor has little or no additional effect. These results indicate that both receptors receive directional information which guides cell migration. Migration can proceed to some extent if only one receptor receives nonuniform (directional) signaling, consistent with a partially redundant guidance function (Duchek, 2001b).

Pvr affects actin accumulation in follicle cells through Myoblast city (Mbc) and Rac. In addition, both Mbc and Rac are required for normal border cell migration. Together, these observations suggest that Pvr signaling controls actin accumulation via Mbc and Rac in migrating border cells. Other receptor tyrosine kinases may also use this signaling module to guide cell migration in vivo. mbc was first identified in Drosophila based on its requirement in myoblast fusion. Mbc has since been implicated in multiple processes requiring cytoskeletal reorganization, and is intriguingly expressed in early germ cells of the embryo, which undergo a guided migration. Genetic data indicate that mbc, as well as the C. elegans homolog ced-5, acts as an upstream activator of Rac. No GTP-GDP exchange activity has been shown for Mbc/DOCK180/CED-5, but Mbc/DOCK180/CED-5 interacts with nucleotide-free Rac, indicating that it plays a role in activation or localization of Rac. The small adaptor protein Crk interacts specifically with Mbc/DOCK180/CED-5 in all three systems. In mammalian cells, Crk and another adaptor protein, p130-CAS, have been shown to regulate cell migration in a Rac-dependent manner. Crk, CAS, and DOCK180 regulate membrane ruffling in a Rac-dependent manner. In C. elegans, CED-5, CED-2 (Crk), and CED-10 (Rac) are required for normal distal tip cell migration as well as cell engulfment, but the receptors regulating this behavior are not known. Cell engulfment (phagocytosis) by mammalian 293T cells involves the alpha_vß₅ integrin receptor which, in an unknown manner, can stimulate the formation of a p130-CAS-Csk-Dock180 complex and also activation of Rac1. Thus, Mbc/DOCK180/CED-5 and Rac are linked in a well conserved signaling module that affects cell behavior, including migration. With the Drosophila Pvr receptor identified, it should now be possible to determine how this guidance receptor affects Mbc and Rac (Duchek, 2001b and references therein).

Signaling through Mbc and Rac is unlikely to be the only effect of the guidance receptors in border cells. mbc null clones give a phenotype which is stronger than loss of signaling from either receptor alone, but not as severe as loss of both Pvr and Egfr activities. Egfr acts partially redundant with Pvr in guiding border cells, but preliminary evidence suggests that Egfr may act differently from Pvr. Pvr may also have additional effects, given that the dominant effect of activated Pvr on the actin cytoskeleton is strongly attenuated but not abolished in mbc null clones. Thus, the receptor pathways may be only partially overlapping, and other effectors are likely to contribute to the complicated task of guiding cell migration in vivo. Many candidate signaling molecules have been tested for their requirement in border cell migration: MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, DOCK, Trio, and Pak. None of these is (individually) required; thus, Mbc and Rac remain the only identified downstream signaling effectors in this context. A number of other genes have been shown to be important for border cell migration, but these are either transcription factors and modulators thereof (which are likely to affect cell fate) or components of the basic cellular machinery for movement/adhesion (Duchek, 2001b and references therein).

Receptor tyrosine kinases serve multiple roles during development. The ability of Pvr to activate the MAP-kinase pathway may be important for control of cell growth and differentiation in other tissues, as is the case for Egfr. Both Egfr and Pvr retain the ability to activate the MAP-kinase pathway when serving the guidance receptor function in border cells, indicating that they can simultaneously display multiple signaling properties. The mammalian PDGF receptors and VEGF receptors also have multiple functions during development and in tissue culture cells, including effects on proliferation and on cell migration. Which pathways downstream of these receptors are critical for which function, and how cell type specific responses are generated, remains an important question. Studies of mice with targeted mutations in specific tyrosines of PDGFR-ß indicate that the requirements for specific docking sites in vivo are not easily predicted from the effect of the same mutations in tissue culture cells (Tallquist, 2000). There may be more compensation and redundancy in vivo, or the importance of different pathways may simply differ in vivo and in tissue culture. In either case, such findings underscore the importance of in vivo analysis. Guidance signaling has been analyzed in a simple and well defined cell migration process in vivo. Based on analyses of mutations in signaling pathway components, the essential involvement of some pathways can be ruled out and another putative pathway (Mbc-Rac) downstream of Pvr (and Egfr) can be implicated. There may be some redundancy in downstream pathways leading to guided border cell migration. In the case of MAP-kinase pathway and PI3K, both loss-of-function and gain-of-function (constitutive activation) mutations were investigated. If a signaling molecule was instructive but redundant, then ubiquitous activation would probably have some effect. However, border cell migration was unaffected, arguing that these signaling molecules do not play instructive roles (Duchek, 2001b).

It is intriguing that, even in this simple cell migration system, there is substantial redundancy between the guidance cues (and between the guidance receptors). It is not a priori obvious that these two different types of receptor tyrosine kinases should show such overlap in function. Redundancy in biological functions of receptor tyrosine kinases is likely to be even more prominent in mammalian systems, which have multiple receptors of each type. Subtle effects of individual factors and genetic redundancy are more the rule than the exception in analysis of axon guidance. It is interesting to speculate that partial reliance on multiple signals is biologically advantageous for continuously and subtly modulated processes such as guidance, as compared to all-or-none cell fate determination switches (Duchek, 2001b).

PDGF/VEGF receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration>

During collective migration, guidance receptors signal downstream to result in a polarized distribution of molecules, including cytoskeletal regulators and guidance receptors themselves, in response to an extracellular gradient of chemotactic factors. However, the underlying mechanism of asymmetry generation in the context of the migration of a group of cells is not well understood. Using border cells in the Drosophila ovary as a model system for collective migration, this study found that the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) is required for a polarized distribution of recycling endosome and exocyst in the leading cells of the border cell cluster. Interestingly, PVR signaled through the small GTPase Rac to positively affect the levels of Rab11-labeled recycling endosomes, probably in an F-actin-dependent manner. Conversely, the exocyst complex component Sec3 was required for the asymmetric localization of RTK activity and F-actin, similar to that previously reported for the function of Rab11. Together, these results suggested a positive-feedback loop in border cells, in which RTKs such as PVR act to induce a higher level of vesicle recycling and tethering activity in the leading cells, which in turn enables RTK activity to be distributed in a more polarized fashion at the front. Evidence is also provided that E-cadherin, the major adhesion molecule for border cell migration, is a specific cargo in the Rab11-labeled recycling endosomes and that Sec3 is required for the delivery of the E-cadherin-containing vesicles to the membrane (Wan, 2013).

It has been proposed that repeated cycles of endocytosis of RTKs (or active RTKs) and recycling of them back to the membrane would effectively concentrate active RTK in the front of the migrating border cells. However, if the levels of endocytic recycling remain uniform in all the outer border cells during migration, a fast amplification of RTK activity levels between front and back would be difficult to achieve. This study shows that there is a polarized endogenous distribution of the recycling endosome and exocyst in the leading border cells within the migrating cluster, which could conceivably make such amplification faster and more efficient in the leading cells. It was also shown previously that Sec15-GFP has an asymmetric localization at the front, when it is overexpressed in border cells. Along their migrating route, the border cells often tumble or rotate as a cluster, resulting in position changes such as front cells becoming lateral and back cells and vice versa. In such a scenario, a fast and robust amplification process would be essential to relocalize active RTKs. Indeed, this study found that overexpressing Sec3 or Rab11-GFP, but not Sec5-GFP, in a single cell clone within a mosaic border cell cluster significantly promotes the likelihood of such a cell being positioned at the leading position, suggesting that this cell utilizes its increased recycling and tethering to amplify and relocalize active RTKs faster and more efficiently than other wild-type neighbor cells. The difference in promoting effect from Sec3 and Sec5 is interesting, suggesting that when overexpressed the Sec3 subunit is more able to enhance the overall exocyst function than Sec5. This is consistent with a Sec3 study in budding yeast, which shows that as a unique subunit of exocyst Sec3 serves as a spatial landmark on the bud tip to recruit a subcomplex (comprising seven subunits) of exocyst containing all subunits but Sec3. Only when the subcomplex along with the associated vesicle arrives at the bud tip, can Sec3 be joined with it to form a fully functional tethering complex (Wan, 2013).

The next question is how the polarized distribution of recycling and tethering activity is initiated in border cells. This study demonstrated that this was likely to be induced by the guidance receptors in response to the external gradient of guidance cues, as removing guidance signaling by DN-PVR and DN-EGFR expression abolished Rab11 and Sec5 polarized distribution, and DN-PVR expression alone markedly reduced the polarization. These data suggested the presence of a positive feedback loop of active RTKs-endocytic recycling-active RTKs in border cells, as Rab11 and exocyst components (Sec3 and Sec15) were shown to be conversely required for polarized pTyr or active RTK localization at the front. Interestingly, this study found that PVR signals downstream through Rac and then polymerized actin to promote recycling endosome levels, providing mechanistic details to this feedback loop. Interestingly, it was recently shown that Rab11 interacts with Rac and actin cytoskeleton regulator moesin during border cell migration. Furthermore, this study found that strong Rab11 stainings were proximal to or partially overlapping with strong F-actin staining in the leading edge of wild-type border cells and around the ectopic F-actin regions in the λ-PVR, RacV12 or twinstar- RNAi expressing follicle cells and border cells. F-actin appears to be the direct cause rather than the effect of recycling endosome accumulation, because manipulating its levels by Lat-A or twinstar RNAi leads to either up- or downregulation of the levels of recycling endosome. However, the possibility cannot be ruled out that Rac can somehow act on recycling endosome-associated regulators directly (independently of F-actin) to affect their function. It was previously shown that actin polymerization is required for recycling of cargo back to plasma membrane, possibly through F-actin serving as a track for the movement of vesicles. However, how F-actin induces recycling endosome formation and organization is not clear and remains to be elucidated (Wan, 2013).

It was previously proposed that recycling of active RTKs needs to be directional (toward the front) to achieve polarized RTK activity. If active RTKs in the leading edge are endocytosed and then recycled to new regions in the membrane, RTK activity would be delocalized. What causes the recycling to be directed toward the front membrane is not clear. The proposed feedback loop via F-actin suggests that the active PVR (RTK) in the leading edge could locally induce higher levels of recycling endosome through Rac and enhanced actin polymerization (by Rac). As a result, the directional recycling could be achieved with the localized actin filaments serving both as a recycling endosome inducing agent and as tracks for movement of vesicles (carrying active RTKs) toward the front membrane, which prevents the active RTKs from being recycled to elsewhere and becoming delocalized. Indeed, inhibiting actin polymerization in the border cells by Lat-A treatment abolished both the polarized F-actin and the elevated Rab11 stainings proximal to F-actin, which are normally present in the leading edge of the wild-type cluster. Lastly, this work also provides some insight into the kinds of cargo that are recycled during border cell migration. E-cadherin is a specific cargo. E-cadherin is the major adhesion molecule required for border cell migration, whereas integrin plays only a minor role and is not required in border cells (Wan, 2013).

These finds suggests that cycles of endocytosis and recycling of E-cadherin could promote the dynamic assembly and disassembly of E-cadherin-mediated adhesion on the substrate (nurse cell E-cadherin), similar to how the turnover of integrin at the focal adhesion is regulated by endocytic recycling in mammalian cells. Interestingly, elevated intracellular E-cad stainings tended to be localized below the cell membrane that juxtaposes nurse cell membrane, suggesting that E-cadherin is normally delivered to or recycled back to this membrane region by Rab11 and exocyst during adhesion and migration. Another important candidate cargo to be determined is PVR. However, no significant colocalization was detected between Rab11 with PVR or active PVR with the previously reported PVR or pPVR antibody. Therefore, the definitive role of PVR or active PVR as a cargo for recycling still awaits further determination (Wan, 2013).

Coordination among multiple receptor tyrosine kinase signals controls Drosophila developmental timing and body size

In holometabolous insects, metamorphic timing and body size are controlled by a neuroendocrine axis composed of the ecdysone-producing prothoracic gland (PG) and its presynaptic neurons (PGNs) producing PTTH. Although PTTH/Torso signaling is considered the primary mediator of metamorphic timing, recent studies indicate that other unidentified PGN-derived factors also affect timing. This study demonstrates that the receptor tyrosine kinases anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), function in coordination with PTTH/Torso signaling to regulate pupariation timing and body size. Both Alk and Pvr trigger Ras/Erk signaling in the PG to upregulate expression of ecdysone biosynthetic enzymes, while Alk also suppresses autophagy by activating phosphatidylinositol 3-kinase (PI3K)/Akt. The Alk ligand Jelly belly (Jeb) is produced by the PGNs and serves as a second PGN-derived tropic factor, while Pvr activation mainly relies on autocrine signaling by PG-derived Pvf2 and Pvf3. These findings illustrate that a combination of juxtacrine and autocrine signaling regulates metamorphic timing, the defining event of holometabolous development (Pan, 2021).

Body size is one of the most important traits of a multicellular organism. In species whose growth is determinate, the body growth of an individual is largely completed when it matures into an adult. A good example of determinate growth is found among holometabolous insects, such as the fruit fly Drosophila melanogaster. During development, the size of a Drosophila larva increases 100-fold during its three molts, but it does not change after metamorphosis, the developmental stage that transitions the juvenile larval form into the sexually mature adult fly. Therefore, the control of metamorphic timing is a key factor that regulates final body size (Pan, 2021).

In the past decades, numerous studies in Drosophila and other holometabolous insect species have demonstrated that the onset of metamorphosis is regulated through a neuroendocrine signaling axis composed of two central information processing nodes: the prothoracic gland (PG), which produces the metamorphosis inducing steroid hormone ecdysone (E), and a bilateral pair of brain neurons, the PG neurons (PGNs), that innervate the PG and release the neuropeptide PTTH that stimulates E production. After release into the hemolymph, E is taken up by peripheral larval tissues through a specific importer (EcI) and then converted into its active form, 20-hydroxyecdysone (20E), by the enzyme Shade. Subsequently, 20E stimulates metamorphosis via activation of the EcR/Usp receptor complex and stimulation of tissue-specific downstream transcriptional cascades (Pan, 2021).

In this scheme, PTTH functions as a trophic hormone to stimulate PG growth and E synthesis. In PG cells, PTTH binds to Torso, a receptor tyrosine kinase (RTK) family member, and stimulates the E biosynthetic pathway via Ras/Erk signaling. As the two central nodes on the neuroendocrine axis, both the PG and the PGNs receive additional diverse internal and external signals to modulate their output appropriately. For instance, the PG cells respond to insulin signals reflecting the general nutritional state. In addition, systemic bone morphogenetic protein (BMP) signals help coordinate metamorphosis with appropriate imaginal disc growth. The PGNs in turn, receive presynaptic inputs from various upstream neurons that regulate circadian and pupation behaviors. They also respond to tissue damage signals to delay maturation onset until the damage is resolved (Pan, 2021).

Although it is widely accepted that PTTH is the key neuropeptide that triggers developmental maturation in holometabolous insects, several studies indicate that additional timing signals are also likely. The first suggestion that PTTH is not the sole prothoracicotropic signal came from PGN ablation studies in Drosophila where it was found that up to 50% of animals with no PGNs still undergo metamorphosis, but after a prolonged ~5-day developmental delay. Subsequently, it was found that genetic null mutations in the Drosophila PTTH gene only produced a 1-day developmental delay and had little effect on viability. In this case electrical stimulation of the mutant PGNs restored proper timing while inactivation produced a more substantial 2-day delay. Ptth null mutants have also been generated in Bombyx mori, and while most animals arrest development at late larval stages, a fraction still escape and produce adults. Taken together, these studies strongly indicated that the PGNs produce additional timing signals besides PTTH (Pan, 2021).

RTK family receptors have been speculated to mediate the additional PGN signal, since blocking the Ras/Erk pathway in the PG causes strong developmental defects, phenocopying the PGN ablation model rather than the ptth mutant. Epidermal growth factor receptor (Egfr) has recently been implicated in regulating PG tissue growth, E synthesis, and secretion. However, the Egfr pathway is activated by autocrine signals from the PG, which does not involve the activity of PGNs. In the present study, two additional RTK family receptors, anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), were identified that play important roles in the PG controlling metamorphic timing. Interestingly, the Alk ligand Jelly belly (Jeb) and Pvr ligand Pvf3 are both expressed in the PGNs, verifying that the prothoracicotropic function of PGNs is mediated by multiple signaling molecules, while Pvf2 and Pvf3 are also expressed in the PG itself and likely provide additional autocrine signals that also contribute to metamorphic timing control (Pan, 2021).

In previous studies, RTKs, that is, Torso, and Egfr, have been demonstrated to be crucial in the PG for the control of pupariation and body size. This work identified two additional RTKs, Alk and Pvr, that are also required for proper timing and body size control. Suppression of either Alk or Pvr compromises E synthesis in the PG, delays pupariation, and increases pupal size, while moderate activation of Alk or Pvr accelerates development. The biological functions of Alk/Pvr in the neuroendocrine pathway are similar to those of the other RTKs, indicating likely signal coordination among the receptors. Downstream signaling from Torso, Egfr, Alk, and Pvr all involve activation of Ras/Erk signaling, while InR and Alk can also stimulate the PI3K/Akt pathway. Consistent with the signaling pathway convergence, suppression of Alk and Pvr simultaneously or suppression of Alk/Pvr in ptth mutants exhibits prolonged delay of developmental timing and larger pupal size. In addition, activation of Alk/Pvr rescues the developmental defects of ptth mutants, while activated Alk rescues the delay of InRDN overexpression. In total, both the downstream signaling pathway convergence and the additive effects of receptor activation/suppression support the coordination of signaling among these RTKs (Pan, 2021).

Cellular level coordination of receptor-mediated signals is very common during development. The PG is a good example of this coordination, which integrates a large variety of signals, such as insulin, PTTH, Hedgehog, Activin, BMP, serotonin, and octopamine, to precisely control hormonal output. The coordination among receptors of the same class is of special interest. At least five RTKs (InR, Torso, Egfr, Alk, and Pvr) are expressed in the PG, all of which activate the Ras/Erk pathway. Although PTTH/Torso has been considered the key tropic signal for PG function, it appears that three of the other RTKs can partially replace Torso to maintain some level of PG E production. Loss of either the Torso, Alk, or Pvr signal causes developmental delay but does not block pupariation. Even considering that loss of Egfr in the PG causes arrest at the L3 stage, Egfr is still dispensable during the first two molts, which also require production of E pulses by the PG. These observations lead to an open question: why does the PG utilize multiple signals that appear to function redundantly (Pan, 2021)?

An obvious possibility is that multiple timing signals provide both robustness and flexibility in response to variable developmental conditions. For example, given a choice of diets, Drosophila larvae chose one that maximizes developmental speed over other life-history traits. This is not surprising given the ephemeral nature of rotting fruit, a primary food source for Drosophila. Thus, multiple signals may enable larvae to maximize developmental speed. Another possibility is that the different signals contribute to different temporal aspects of the developmental profile. For example, perhaps none of the receptors alone can achieve a strong enough Ras/Erk activation in late-stage larva that meets the demand for the large rise in E production that triggers wandering and initiation of pupation. Interestingly, the expression of Egfr, Alk, and Pvr all increase remarkably during the late L3 stage when both Halloween gene expression and E synthesis ramps up, suggesting that the three receptors may function as supplements to Torso in order to achieve robust Ras/Erk activation and stimulation of E production (Pan, 2021).

Yet another possibility is that in addition to Ras/Erk signaling, each receptor may induce other downstream pathways. For instance, it has been previously reported that regulated autophagy induction in the PG is a key mechanism that prevents precocious non-productive pupation by limiting E availability if larva have not achieved critical weight (CW) (Pan, 2019). In that report, it was also demonstrated that after CW, autophagy inducibility is greatly repressed. This makes sense from a developmental perspective because if food becomes limiting after CW is achieved, it is likely disadvantageous to slow development down by limiting E production. Therefore, a mechanism to shut down autophagy inducibility after attainment of CW may be beneficial and, in this study, it was found that Alk activation is, in part, responsible for shut down of autophagy activation in the PG after the CW nutrient checkpoint has been surpassed (Pan, 2021).

Manipulations of Alk and Pvr, but not Torso, signaling in the PG led to the discovery that Jak/Stat activation can also affect developmental timing. A distinct feature of Alk and Pvr is that they can exert opposite effects on development likely depending on the activation strength. Weak activation of Alk or Pvr in the PG facilitates pupariation, while strong activation results in the arrest of development at various larval stages due to Jak/Stat activation. Using a weak spok-Gal4 driver led to overgrowth of the PG and to atypical morphology. Tissue overgrowth is commonly observed when either PI3K/Akt or Ras/Erk is hyperactivated in the PG; however, neither pathway induces atypical morphological change in the overgrown PGs or developmental arrest, which was observed when Alk or Pvr are hyperactivated, especially with the strong phm-Gal4 driver. Since suppression of Jak/Stat rescues the developmental arrest caused by phm-Gal4-driven Alk/Pvr hyperactivation, it appears that Jak/Stat signaling is the key factor that mediates the side effect of Alk/Pvr activation on PG morphology and developmental timing. At lower levels of activation as found in the spok>AlkCA and spok>PvrCA, many larvae still manage to pupariate, suggesting that larvae can tolerate a certain level of ectopic Jak/Stat signaling caused by Alk/Pvr activation. What goes wrong at a high level of activation of Jak/Stat is still not clear (Pan, 2021).

At present, it is not known what the endogenous late Jak/Stat signal contributes in terms of PG function since knockdown with available reagents did not produce a significant phenotype. In Drosophila, the canonical Jak/Stat signaling pathway is commonly induced by a group of cytokines including unpaired 1-3 (Upd1-3) via their cognate receptor Domeless (Dome). However, it has also been reported that Torso and Pvr are capable of inducing Jak/Stat activation in some circumstances. Although induction of Jak/Stat signal by overexpressing wild-type Torso was not observed in the PG, this might be due to a weaker activation using wild-type Torso overexpression versus gain-of-function tor^Y9 and tor^RL3 mutants as used in the previous study. Since this study observed Dome expression and endogenous activation of the 10xStat92E-GFP reporter in late L3 PGs, it is assumed to be likely to play some role at this stage. Whether the Jak/Stat activation is through Alk/Pvr or via reception of canonical Upd/Dome signals is not clear. Interestingly, note that Upd2 is secreted from the fat body into hemolymph and therefore may provide a nutrient storage signal to the PG that could be an important regulator of developmental timing, perhaps under certain types of non-standard laboratory growth conditions. It has also been recently demonstrated that inflammation-triggered release of Upd3 acts on the PG to produce developmental delay, indicating that the Jak/Stat pathway may be an important sensor for imbalance of various types of physiological processes (Pan, 2021).

Since its discovery, PTTH has been recognized as the most important prothoracicotropic neuropeptide that triggers metamorphosis in holometabolous insects. In some species, such as Bombyx mori, additional prothoracicotropic neuropeptides such as orcokinin and FXPRL-amide peptides have been discovered; however, PTTH, insulin-like peptides (Ilps), and serotonin are the only known brain-derived PG tropic hormones in Drosophila. Nevertheless, analysis of the Drosophila ptth null mutant phenotype verses PGN ablation and PGN electrical manipulation provided evidence that there are other tropic signals derived from the Drosophila PGNs. The observations described in this study demonstrate that the Alk ligand Jeb and the Pvr ligand Pvf3 are produced in the PGNs. Knockdown of jeb in the PGNs causes delay of pupariation and increased pupal size, phenocopying the phm>AlkRNAi animals and showing that the PGNs are the major source of Jeb that functions in the PG. Depletion of Pvf3 in the PGNs does not significantly affect developmental timing, which is not a surprise since it was found that Pvf2 and Pvf3 are also produced in the PG itself. Overexpression of Jeb or Pvf3 in the PGNs did not influence timing either, indicating that the neural activity of PGNs and/or the temporal regulation of Alk/Pvr expression plays the dominant role in the regulation of signaling by these factors. It is also pointed out that the combined knockdown of both ptth and jeb or ptth, jeb, and Pvf3 in the PGNs still does not produce the ~4- to 5-day developmental delay exhibited by larvae in which the PGNs are ablated, likely signifying that the additional developmental delay produced by PGN ablation is due to elimination of some other non-RTK-mediated neuropeptide signals (Pan, 2021).

Besides the well-established role of the PGNs in regulating developmental timing and body size, several recent studies also indicate that autocrine signaling within the PG itself provides important developmental regulatory cues. This signaling was first documented for biogenic amine signaling but more recently was extended to include the RTK Egfr and its ligands Vein and Spitz. Interestingly, the expression levels of Vein and Spitz in the PG increase in middle to late L3 and may not contribute to CW determination, but instead they respond to it to form part of a E feedforward circuit that helps ramp up hormone production during late L3 in anticipation of the large pulse that drives pupation. Similarly, since expression of both Pvf2 and Pvf3 was observed in the late L3 PG, and since knockdown of Pvf2 and Pvf3 simultaneously in the PG causes delay of pupariation and larger pupal size, these ligands together with their receptor Pvr also appear to form an autocrine signaling pathway. Expression of Pvf2/3 has also been observed in other tissues/cell types such as fat body, salivary gland, and hemocytes. Whether these sources also provide some input to the PG is not clear. This study also found that overexpression of Pvf2 or Pvf3 did not cause accelerated development. This is in stark contrast to the case of Egfr signaling in which overexpression of Vein or Spitz advances pupariation significantly. This finding indicates that the activity of Pvr signaling may depend on the expression of Pvr receptor and/or the release of ligands, rather than ligand expression. Endogenous Pvf2 expression is limited to the late L3 stage, yet Pvf3 is constitutively expressed in the L3 stage. The biological significance of the differentially regulated Pvf ligand expression is still an open question. It is noteworthy that there are three Pvr isoforms produced by alternative splicing among the exons coding the ligand-binding domain. Thus, reception of different Pvf ligand signals could very much depend on the levels and timing of receptor isoform expression in the PG. Lastly, it is noted that neither Alk nor Pvr accumulates to substantial levels on the PG membrane until after CW. Thus, similar to Egfr signaling, their primary functions likely control post-CW events. What regulates the post-CW membrane localization of these receptors is not yet clear, but it is interesting to speculate that the process might be one of the first downstream responses to surpassing the CW checkpoint that prepares the PG gland for a major acceleration in hormone production (Pan, 2021).

REGULATION

Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors

Blood progenitors within the lymph gland, a larval organ that supports hematopoiesis in Drosophila melanogaster, are maintained by integrating signals emanating from niche-like cells and those from differentiating blood cells. The signal from differentiating cells has been termed the 'equilibrium signal' in order to distinguish it from the 'niche signal'. Earlier work showed that Equilibrium signaling utilizes Pvr (the Drosophila PDGF/VEGF receptor), STAT92E, and Adenosine deaminase-related growth factor A (ADGF-A). Little is known about how this signal initiates during hematopoietic development. To identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling, a genetic screen was performed that identified bip1 (bric a brac interacting protein 1) and Nucleoporin 98 (Nup98) as additional regulators of the equilibrium signal. The products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr (Mondal, 2014: PubMed).

The screen described in this study identified Nup98 as a potential equilibrium signaling component because its knockdown in differentiating cells specifically causes a loss of progenitors cells. Although Nup98 is widely known as a general component of the nuclear pore complex, recent work has demonstrated that Nup98 and other nuclear pore components such as Sec13 and Nup88, can regulate gene expression through the binding of target promoters. Moreover, chromatin immunoprecipitation experiments identified bip1, RpS8, and the equilibrium signaling genes Pvr and STAT (STAT92E) as in vivo Nup98 regulatory targets. Consistent with a function in regulation of equilibrium signaling genes, Nup98 knockdown specifically in differentiating cells of lymph glands causes a strong reduction in Pvr expression. By contrast, RNAi knockdown of the nucleoporin Sec13 in differentiating cells has no effect on the maintenance of progenitor cells or Pvr expression underscoring the specific role of Nup98 in Pvr expression control. Furthermore, the close genetic relationship between Nup98 and Pvr is illustrated by the fact that single-copy loss of these genes in combination causes extensive loss of progenitor cells to differentiation. Interestingly, overexpression of bip1 in Nup98 RNAi lymph glands is sufficient to restore Pvr protein expression and to suppress the loss of progenitors to differentiation (Mondal, 2014).

Knockdown of bip1, Nup98, or RpS8 in differentiating cells each causes a strong reduction in Pvr expression in the lymph gland. The interpretation of this common phenotype is that each gene works in the equilibrium signaling pathway to control Pvr expression, although an alternative hypothesis is that the loss of Pvr expression is a common feature of highly differentiated lymph glands and is not specifically related to the function of these genes. To test this, Pvr expression was examined in collier (col) mutant lymph glands, which lack niche signaling and are strongly differentiated by late larval stages, and was found to be normal, compare with Pvr expression in wild-type cortical zone differentiating cells. Thus, Pvr requires bip1, RpS8, and Nup98 for proper developmental expression in the lymph gland (Mondal, 2014).

Several genetic screens, including overexpression and enhancer/suppressor screens of mutant or tumor phenotypes, have been conducted in the fly hematopoietic system; however, the screen described in this study represents the first loss-of-function screen targeting normal developmental mechanisms throughout the lymph gland. This was accomplished with the development and use of the pan-lymph gland expression tool HHLT-gal4 to drive UAS-mediated RNAi, which identified 20 different candidate genes that cause a loss of progenitor cells when knocked down within the lymph gland. From subsequent analyses using lymph gland zone-restricted Gal4 driver lines, a model is proposed in which Bip1, RpS8, and Nup98 are required in differentiating blood cells upstream of Pvr to control its expression and function in the equilibrium signaling pathway that maintains blood progenitors within the lymph gland. Future analyses will be required to identify additional components of this important signaling pathway and to provide more information about how equilibrium signaling interacts with other pathways in the control of blood cell progenitor maintenance, cell fate specification, and proliferation (Mondal, 2014).

The Pvr receptor, with its numerous developmental roles, is arguably one of the most important members of the Drosophila RTK family, yet most of what is known about Pvr stems from analyses of how it works in the context of intracellular signaling. Little is known about how Pvr gene or protein expression is regulated. Importantly, the work described in this study sheds new light upon this issue by demonstrating a role for bip1, RpS8, and Nup98 in the regulation of Pvr expression. The data and that of others suggest that this regulation of Pvr is likely taking place at the gene level, although other mechanisms are also possible. Ribosomes are required for protein translation, however specific ribosomal components or subunits may selectively stabilize transcripts and/or mediate preferential translation, while nucleoporins control both nuclear entry of regulatory proteins and the exit of mRNAs to the cytoplasm, and specific subcomponents are known to exhibit differential functions in this regard. Thus, RpS8 and Nup98 may selectively affect Pvr expression post-transcriptionally through transcript stabilization, transport, and translation. Although the specific mechanisms of molecular control of Pvr expression by bip1, RpS8, and Nup98 remain to be determined, their function is clearly critical in mediating proper equilibrium signaling and, therefore, proper blood progenitor maintenance within the lymph gland. The finding that bip1 regulates Pvr expression in the context of hematopoietic equilibrium signaling represents the first functional association for bip1 in Drosophila. The predicted Bip1 protein exhibits only one recognizable structural sequence, namely a THAP domain that contains a putative DNA-binding zinc finger motif. The results suggest that Bip1 behaves as a positive regulator of Pvr transcription, but whether this occurs directly through Bip1 interaction with the Pvr locus will require further investigation (Mondal, 2014).

Understanding how progenitor cell maintenance and homeostasis is controlled over developmental time is crucial for understanding normal cellular and tissue dynamics, especially in the context of ageing or disease. The identification of Bip1 and Nup98 as regulators of hematopoietic progenitors in Drosophila may be indicative of important conserved functions of related proteins within the vertebrate blood lineages similar to what has been shown previously for GATA, FOG, and RUNX factors. THAP-domain proteins are conserved across species and have been reported to have a variety of important functions in mammalian systems, including maintenance of murine embryonic stem cell pluripotency. What role, if any, THAP-domain proteins have in vertebrate blood progenitor maintenance (or hematopoiesis in general) remains to be established. Likewise, Nup98 has not been implicated in any normal hematopoietic role despite being a well-studied protein in other contexts (Mondal, 2014).

With regard to the diseased state, mutations in the human THAP1 gene have been associated with dystonia, a neuromuscular disorder that causes repetitive, involuntary muscular contraction, and THAP1/Par4 protein complexes have been shown to promote apoptosis in leukemic blood cells in various experimental contexts in vitro. Chromosomal translocations that generate Nup98 fusion proteins have been implicated in numerous human myelodysplastic syndromes and leukemias, further underscoring the need to explore Nup98 function in the hematopoietic system. Therefore, the study of bip1 and Nup98 in Drosophila, a powerful molecular genetic system, will likely be of benefit to understand the function of related vertebrate genes in normal and disease contexts (Mondal, 2014).

Signaling downstream of Pvr

Similar to the Drosophila Egfr and to the mammalian PDGFR family, stimulation of PDGF- and VEGF-receptor related (Pvr) activates the MAP-kinase pathway in Schneider cells as well as in border cells. However, it has been shown, by loss-of-function and gain-of-function experiments, that MAP-kinase signaling does not affect border cell migration. In addition, no effect of phospholipase C-gamma (PLC-gamma) or phosphatidylinositol 3' kinase (PI3K) has been demonstrated on this migration, using loss-of-function mutants (PLC-gamma) or border cell expression of dominant negative and dominant activated forms (PI3K). This was somewhat unexpected, since PLC-gamma and PI3K have been implicated in motility and guidance effects of RTKs (in particular PDGFR) in tissue culture cells. To address how Pvr signaling might be affecting cell migration in vivo, the effect of Pvr signaling on cell morphology and cytoskeleton was tested. In border cells as well as in other follicle cells, expression of lambda-Pvr has a dramatic effect on the actin cytoskeleton. Massive F-actin accumulation, actin-rich extensions, and changes in cell shape were produced in lambda-Pvr expressing follicle cells. The normal cells have modest cortical F-actin accumulation. This result was likely to be relevant to the guidance function of Pvr, because direct control of F-actin accumulation would allow receptor activation to control cell migration (Duchek, 2001b).

The actin cytoskeleton has been shown to be affected by small GTPases of the Rho superfamily in many systems, with the exact effects depending on the cellular context. In the border cell migration system, Rac is an attractive candidate for mediating the effect of activated Pvr, since dominant negative Rac (RacN17) has been shown to inhibit border cell migration (Murphy, 1996). Epistasis experiments could not be done by quantifying border cell migration because activated Pvr and dominant negative Rac have the same effect. Instead, whether Rac is required for the effect of Pvr on the actin cytoskeleton in follicle cells was tested. Coexpression of dominant negative Rac suppresses the effect of activated Pvr on the actin cytoskeleton. In addition, follicle cells expressing activated Rac (RacV12) have dramatic accumulation of F-actin, resembling that caused by activated Pvr. Finally, if Rac were directly downstream of Pvr, one would expect activated Rac to inhibit border cell migration, as observed for the activated receptor. Although a previous study reported that activated Rac does not affect border cell migration (Murphy, 1996), this was reexamined using the slboGal4 driver and it was found that activated Rac completely blocks border cell migration. These results are consistent with a role of Rac in the guidance pathway downstream of Pvr (Duchek, 2001b).

In mammalian tissue culture cells, PDGF stimulation can cause Rac-dependent F-actin accumulation, suggesting that the effect observed in follicle cells may reflect a conserved pathway. PI3K has been implicated as a mediator of the effect of PDGFR on Rac in Swiss 3T3 cells. However, PI3K does not appear to play a key role in guidance of border cell migration as discussed above. To investigate how Pvr might lead to activation of Rac, two groups of Drosophila mutants were tested for their effect on border cell migration: mutants in genes shown to be downstream of receptor tyrosine kinases in other contexts, and mutants linked to Rac activation. Most mutations were homozygous lethal, so their effect in border cells was tested by generating mutant clones in a heterozygous animal (mosaic analysis). Of the 8 genes tested, only myoblast city (mbc) has a detectable effect on border cell migration. Mbc is homologous to mammalian DOCK180 and C. elegans CED-5. Mbc/DOCK180/CED-5 acts as an activator of Rac (Kiyokawa, 1998; Nolan, 1998; Reddien, 2000, cited in Duchek, 2001b).

mbc has been independently identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. slbo mutant border cells migrate poorly. Increased expression of mbc in slbo mutant border cells improves their migration, suggesting that mbc has a positive role in promoting border cell migration. Mbc protein is detected in follicle cells, including border cells, and is overexpressed upon induction of the EP element EPg36390 located upstream of mbc. Removing mbc function from border cells by generating mutant clones causes severe delays in their migration. At stage 10, when 100% of control (GFP) clones have reached the oocyte, only 10% of mbc mutant border cell clusters have done so, and these are the oldest egg chambers. Thus, mbc is not absolutely required for border cell migration, but, contrary to the other genes implicated in RTK and Rac signaling, loss of mbc function severely impairs this cell migration (Duchek, 2001b).

To test whether Mbc can act downstream of Pvr, focus was placed on the effect of lambda-Pvr on F-actin accumulation in follicle cells. In order to obtain mbc mutant clones in egg chambers that express lambda-Pvr (constitutively active Pvr), the experiment was done slightly differently from that for dominant negative Rac. Expression of lambda-Pvr under control of slboGal4 causes disruption of centripetal cell morphology and abnormal actin accumulation. When follicle cells are mutant for mbc, this effect is strongly attenuated, indicating that Mbc acts downstream of Pvr. Taken together, these results suggest that Pvr affects guidance of border cell migration, at least in part, by signaling through Mbc to Rac, which then controls F-actin accumulation (Duchek, 2001b).

Invasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells (BCs) exit the follicular epithelium to perform a stereotypic, invasive migration. The ETS transcription factor Yan is required for border cell migration and Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor tyrosine kinase pathways (Egfr and Pvr) in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. These results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells (Schober, 2005).

Interestingly, Yan expression levels gradually decrease as BCs move along an increasing PVR/EGFR activity gradient. Yan has been shown to be phosphorylated by the EGFR-MAPK pathway, which triggers its nuclear export and protein degradation. Consistent with these previous studies, expression of dominant-active PVR and EGFR in BCs blocks BC migration and abrogates Yan protein expression, whereas yan transcript or enhancer trap expression is still detectable. Expression of activated Ras and Raf similarly induced Yan downregulation, consistent with an involvement of the canonical Ras/MAPK pathway in mediating PVR/EGFR signaling. It is noted, however, that although BC migration is significantly delayed upon ectopic expression of activated Ras, activated Raf hardly affects their ability to migrate. The basis of this difference, which might be due to complex feedback loops between the implicated signaling pathways, is unclear at the present time and will need to be investigated further (Schober, 2005).

awd, the Homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion

Border cell migration during Drosophila oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. This study shows that the metastasis suppressor gene homolog Nm23/awd (abnormal wing discs) is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin (Shibire or Shi). The functional relationship between awd and shi is highly specific and almost exclusive in the endocytic pathway. The functional relationship between Nm23/Awd and dynamin has prompted the suggestion that Nm23/Awd is a GTP supplier for dynamin, a GTPase. Nonetheless, the putative antimetastasis activity of Nm23/Awd has never been demonstrated in a physiologically relevant metastasis or epithelial to mesenchymal transition model. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion (Nallamothu, 2008).

This report describes a novel role of a negative regulator of directional migration in border cells. Specifically, the significance of developmentally regulated loss of Awd expression in border cells during their active migratory phase was studied. Ectopic expression of Awd effectively blocks border cell movement, suggesting that Awd is involved in modulating the directional movement of the border cell complex. Conversely, a high level of Awd expression was observed in the nonmigrating follicle cells, possibly promoting rapid turnover of surface receptors to prevent ectopic cell migration. Indeed, loss of awd in these cells and in premigratory border cells resulted in up-regulation of Pvr and stalling of border cells, consistent with the phenotype of overactive Pvr signaling reported previously observation of overexpression of wild-type Pvr. The results show that the function of Awd is to promote down-regulation of the surface receptors such as Pvr and Dome, in collaboration with Shi/dynamin, thereby regulating the chemotactic signal strength. Although the function of Awd has been linked to dynamin (Dammai, 2003; Krishnan, 2001), this report is the first on the relevance of the Nm23/Awd antimetastasis function in an analogous developmental model. This study has demonstrated that border cell migration is stalled by both ectopic expression of Awd in the migrating cells and knockdown of Awd in premigrating cells, although through opposite consequences of reducing and increasing Pvr expression, respectively. This is consistent with the published finding that both loss of function and gain of function in Pvr signaling can disrupt border cell migration, due to loss of chemotactic signal or overwhelming signal, respectively. It was proposed and subsequently demonstrated by time-lapse microscopy that border cells that are oversaturated with Pvf signaling spin around without moving forward, consistent with the overborne, nondirectional chemotactic signaling response; while pvr loss-of-function border cells do not move. That is, although the phenotypic outcomes are the same, the cellular behaviors of the two genetic conditions are just the opposite (Nallamothu, 2008).

At this time, the cellular events that precisely down-regulate Awd expression in migrating border cells remain unknown. However, the observations suggest that the regulatory mechanism, besides potential transcriptional regulation, could at least in part be posttranscriptional. For example, the slbo-GAL4 driver can usually induce very high levels of ectopic expression, as evidenced by the expression of UAS-{lambda}pvr in this study. However, with UAS-awd (without the endogenous 3' untranslated region), it was possible to achieve at best a level equal to the endogenous one in nearby follicle cells and very often much lower (Nallamothu, 2008).

The histidine-dependent phosphotransferase activity of Nm23/Awd has functional correlation with the production and usage of GTP and the Awd-GTP link is worth noting since dynamin is a GTPase. In a classic study to identify components of eye color pathway, one peculiar, otherwise healthy mutant caused dominant lethality in the viable eye color prune null mutant background (Sturtevant, 1956). This dominant conditional lethal allele was named Killer-of-prune (K-pn) and turned out to be a missense mutant allele of awd. This is highly interesting because the Drosophila eye pigmentation is determined by pteridines that is also a precursor of essential enzyme cofactors. The rate-limiting enzyme in pteridine biosynthesis is GTP cyclohydrolase, which uses substrate GTP to generate dihydroneopterin triphosphate. It was suggested that the Prune protein, which contains pyrophosphatase activity, stabilizes or promotes Nm23/Awd multimeric protein activity by channeling the phosphate. It is possible that Awd and Prune proteins together form a relay system for generating GTP. Therefore, the K-pn mutation of awd in the prune mutant background renders the phosphate transfer function of the Prune-Awd protein complex even less stable. Indeed, among the myriad of interacting proteins of Nm23 in mammalian cells, many are related directly or indirectly to the GTPases, such as Arf6, TIAM1 (a guanine exchange factor for Rac), Lbc (a guanine exchange factor for Rho), and Rad. Whether or not these GTPase-related functions hold true requires further in vivo investigation. Recently, the lysophosphatidic acid receptor EDG2 was found to be overexpressed in Nm23-H1 mutant metastatic breast cancer cells, which can account for the metastatic activity of this cell line. However, whether the up-regulation is a direct or downstream effect of Nm23 loss of function is not clear. It therefore remained to be determined whether the similar receptor down-regulation mechanism by Awd observed in this report is applicable to EDG2 regulation (Nallamothu, 2008).

It should be noted, however, that although the observed genetic interaction between awd and shi suggests that Awd may promote the endocytic activity of Shi/dynamin, it is formally possible that Awd may promote protein turnover that is downstream of the initial endocytic event. On this note, it is also worth considering other activities of Nm23/Awd. The results showed that substitution of the active-site histidine residue that is critical for the nucleoside diphosphate kinase activity could not stall border cell migration. This is consistent with previous finding that this residue is required for rescuing the enhancer of shi phenotype (Krishnan, 2001). Curiously, this residue is not required for suppressing the in vitro motility (assayed by Boyden chamber) of the metastatic breast cancer cells. However, the histidine substitutions employed in the two systems are different (phenylalanine in human versus alanine in fly). It is therefore difficult at this time to draw a direct comparison. In contrast, human mutants that affect the histidine-dependent protein kinase activity failed to suppress the in vitro motility of the cancer cells. So far, very few Nm23 protein kinase targets have been identified and none verified in physiological settings. Nonetheless, the protein kinase activity may be of specific functional significance since the range of targets is likely limited, so that specific pathways that contribute to metastasis may be identified more readily. The border cell migration model describe here should be used in future studies to test the functions of Nm23/Awd based on the above-mentioned human mutations (Nallamothu, 2008).

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. This study shows that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, it was demonstrated that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, these data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration (Assaker, 2010).

During migration, the cell needs to rearrange its cytoskeleton, its plasma membrane content, and its interaction with other cells. Many of these features can be controlled by vesicular trafficking. For example, Integrins, Cadherins, and other cell-cell or cell-matrix attachment proteins are transmembrane proteins tightly regulated by trafficking. Furthermore, the distribution of proteins and lipids at the plasma membrane is directly controlled by vesicular trafficking, as well as the localization of some actin remodeling proteins. During the process of border cell migration, endocytosis has been shown to regulate the polarity of RTK activity. This paper shows that the endocytic process plays a role in regulating the spatial localization of RTK activity by trafficking through the recycling endosome and by the polarized redelivery of endocytosed material to the plasma membrane (Assaker, 2010).

The key endocytic proteins previously involved in border cell migration - Sprint, Cbl, and Shibire - regulate the polarization of RTK activity during border cell migration. Different possible mechanisms are proposed to explain their action and have not been addressed in this landmark article (Jékely, 2005). Recently, it was shown that both the degradative pathway and the recycling pathway might be involved in this process. Thus, at least two models, which are not mutually exclusive, could explain the role of endocytosis in establishing this polarity. First, active RTKs could be endocytosed and degraded when diffusing away from the leading edge. Second, polarized recycling of endocytosed active RTKs could concentrate these active receptors at the leading edge. From the current experiments it can be concluded that the recycling of active RTKs or of a cofactor at the plasma membrane is necessary for border cell migration. Furthermore, it was demonstrated that the slow recycling route, through the recycling endosome, is used and that polarized redelivery at the plasma membrane is mediated by the exocyst subunit sec15 (Assaker, 2010).

It seems logical to think that RTKs, or active RTKs, are the cargo transported through this endocytic cycle. However, the identity of the protein being recycled remains to be determined. Indeed, there is no indication that RTKs are recycled. Immunofluorescence staining of the EGFR was performed. The signal obtained was diffuse and inconclusive in both control and rab11^SN-expressing border cells. In addition, if active RTKs were trafficking through the recycling endosome, the pTyr in endocytic vesicles would be expected to be marked by Rab11. However, such a colocalization has never been observed. These data do not rule out a potential recycling of RTKs, because they could be present in the recycling endosome in quantities below detection levels or in an inactive form. However, the data suggest that the main cargo of this trafficking cycle is of another nature. This cargo could be a plasma membrane diffusion barrier, because polarized cells, such as epithelial cells and neurons, maintain different membrane domains, which rely on such barriers: the tight junctions and the axon hillock, respectively. Moreover, diffusion barriers have been proposed to define plasma membrane domains in migrating cells. These diffusion barriers appear to be linked to the actin cytoskeleton, but their exact nature is unknown. Because E-cadherin is involved in cell migration, it would have been an ideal candidate to play such a role, but it is unaffected by ^rab11SN expression (Assaker, 2010).

Another possibility is that endocytosis acts indirectly. For example, it might regulate key components of the plasma membrane or of the cytoskeleton. Recent evidence has shown that endocytosis and recycling can play a critical role in creating a positive feedback loop during polarity establishment in the budding yeast. In this particular case, endocytosis is critical for the localization of regulators of small GTPases of the Rho family. Furthermore, mammalian Tiam1, a GDP-to-GTP exchange factor (GEF) for Rac, has been shown to localize to endosomes, leading to the loading of active Rac at the plasma membrane through an endocytic-recycling cycle. Interestingly, a robust genetic interaction has been found between Rab11 and Rac1, which directs border cell migration (Wang, 2010). Until now, two Rac-GEFs, Myoblast city and Elmo, have been involved in border cell migration, but not the Drosophila Tiam1 homolog still life. Further studies will be necessary to determine if Rac1 is the main cargo of the endocytic-recycling cycle that regulates border cell migration (Assaker, 2010).

In the past few years, trafficking via the recycling endosome has been involved in the establishment or rearrangement of cell polarity in various events. In particular, a role for the recycling endosome has been observed when a rapid and dramatic rearrangement of the cell organization is required, including cellularization, cell-cell boundary rearrangement, asymmetric cell division, and cell migration. Trafficking through the recycling endosome is an ideal mechanism to polarize a cell rapidly, because it hijacks material already available in the cell at a new location. Furthermore, it is a very efficient mechanism to reinforce polarity by feedback loops. Similarly the exocyst plays a key role in the majority of these cell polarizations. In the case of cell migration, the recycling endosome may transform the diffuse extracellular gradient of RTK ligand into a robust intracellular polarization of RTK activity that is crucial for directed migration (Assaker, 2010).

There is much evidence that the function of the recycling endosome in the regulation of directed migration is conserved in mammals. The mammalian homologs of Drosophila Rab11 are Rab11A and -B and Rab25. They have been directly implicated in the migration of cancerous cells and in the formation of metastasis, a cell migration event resembling border cell migration. Rab11 effectors are also involved in mammalian cell migration. More specifically, PDGF receptor-dependent cell migration has been shown to be regulated by endocytosis in a mammalian cell culture assay and the recycling endosome has been indirectly implicated in the regulation of migration guided by the EGFR. Given the involvement of the recycling endosome in so many processes, targeting its function to reduce metastasis is unlikely to be efficient. However, identifying the main cargo of this recycling cycle could help identify more specific targets for drugs blocking the formation of metastasis (Assaker, 2010).

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

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

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

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

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

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

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

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

ECM stiffness regulates glial migration in Drosophila and mammalian glioma models

Cell migration is an important feature of glial cells. This study used the Drosophila eye disc to decipher the molecular network controlling glial migration. Glial motility was stimulated by pan-glial PDGF receptor (PVR) activation, and several genes acting downstream of PVR were identified. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of integrin (see Myospheroid) results in ECM softening. Moreover, it was shown that lox expression is regulated by integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration (Kim, 2014).

Protein Interactions

Schneider cells were used to determine whether Pvf1 could bind to and activate Pvr. The anti-Pvf1 antibody detected a specific band of about 36 kDa in conditioned medium from Schneider cells. This appears to be secreted Pvf1, since a stronger signal at the same position was observed in conditioned medium from Schneider cells transfected with an expression construct for Pvf1 (pRm-Pvf1). For binding studies, a Pvf1-Alkaline Phosphatase (AP) fusion protein was produced in Schneider cells and harvested in conditioned medium. The Pvf1-AP fusion protein binds to Schneider cells in a Pvr-dependent manner, since binding is significantly decreased by pretreatment of the cells with Pvr dsRNA. The remaining binding of Pvf1-AP to cells may be nonspecific sticking or binding to other proteins. To look at consequences of Pvf1 binding to Pvr, MAP-kinase activation was monitored in recipient cells by anti-diphospho-ERK (anti-dpERK) staining. MAP-kinase is activated by conditioned medium containing Pvf1. This activation is abolished by prior treatment of the cells with Pvr dsRNA. Maximal activation was observed in 10 min. Thus, Pvf1 binds to Pvr, and Pvr activates the MAP-kinase pathway in Schneider cells (Duchek, 2001b).

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).

Hrs mediates downregulation of Pvr and other signalling receptors in Drosophila

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).

Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homolog, Vps27p, also have a UIM and bind to ubiquitin. The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Jékely, 2003 and references therein).

To determine whether Hrs is generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the Drosophila ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation at the cell cortex could also be observed. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).

An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27. Genetic studies in mice and Drosophila (Lloyd, 2002) have also shown that cells mutant for hrs have enlarged endosomes, possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes, and can also be used to specifically label endosomes. The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells is compared, an enlargement of FYVE-positive structures is observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).

Hrs affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs that were analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures were also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).

Cortactin modulates cell migration and ring canal morphogenesis downstream of Src and PVR during Drosophila oogenesis

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. Complete loss-of-function mutants of the single Drosophila Cortactin gene have been isolated. Mutants are viable and fertile, showing that Cortactin is not an essential gene. However, Cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In Cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). Accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. Evidence is presented that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the Cortactin gene not being essential for development (Somogyi, 2004).

A number of specific defects were observed in cortactin mutants. One cortactin phenotype is a mild defect in 'dumping', transfer of bulk cytoplasmic material from nurse cells to the oocyte. This phenotype is similar to that observed in Src64 mutants, but is also seen in other mutants. In the case of Src64, the defect is correlated to the presence of Src64 protein at the ring canals and to reduced size of the ring canals. Cortactin is also present at ring canals. To determine whether cortactin affects ring canal morphogenesis, their size was quantified at stage 10. Ring canal size is significantly reduced in the cortactin mutant relative to wild type. Dumping defects usually result in smaller eggs. Eggs from cortactin mutant mothers and from Src64 mutant mothers are on average smaller than wild type. The hatching rate of the eggs from mutant mothers is also decreased. Thus, mutations in cortactin affect the actin rich ring canals, and processes dependent on function of ring canals, in a manner similar to mutations in Src64. Mutations in components of the Arp2/3 complex (Arpc1 or Arp3 mutants) are also known to specifically affect ring canal morphogenesis (Somogyi, 2004).

There were several reasons to suspect that the effect of Cortactin on the actin cytoskeleton could be related to effects of RTKs. Border cell migration is guided by two RTKs, namely PDGF/VEGF receptor (PVR) and EGFR. Also, an activated form of PVR induces robust formation of actin-rich extensions in follicle cells in a Rac dependent manner. Finally, mammalian Cortactin has been suggested to act as a link between RTKs such as PDGF receptor and the actin cytoskeleton. Therefore the effect of PVR signaling on Cortactin protein was examined in the follicular epithelium. Overexpression of wild type PVR is sufficient to increase signaling in follicle cells slightly, resulting in a small increase in F-actin accumulation in the cell. This is most visible at the basal F-actin network. PVR overexpression also results in clear recruitment and/or stabilization of Cortactin at the cell cortex. Cortactin protein is not simply recruited by the increased amount of F-actin; the subcellular localization of Cortactin is distinct from that of F-actin. In addition, the level and the localization of other actin-associated proteins such as moesin and alpha-spectrin are not visibly affected by PVR overexpression. Expression of a constitutive active form of PVR (lambda-PVR) results in more robust F-actin accumulation but also disruption of the normal cell shape. The activated receptor is not restricted to the cell cortex but is present in vesicles throughout the cell. The constitutive active PVR also induces accumulation of Cortactin throughout the cell. Thus, PVR activation in follicle cells affects the accumulation and subcellular localization of Cortactin protein, primarily resulting in more Cortactin at the cell cortex of normal epithelial cells (Somogyi, 2004).

The ability of Cortactin to induce F-actin accumulation and filopodia formation in conjunction with the effect of PVR on Cortactin protein suggested that Cortactin might act downstream of PVR with respect to control of the actin cytoskeleton. To determine if this might be the case, an epistasis experiment was performred. The effect of activated PVR (lambda-PVR) on F-actin accumulation and cell shape in follicle cells was scored using three categories of severity. Quantification was done by blindly scoring severity of the phenotype in many egg chambers. In each experiment follicle cells that were mutant for cortactin were compared to a control, wild type background. A small, but statistically significant decrease was seen in the severity of the lambda-PVR induced phenotypes in the cortactin mutant background. This result is consistent with Cortactin acting downstream of PVR, but also shows that the effect of PVR on the actin cytoskeleton does not strictly require Cortactin. Another factor that appears to act downstream of PVR is the Rac activator Mbc (related to DOCK180 and Ced-5). In the same assay, removal of mbc has a much more pronounced effect. Activation of Rac can cause translocation of Cortactin to the cell periphery in mammalian cells. Thus, cortical Cortactin accumulation could be one of the downstream effects of Rac activation by PVR. In conclusion, Cortactin appears to contribute to the effects of PVR on actin, but is largely redundant with other factors. A contributing, but not essential, function is also consistent with cortactin not being an essential gene (Somogyi, 2004).

Expression of an activated form of Src (Src42^CA) in border cells and other follicle cells shows a phenotype similar to that of activated PVR. It completely blocks border cell migration and disrupts cell shape and the actin cytoskeleton of follicle cells. Src64 has a specific function in the female germ line, which coincides with a function of Cortactin as discussed above. Src42 appears to function more generally in somatic cells and is required for viability. Unfortunately, it is technically not feasible to make Src42 mutant clones to determine whether Src42 function is required in border cells. However, whether Cortactin might act downstream of activated Src42 could be examined by performing an epistasis experiment equivalent to that with activated PVR. Removal of cortactin results in a small, but significant, decrease in the severity of the activated Src-induced phenotype. This is consistent with a role of Cortactin downstream of Src. It also shows that Src can affect the cytoskeleton independently of Cortactin (Somogyi, 2004).

It is perhaps surprising that Cortactin in not an essential gene. Many modulators of essential processes in the cell such as dynamics of the cytoskeleton, cell adhesion or cell signaling are very well conserved in higher eukaryotes. They may add to the robustness and fidelity of the regulation, but they may only be essential to the organism if their absence completely changes the behavior or fate of specific, important cells. In mammals, there are often multiple closely related genes and simple redundancy between these gene products may explain an absence of phenotypes in knockout mice. In Drosophila, this type of simple redundancy is less frequent. For example, there is no evidence for another Cortactin gene in the sequenced Drosophila genome. However, more distantly related genes may have overlapping functions. Subtle phenotypes may also reflect that one process can be regulated in multiple ways. Combining multiple mutations can then be used to genetically help define which genes and pathways overlap in function (Somogyi, 2004).

Cbl and Sprint regulate early steps of RTK endocytosis

Guidance receptors detect extracellular cues and instruct migrating cells how to orient in space. Border cells perform a directional invasive migration during Drosophila oogenesis and use two receptor tyrosine kinases (RTKs), EGFR and PVR (PDGF/VEGF Receptor), to read guidance cues. Spatial localization of RTK signaling within these migrating cells is actively controlled. Border cells lacking Cbl, an RTK-associated E3 ubiquitin ligase, have delocalized guidance signaling, resulting in severe migration defects. Absence of Sprint, a receptor-recruited, Ras-activated Rab5 guanine exchange factor, gives related defects. In contrast, increasing the level of RTK signaling by receptor overexpression or removing Hrs, an endosome-associated, ubiquitin binding protein required for multivesicular body formation and degradation of RTKs, and thereby decreasing RTK degradation, does not perturb migration. Cbl and Sprint both regulate early steps of RTK endocytosis. Thus, a physiological role of RTK endocytosis is to ensure localized intracellular response to guidance cues by stimulating spatial restriction of signaling (Jekely, 2005).

It was reasoned that regulation of RTK turnover might be important to maintain a directional response in border cells, and the effects of mutations likely to affect this process were analyzed. Cbl is a ubiquitin ligase with a conserved role in regulating RTK signaling. Clones of border cells mutant for Cbl are correctly specified, as judged by staining for Slbo, a marker specific for differentiated border cells, but have severe migration defects. To explore the relationship between Cbl and RTK signaling, RTK signaling was manipulated in Cbl mutant border cells. The migration defect in Cbl mutant cells was suppressed by reducing the level of an EGFR ligand (grk/+) and was enhanced by overexpression of either receptor in border cells (UAS-PVR or UAS-EGFR. Note that overexpression of either PVR or EGFR alone has no effect. This indicates that the Cbl phenotype is not due to lack of guidance signaling but instead due to excessive, misregulated RTK signaling (Jekely, 2005).

Consistent with the interpretation that Cbl is required to restrict signaling, the complete failure of many Cbl mutant border cell clusters to migrate resembles the effect of increased guidance receptor signaling due to expression of constitutively active receptors or a strong ligand. In contrast, border cells lacking Pvr and Egfr all eventually initiate migration but never make it to the oocyte. Similar phenotypic effects of manipulating guidance cues have been observed by live imaging of germ cell migration in the zebrafish embryo, a migration guided by a G protein-coupled receptor: migratory cells lacking guidance cues migrate, but randomly, whereas the same cells subject to high uniform guidance cues did not migrate. Thus, border cells lacking Cbl responded as if they were receiving high uniform signaling, a situation that mimics the endpoint of migration (Jekely, 2005).

Mammalian Cbl proteins negatively regulate multiple RTKs by stimulating their ubiquitination and lysosomal degradation. The N-terminal phospho-tyrosine binding domain of Cbl directly binds to activated receptors, and ubiquitin-conjugating enzymes are recruited via the E3 type RING finger. The N-terminal part of Drosophila Cbl also physically interacts with autophosphorylated Pvr. In some assays, these conserved domains are sufficient for mammalian Cbl to regulate Egfr. Cbl can also interact with proteins regulating endocytosis as well as other signaling molecules through its less well conserved C-terminal region. Drosophila Cbl is expressed as two isoforms. Ubiquitous expression of either Cbl-L or Cbl-S, which lacks the C-terminal tail, rescues lethality associated with the Cbl null mutation as well as the migration phenotype. To determine whether E3 ligase activity of Cbl is required, a single cysteine residue essential for this activity was mutated to alanine (Cys-369, corresponds to Cys-381 in human Cbl). The Cbl ring finger mutant was unable to rescue viability of the Cbl mutant or migration of Cbl mutant border cell clones, showing that this function is essential for Cbl activity during migration (Jekely, 2005).

Overall level of RTK activity, in the cell or at the cell cortex, does not need to be precisely controlled to allow migration. Yet Cbl is apparently required to restrict RTK activity. This prompted examination of whether Cbl might affect subcellular localization of RTK signaling at a more refined level. Experiments with Hrs mutants and RTK overexpression suggest that total phospho-tyrosine might be used as a local indicator of RTK signaling. Many proteins are tyrosine-phosphorylated in cells by a number of kinases, but the RTKs have a quantitatively significant effect (direct and indirect). Remarkably, wild-type border cells initiating migration show a clear localization of phospho-tyrosine signal to the front. The front is the side facing the direction of subsequent migration and the source of the ligands, namely the oocyte. Since border cells migrate as a tight cluster of cells, several cells contribute to the front. To further test whether the signal reflected stimulation of endogenous RTKs, a strong Egfr ligand (secreted Spitz) was expressed in border cells to stimulate the endogenous receptor uniformly. This resulted in delocalized phospho-tyrosine signal all over the cortex of the border cells, and, as expected, a block in directed migration (70% nonmigrating clusters). This validated the use of phospho-tyrosine as a reasonable local readout of endogenous RTK activation (Jekely, 2005).

Endogenous Pvr and Egfr are detected at low uniform levels in border cells. Overexpression of Egfr or Pvr results in high level of receptor throughout the cluster; however, the phospho-tyrosine signal remains localized. Thus, local activation is maintained despite RTK overexpression. Consistent with signal location being the critical parameter for guidance signaling, directed migration also proceeds normally upon RTK overexpression. In contrast, border cells mutant for Cbl show a high frequency of delocalized phospho-tyrosine signal. These results indicate that Cbl is important in migrating cells because it is required to restrict RTK signaling spatially within the cell; without Cbl, signaling becomes delocalized. Since Cbl affects RTK endocytosis, whether perturbing endocytosis more generally would have the same effect was tested. Expression of a dominant-negative form of Shibire (dynamin) in border cells initiating migration also caused efficient delocalization of the phospho-tyrosine signal (Jekely, 2005).

The incomplete penetrance of the Cbl phenotype suggests that other molecules might partially compensate for the loss of Cbl. Indirect evidence is available that another potential RTK binding endocytosis regulator called Sprint might have a role in border cells. The mammalian counterpart of Sprint, called RIN1, displays Ras-activated Rab5 guanine nucleotide exchange factor (GEF) activity and can bind Egfr and stimulate Egfr activity. RIN1 also binds and activates the Abelson tyrosine kinase. To analyze the function of Drosophila sprint in vivo, sprint mutants, including a complete loss-of-function mutant, were generated. Despite sprint being the only rin1-related gene in Drosophila, homozygous sprint mutant flies are completely viable and fertile with normal oogenesis. To determine whether Sprint might contribute to regulating RTKs during border cell migration, the cells were challenged by overexpressing Pvr or Egfr in the mutant background. By itself, this overexpression has no effect on migration. In the sprint mutant background, however, RTK overexpression results in significant migration defects and, as for Cbl mutants, a corresponding increase in delocalized phospho-tyrosine signal. This suggests that Sprint might play a role similar to Cbl. Sprint might not be essential under normal conditions due to overlap in function with Cbl. To test this further, border cells mutant for both sprint and Cbl were analyzed. These cells have very severe migration defects and rarely reach the oocyte. For comparison, almost half the Cbl single mutant clusters reach the oocyte. Since sprint has barely any defect on its own, this strong enhancement of the Cbl phenotype is significant. Such a synergistic effect of two null mutants indicates that the gene products function in parallel to regulate the same process (Jekely, 2005).

To understand more about how Sprint might function in vivo, an antibody was generated that detects endogenous Sprint. In a pattern strikingly similar to the wild-type polarized phospho-tyrosine signal, endogenous Sprint was detected at the front of border cells initiating migration. This is consistent with Sprint being recruited to active RTKs. This was confirmed by the ability of overexpressed Egfr or Pvr to recruit endogenous Sprint. In overexpression experiments, it was also found that Sprint has the characteristics expected from its homology to RIN1: Sprint binds Ras-GTP recruited Abelson kinase to the cell cortex and associates with endocytic vesicles. Finally, endogenous Sprint accumulates at the apical cortex of follicle cells, contacting the oocyte, upon transient block of endocytosis. Since endogenous Egfr and Pvr ligands come from the oocyte, this supports the idea that Sprint dynamically associates with early endocytosis of RTKs at the cell cortex. Taken together with the genetic analysis, it is concluded that Cbl and Sprint both serve to maintain RTK signaling localized for guidance, although they stimulate early endocytosis events in molecularly unrelated ways (Jekely, 2005).

Regulators such as Cbl and Sprint might be recruited directly to activated and autophosphorylated RTKs or might bind indirectly, via phosphorylated adaptor proteins. A yeast two-hybrid assay was set up to detect possible direct, phosphorylation-dependent binding. The intracellular domain of Pvr is able to autophosphorylate in yeast and bind SH2 and PTB domain proteins. Binding was detected of both Cbl and Sprint to Pvr, but not a kinase-dead Pvr mutant. Potential docking tyrosines in Pvr were systematically mutated. Mutation of 16 or 14 (YF14) tyrosines results in strong decrease in binding of Cbl and Sprint. To map the binding sites, 5 tyrosines at a time were 'added back' to the YF14 mutant. Although there was no overlap in tyrosines, two of the resulting constructs regained full binding to Cbl and Sprint, indicating that both proteins have more than one direct binding site on Pvr. One construct (YFc) did not bind either Cbl or Sprint directly and therefore seemed to be a potentially useful tool to study the role of their direct binding to Pvr in vivo (Jekely, 2005).

To determine the signaling potential of each Pvr mutant in vivo, the mutations were placed in the context of a constitutively active form of Pvr (λ-Pvr) to induce full, unregulated activation. The ability to block border cell migration and induce F-actin accumulation was monitored. The activity of YF14 was strongly reduced, but each add back mutant had only slightly reduced activity relative to wild-type despite missing nine potential docking tyrosines. Thus, each of the add back mutants was still capable of signaling to affect migration and guidance when artificially activated (Jekely, 2005).

To determine whether the YFc mutations affected receptor regulation, they were placed in the context of full-length Pvr. From transgenes, Pvr and Pvr-YFc were expressed in the ovary at similar levels. As expected, the ability to activate signaling in border cells (measured by anti-dpERK staining) was quantitatively reduced in Pvr-YFc compared to Pvr. This result was confirmed using the sensitive MAP kinase-activated reporter gene kekkon-lacZ. However, expression of Pvr-YFc but not Pvr caused border cell migration defects. Although the frequency of defects was low, finding a gain-of-function activity at all was significant, given that the signaling strength of Pvr-YFc was reduced compared to Pvr. The migration defects were qualitatively similar to those of Cbl mutants and distinct from dominant-negative effects, which even in their strongest form cause migration delays but not arrest. Uniform expression of the ligand PVF1 did not further affect the phenotype of Pvr-YFc, indicating that this form of Pvr that cannot bind Cbl and Sprint had already lost its spatial information. Consistent with this, expression of Pvr-YFc also induced a delocalized phospho-tyrosine signal at a frequency corresponding to the migration defects. This analysis of Pvr itself further indicates that the phenotypes of Cbl and sprint mutant border cells are due to their effects on RTK signaling: recruitment of Cbl and Sprint to Pvr serves to regulate Pvr guidance signaling, specifically to keep it localized (Jekely, 2005).

Thus, Cbl and Sprint are required to keep RTK signaling properly localized. To show this, phospho-tyrosine was used as a read-out of local RTK signaling. Although this reagent is not uniquely specific for the active receptor, the visualized effects of Pvr or Egfr overexpression, Hrs mutation, ligand misexpression, as well as Sprint colocalization, validates its utility in visualizing the high level of local receptor activity found at the leading edge of migrating border cells. The requirement for Cbl and Sprint suggests that the cellular activity required for signal restriction is receptor endocytosis, which is supported by experiments with dominant-negative dynamin. Thus, in this physiological context of guidance by RTKs, receptor endocytosis serves not to downregulate active receptors, but to ensure their correct spatial localization (Jekely, 2005).

The proposed role of RTK endocytosis regulators should be seen in the context of what is already know about RTK signaling and regulation. Signaling from RTKs is initiated upon transphosphorylation of activating tyrosines and docking tyrosines, the latter generating binding sites for PTB and SH2 domain proteins. Receptor activation is elicited by binding of activating ligand but can also occur if two receptor molecules contact each other productively for other reasons. The likelihood of ligand-independent activation depends on receptor density, and hence overexpressed receptors may have ligand-independent activity in addition to responding more strongly to ligands. Inactivation of receptors is therefore critical for proper signaling in the cell. Phosphatases inactivate receptors by catalyzing the reverse reaction of the activation. Phosphatases are very abundant in cells and may be constitutively active. Local inactivation of phosphatases is one mechanism that can lead to spreading of an initially localized RTK signal. In addition, signaling can be inactivated by endocytosis, which leads to degradation of activated receptors, stimulated by molecules such as Cbl and Sprint/RIN1 and at a later step by Hrs (Jekely, 2005).

Most studies of induced endocytosis, in order to give maximal experimental resolution, have been done in tissue culture cells with acute stimulation by high levels of ligand. In tissues, which have steady and modest levels of ligand and a complex, multicellular environment, the role of endocytosis in RTK regulation is less well understood. For example, Egfr signaling is mildly increased in Cbl mutant follicle cell clones, but so mild that even in Cbl, sprint double mutant follicle cells, there are no detectable changes in levels of Egfr, Pvr, or phospho-tyrosine. However, the effects on border cell migration are striking. Receptor proteins do turn over in the tissue and at least some of this turnover is blocked in Hrs mutant cells. However, under physiological conditions in the ovary, the Hrs-dependent degradation is not dependent on ligand and is not required for guided migration. These results show that the physiological role of Cbl and Sprint in border cell guidance is not to control receptor degradation and/or to turn off signaling, but instead to keep the signal localized (Jekely, 2005).

It is becoming appreciated that endocytosis of signaling receptors is not simply a matter of signal attenuation and receptor removal. It was found that RTK endocytosis differentially affected signaling through different pathways, suggesting (1) that signaling can happen in different compartments and (2) that the process of endocytosis could be used to differentially regulate signaling outcomes. For TGF-β signaling, the process of endocytosis actively brings receptors to internal signaling. This study suggests a third role for early aspects of receptor endocytosis in signaling: to keep active signaling complexes localized in the plane of the membrane. This activity prevents signaling from becoming uniform and therefore uninformative about the spatial distribution of the ligand (Jekely, 2005).

How do Cbl and Sprint spatially restrict signaling? They may prevent signaling from becoming delocalized by restricting lateral movement of activated receptors or lateral spread of RTK activation. Microdomains of active RTKs on the plasma membrane or in endocytic pits could maintain activity, whereas they would be inactivated at other places by ubiquitous phosphatases. Alternatively, recycling of activated receptors to new regions of the cell membrane could delocalize signaling. Normally, this recycling might be prevented by Cbl and Sprint activity by routing active RTKs to degradation via the proper endosome compartment (without requiring Hrs). For these two scenarios, however, it is not obvious why physically blocking endocytosis (Shibire dominant-negative) should also delocalize signaling. Shibire/dynamin is a general effector of endocytosis (required for cell viability), and interfering with it therefore is a more blunt tool than manipulating Cbl and Sprint or mutating Pvr. But the effects are unambiguous. This leads to a third hypothesis, whereby endocytosis of active RTKs allows their redelivery or recycling to regions of higher signaling. Endocytosis and plasma membrane redelivery of active proteins contributes to polarization in yeast, another case of controlling spatial information. Obviously, further analysis will be needed to fully explore these cellular mechanisms in vivo. In any case, at sufficiently high level of receptor expression and activation, the regulatory mechanism may collapse. Indeed, when a C-terminally tagged Egfr was expressed at extremely high levels in border cells, migration and phospho-tyrosine staining were perturbed in a manner similar to what was observed for Cbl mutant clones. Like many regulatory mechanisms, the spatial restriction imposed by Cbl and Sprint works effectively within a certain range of input, emphasizing the need for understanding the mechanism of regulation at a physiological range in vivo (Jekely, 2005).

The role of early endocytosis regulators in spatial signal regulation described in this study is clearly physiologically relevant. But how general might it be? The regulation is likely to be relevant when RTKs are used for spatially resolved signaling. RTKs can act as canonical guidance receptors to detect specific ligands. In addition, mammalian Cbl is required for integrin-dependent migration of macrophages and osteoclasts in vitro and in vivo. This requirement was suggested to reflect an active signaling role of Cbl downstream of integrins, but it could also reflect a role for Cbl in localizing signaling analogous to what has been seen in border cells. There is ample evidence for cross-talk between integrins and RTKs, which in turn are regulated by Cbl. That RTKs can be activated in the absence of cognate ligand by high receptor density or by cross-talk from other pathways such as integrins might seem at odds with their serving as guidance receptors. But with effective regulatory mechanisms to maintain localized signaling, this excitable signaling system may help migrating cells obtain sufficient sensitivity to read guidance cues over a large dynamic range (Jekely, 2005).

A key issue in guidance signaling is that migrating cells must achieve a polarized output despite having to respond, over a large dynamic range, to subtle concentration differences of an attractant or repellant from one end of the cell to the other. One way to achieve this is to amplify the initial signal difference between stimulation of receptors at the front and back of the cell. The use of PI3 kinase and PTEN phosphatase, two antagonistic enzymes that are reciprocally regulated in Dictyostelium chemotaxis, may be an example of this. Alternatively, guidance cues may simply bias a separate, preexisting intrinsic polarity in the migrating cells. Finally, guidance signaling and intrinsic polarity may interact dynamically to reinforce one another during directed migration. The net outcome is a robust difference between the front and the back of the cell, allowing migration. It is interesting to consider that RTK endocytosis may also function to enhance the difference between signaling in the front and back of migratory border cells. Although the gradient of RTK ligands around border cells can not be measured, it is very unlikely to be as steep as the observed difference in phospho-tyrosine staining. Enhancement of a signaling differential can occur at different levels. Binding of Cbl and Sprint to activated RTKs and recruitment to membrane subdomains may effectively concentrate activated receptors. Due to the density dependence and positive feedback in RTK activation, local activity will then be increased, whereas global inactivation by phosphatases could ensure that signaling is reduced elsewhere. It is suggested that spatial organization of signaling may be controlled by endocytosis and redelivery of active receptors. Such active turnover processes can be used as an effective mechanism to increase signaling differentials within a cell, leading even to spontaneous, or self-organized, polarity. A distinct cell front and cell rear and hence short-term productive migration is often seen in migrating cells even without perception of localized guidance cues, indicating intrinsic polarity. Most migrating cells may need to integrate intrinsic polarity and external guidance. Using a regulatory principle that can produce both intrinsic polarity and local response to guidance cues would provide an elegant means to achieve this (Jekely, 2005).

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. A unique embryonic tracheal tubulogenesis phenotype has been described caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. This study demonstrates that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, it was shown that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. It was also shown that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways (Jeon, 2012).

The Drosophila tracheal system is an intricate branched network of air-filled tubes that delivers oxygen to tissues. Tube formation in the tracheal system involves complex morphogenetic events that differ between tube types. Multicellular tubes have lumens that are surrounded by the apical surfaces of several cells. Unicellular tubes are formed by rolling up of single cells to form junctions with themselves. Seamless tubes are intracellular structures within terminal cells. Many genes have been identified that affect the formation and morphology of tracheal tubes (Jeon, 2012).

The absence of the two Type III RPTPs, Ptp4E and Ptp10D, changes the geometries of the tubes in unicellular and terminal branches, so that they form spherical cysts in place of continuous tubular lumen. The phenotype involves a loss of negative regulation of the Egfr RTK, and Ptp10D physically associates with Egfr ( Jeon, 2012).

One of the mammalian Ptp4E/Ptp10D orthologs, PTPRJ (DEP-1), is a direct regulator of multiple growth factor receptor TKs. This led to a test of the hypothesis that Btl and Pvr, the other two Drosophila growth factor receptor TK orthologs that are expressed in embryonic tracheae, are also required for the Ptp4E Ptp10D phenotype (Jeon, 2012).

A quantitative analysis of cyst size at the TC/LT junction showed that tracheally expressed DN mutants of Egfr, Btl, and Pvr all suppress the Ptp4E Ptp10D phenotype almost to wild-type, suggesting that dysregulation of all three RTKs is required for the replacement of linear tubes by spherical cysts. Also, CA mutants of each RTK enhance the phenotype, producing enlarged cysts. These effects cannot be produced by all RTKs, since expression of a CA mutant of InR did not enhance the phenotype (Jeon, 2012).

If the Ptp4E Ptp10D cyst phenotype is the consequence of simultaneous deregulation of all three RTKs, it might be possible to generate the phenotype in a wild-type background by expressing multiple RTK CA mutants. This did not work, even when all three CA mutants were expressed at once. Possible explanations include: 1) because PTP activity normally dominates over RTK activity, the effect on PTyr levels of removing negative regulation by the RPTPs is much greater than that produced by expressing CA RTK mutants in the presence of the RPTPs; 2) other TKs are important for the phenotype, and their activities must also be increased; 3) the RPTPs have PTP-independent activities as adhesion molecules, and generation of the phenotype requires both elevation of RTK activity and the absence of the PTP-independent functions of the RPTPs (Jeon, 2012).

Whether Ptp4E and Ptp10D both regulate all three RTKs was investigated. If they have specificity for particular RTKs, one might be able to generate the cyst phenotype by removing only one RPTP in the presence of a CA RTK mutant. Such combinations were made for Egfr (Ptp4E, Btl>Egfr-CA and Ptp10D, Btl>Egfr-CA), but neither of them had cysts. Since the PTP domains of Ptp4E and Ptp10D are 89% identical, it is likely that they have the same enzymatic targets. The idea that the RPTPs have redundant functions is also consistent with the observation that Ptp4E and Ptp10D single mutants have no detectable phenotypes, while the double mutant is lethal and has both tracheal and nervous system defects (Jeon, 2012).

When each of the DN RTK mutants was expressed together with a CA mutant of one of the other RTKs in the Ptp4E Ptp10D background, the DN mutant was now unable to suppress the phenotype back to wild-type. Instead the phenotype returned to the strength of unmodified Ptp4E Ptp10D mutants. This shows that if the activity of one RTK is sufficiently elevated, it can replace the requirement for another RTK. Thus, none of the three RTKs is uniquely required to generate the phenotype. Rather, the formation of tubes vs. cysts is controlled by the total amount of activity of certain RTKs in tracheal cells. This implies that the downstream pathways whose increased activity causes cyst formation use SH2-domain effectors that can bind to PTyr sites on any of the three RTKs (Jeon, 2012).

It is interesting that the three RTKs can substitute for each other in regulating the tube vs. cyst decision when they are deregulated by loss of the RPTPs, since the RTKs do not seem to have interchangeable activities in wild-type tracheal cells (or in other tissues). Loss or gain of Btl function produces defects in primary, secondary, and terminal tracheal branching. Loss of Egfr function produces a much more subtle tracheal phenotype affecting tissue integrity. Maintenance of tissue integrity requires signaling through the MAP kinase pathway downstream of Egfr, but is unaffected by reduction of MAP kinase signaling downstream of Btl (Jeon, 2012).

These findings can be explained by the fact that growth factor receptor TKs are usually in an inactive state, due to insufficient levels of ligands and to negative regulation by PTPs. They become active only when they come into contact with elevated levels of their ligands at specific times and places. The activities of Type III RPTPs that dephosphorylate the RTKs might also be transiently reduced at some of these times and places, possibly through interactions of their XC domains with as yet unidentified ligands. As a consequence of the tight control of RTK activity, only those downstream signaling pathways that are most responsive to a particular RTK are likely to be activated by that RTK at any time in wild-type embryos, and the outcomes of signaling through these pathways may also be controlled by the subcellular distributions of ligands, RTKs, RPTPs, and downstream effectors. By contrast, in the absence of Ptp4E and Ptp10D, basal levels of RTK ligands may be able to drive all of the growth factor RTKs to a high level of activity, resulting in strong signaling through all of the downstream pathways they can control. Loss of negative regulation might also cause delocalization of signaling, so that effectors whose activity is normally restricted to particular parts of the cell become activated in a cell-wide manner. Under these conditions, a reduction in the activity of any one of the RTKs by a DN mutant will decrease signaling through multiple downstream pathways. Adding a CA mutant of another RTK can then turn signaling through all of these pathways back up, compensating for the effects of the DN mutant (Jeon, 2012).

The ability of RTKs to substitute for each other in control of cyst formation is conceptually similar to cell transformation through elevation of RTK signaling. RTK activity in cultured cells is tightly controlled, and only a few endogenous RTKs are normally involved in cellular responses to the mitogenic growth factors in their culture medium. Many RTKs can signal through the Ras/MAP kinase pathway, however, and elevated Ras/MAP kinase transduction is sufficient to cause transformation of established cell lines. Thus, oncogenic (CA) mutants of a variety of RTKs are able to transform fibroblastic cell lines when expressed at high levels, regardless of whether the endogenous versions of the RTKs are normally used to regulate proliferation in those cell lines (or are even expressed there) (Jeon, 2012).

The observations on the interchangeability of the RTKs suggests that the decision to form cysts rather than tubes in Ptp4E Ptp10D mutants is very sensitive to the levels of PTyr on the effector binding sites on autophosphorylated RTKs, and that cysts appear when total PTyr rises above a critical threshold. This conclusion is based on the complete suppression of the Ptp4E Ptp10D cyst phenotype that is produced by expression of any of the three DN mutants, even though each DN would eliminate only about 1/3 of total PTyr on the RTKs, if they have roughly equal activities. In wild-type embryos, negative regulation by the RPTPs keeps RTK signaling well below this threshold, so the system is insulated against random fluctuations in phosphorylation or downstream signaling (Jeon, 2012).

The Ptp4E Ptp10D GB cyst number phenotype is completely suppressed by Btl-DN, but not suppressed at all by Egfr-DN and only slightly by Pvr-DN. This might be taken as evidence that elevation of Btl activity is uniquely required to replace GB tubes with cysts. However, when Btl-DN is competed against Pvr-CA, it is only able to suppress the Ptp4E Ptp10D, Btl>Pvr-CA phenotype back to that of unmodified Ptp4E Ptp10D, indicating that Btl can be replaced by Pvr if its activity is sufficiently elevated. These findings can be explained if Btl activity is much higher than Egfr or Pvr activity in GB cells, so that a DN mutant that knocks down endogenous Btl activity by a DN mutant has a greater effect on phosphorylation of effector binding sites than mutants that reduce Egfr or Pvr activities. The activity of CA RTK mutants is independent of the endogenous levels of RTKs, so the Pvr CA mutants could still reverse the effect of Btl-DN even if Pvr-DN has no effect on its own (Jeon, 2012).

To evaluate whether the MAP kinase signaling pathway is involved in the determination of tube geometry, a CA mutant of Phl, the Drosophila Raf kinase, was expressed in the Ptp4E Ptp10D background. Phl-CA enhances the TC/LT phenotype almost as strongly as Egfr-CA. When Phl-CA is combined with Egfr or Btl DN mutants, the phenotype is suppressed back to that of unmodified Ptp4E Ptp10D. Since these RTKs are upstream of Raf, the fact that suppression occurs suggests that pathways other than the MAP kinase pathway are required to generate the phenotype. However, these other pathways may be stimulated by elevation of MAP kinase signaling, since one would expect suppression back to a near-wild-type phenotype if they were completely independent of the MAP kinase pathway (Jeon, 2012).

The involvement of Rho GTPases was tested by expressing DN mutants of Rho1, Rac1, and Cdc42 in the Ptp4E Ptp10D background. Rho1-DN and Rac1-DN completely suppress the TC/LT phenotype, and Cdc42-DN and a tracheally expressed Rho1 RNAi construct produces partial suppression. The DN mutant data do not necessarily show that Rho and Rac are both required for generation of the phenotype. DN mutants may occlude binding of wild-type Rho family GTPases to their GEFs, some of which can act on both Rho and Rac. Therefore high-level expression of Rac-DN might inhibit Rho activation, and vice versa. However, the suppression of the TC/LT phenotype by Rho1 RNAi, which is a specific inhibitor, implicates Rho1 itself in the phenotype. Rho1-CA enhances the Ptp4E Ptp10D phenotype, producing cysts on most DBs (Jeon, 2012).

The differences in the abilities of the three RTK DN mutants to suppress the effects of Phl-CA and Rho-CA may provide clues to the pathways that act downstream of these kinases. The MAP kinase and Rho pathways are not necessarily independent, since Ras/MAP kinase pathway activation can increase Rho-GTP in some cell lines. Also, each of the three RTKs is likely to activate both pathways to some extent, as well as many other downstream pathways. Defining the specific outputs of the MAP kinase and Rho pathways that control tracheal tube geometry and identifying other RTK pathways that regulate tube formation is likely to require genome-wide screens for suppressors and enhancers of the Ptp4E Ptp10D phenotype (Jeon, 2012).

GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Using a multidisciplinary approach, this study identified the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). Elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. These results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements (Fernandez-Espartero, 2013).

Directed cell migration plays a crucial role in many normal and pathological processes such as embryo development, immune response, wound healing and tumor metastasis. During development, cells migrate to their final position in response to extracellular stimuli in the microenvironment. To migrate towards or away from a stimulus, individual cells or groups of cells must first achieve direction of migration through the establishment of cell polarity. Guidance cues, such as growth factors, control cell polarization through the regulated recruitment and activation of receptor tyrosine kinases (RTKs) to the leading edge. A key event downstream of RTK signalling in cell migration is the localization of activated Rac at the leading edge. However, little is known about the mechanisms by which external cues regulate Rac activity during cell migration. Rac is activated by GTP exchange factors (GEFs), which facilitate the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) states. Thus, GEFs appear as excellent candidates to regulate the cellular response to extracellular cues during cell migration (Fernandez-Espartero, 2013).

Among the different Rac GEF families characterized so far, the Vav proteins are the only ones known to combine in the same molecule the canonical Dbl (DH) and pleckstrin homology (PH) domains of Rac GEFs and the structural hallmark of tyrosine phosphorylation pathways, the SH2 domain. In addition, Vav activity is regulated by tyrosine phosphorylation in response to stimulation by transmembrane receptors with intrinsic or associated tyrosine kinase activity. These features make Vav proteins ideal candidates to act as signalling transducer molecules coupling growth factor receptors to Rac GTPase activation during cell migration. In fact, a number of cell culture experiments have suggested a role for the Vav proteins in cell migration downstream of growth factor signalling. Thus, the ubiquitously expressed mammalian Vav2 is tyrosine phosphorylated in response to different growth factors, including epidermal (EGF) and platelet-derived (PDGF) growth factors, and its phosphorylation correlates with enhanced Rac activity and migration in some cell types. However, the biological relevance for many of these interactions and the cellular mechanisms by which Vav regulates in vivo cell migration remains to be determined (Fernandez-Espartero, 2013).

The Vav proteins are present in all animal metazoans but not in unicellular organisms. There is a single representative in multicellular invertebrates and urochordata species (such as C. elegans, Drosophila melanogaster and Ciona intestinalis) and usually three representatives in vertebrates. The single Drosophila vav ortholog possesses the same catalytic and regulatory properties as its mammalian counterparts. In addition, the Drosophila Vav is tyrosine phosphorylated in response to EGF stimulation in S2 cells. Furthermore, a yeast two hybrid analysis has shown that the SH2-SH3 region of Vav can bind the epidermal growth factor receptor (EGFR) and the intracellular domain of PVR, PVRi, but not a kinase-dead version of PVRi, suggesting that Vav SH2-SH3-HA::PVRi interactions depend on PVR autophosphorylation. Altogether, these results suggest that the role of mammalian Vavs as transducer proteins coupling signalling from growth factors to Rho GTPase activation has been conserved in Drosophila. Thus, this study took advantage of Drosophila to analyse vav contribution to growth factor-induced cell migration in the physiological setting of a multicellular organism (Fernandez-Espartero, 2013).

The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to study guided cell migration downstream of PVR/EGFR signalling in vivo. Each egg chamber contains one oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (FCs), known as follicular epithelium (FE). The BC cluster is determined at the anterior pole of the FE and it comprises 6-8 outer cells and two anterior polar cells in a central position. BCs delaminate from the anterior FE and migrate posteriorly between the nurse cells until they contact the anterior membrane of the oocyte. BCs use the PVR and the EGFR to read guidance cues, the PDGF-related Pvf1 and the TGFβ-related Gurken, secreted by the oocyte. The Rho GTPase Rac is required for BC migration. The current model proposes that higher levels of Rac activity present in the leading cell determine the direction of migration and that this asymmetric distribution of Rac activity requires guidance receptor input. The unconventional Rac GEF Myoblast city, Mbc, is the only identified downstream signalling effector in this context. However, although genetic analysis have led to propose that the unconventional GEF for Rac, Mbc/DOCK 180, could activate Rac downstream of PVR during BC migration, this has not been formally proven. In addition, Mbc is unlikely to be the only Rac GEF actin downstream of guidance receptors in BCs as the migration phenotype due to complete removal of mbc is not as severe as the loss of both Pvr and Egfr. Thus, other effectors are likely to contribute to the complicated task of guiding BC migration. Many candidate molecules have been tested for their requirement in BC migration, MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, such as DOCK, Trio, and Pak, but none of these is individually required (Fernandez-Espartero, 2013).

Vav proteins were initially involved in lymphocyte ontology. Only recently, cell culture experiments have implicated these proteins in cell migration events downstream of guidance factors. Interestingly, Vav proteins can either promote or inhibit cell migration. In macrophages, Vav is required for macrophage colony-stimulating factor-induced chemotaxis. In human peripheral blood lymphocytes, Vav is involved in the migratory response to the chemokine stromal cell-derived factor-1. Conversely, in Schwann cells, Vav2 is required to inhibit cell migration downstream of the brain-derived neurotrophic factor and ephrinA5. In spite of the knowledge gained from cell culture experiments, the biological relevance for many of the above interactions has remained elusive. In recent years, Vav proteins have started to emerge as critical Rho GEFs acting downstream of RTKs in diverse biological processes. Analysis of Vav2^-/- Vav3^-/- mice revealed retinogeniculate axonal projection defects and impaired ephrin-A1-induced migration during angiogenesis, suggesting a role for Vav in axonal targeting and angiogenesis downstream of Eph receptors in vivo. This study has shown that Vav can act downstream of growth factors receptors to promote BC migration in the developing Drosophila ovary, supporting a role of this family of GEFs in transducing signals from RTKs to regulate cell migration during development (Fernandez-Espartero, 2013).

Analysis of the cellular mechanisms by which Vav regulates cell migration in vertebrates is hampered by the inaccessibility of the cells and the difficulty of visualizing them in their natural environment within the embryo. Thus, it is not yet clear how Vav proteins regulate cell migration downstream of RTKs during development. In this study, by analysing cell movement in their physiological environment, it has been possible to show that Vav is required to control the length, stabilization and life of front cellular protrusions. In addition, disruption of Vav function in vivo was found to result in a decrease in Rac activity at the leading edge. Defective signalling downstream of EGFR/PVR results in defects in the dynamics of cellular protrusion and Rac activation, which are very similar to those observed in vav^-/- BCs. In addition, this study found that ectopic activation of Vav in BCs, as it is the case for PVR/EGFR and Rac, causes non-polarized massive F-actin accumulation. Thus, it is suggested that one of the roles of Vav in directed cell migration downstream of EGF/PVF signals is to remodel the actin cytoskeleton via Rac activation, hence promoting the formation and stabilization of cellular protrusions in the direction of migration. Studies in cultured neurons, have shown that the main role for mouse Vav2 during axonal repulsion is to mediate a Rac-dependent endocytosis of ephrin-Eph. Although endocytosis has been normally shown to be involved in attenuation of RTKs signalling, in BCs it has been proposed to ensure RTKs recycling to regions of higher signalling, thus promoting directed BC movement. This is based on the fact that elimination in BCs of the ubiquitin ligase Cbl, which has been shown to regulate RTK endocytosis, leads to delocalized RTK signal and migration defects. In this context, another possible role for Vav downstream of EGFR/PVR could be to mediate RTK endocytosis, as it is the case during axonal repulsion. Further analysis will be needed to fully explore the molecular and cellular mechanisms by which Vav proteins regulate cell migration in vivo in other developmental contexts (Fernandez-Espartero, 2013).

BC migration is a complex event and activation of EGF/PDGF receptors will most likely engage different GEFs to affect the distinct cytoskeletal changes necessary to accomplish it. In fact, the migration phenotype of BCs mutant for vav is less severe than that of BCs double mutant for both EGFR and PVR. In addition, although reducing Vav function decreases the asymmetry in Rac activity between front and back present in wild-type clusters, it does not eliminate it, as it happens when the function of both guidance receptors is compromised. All these results suggest that there are other GEFs besides Vav that could act downstream of EGFR and PVR to activate Rac. Previous analysis have implicated the Rac exchange factor Mbc/DOCK180 and its cofactor ELMO on BC migration. In this context, Vav and the Mbc/ELMO complex could act synergistically as GEFs to mediate Rac activation to a precise level and/or to a precise location. This awaits the validation of the Mbc/ELMO complex as a GEF for Rac in BCs. In the future, it will be important to determine how the different GEFs contribute to Rac activation, which specific downstream effectors of Rac they activate, and ultimately what cellular aspects of the migration process they control (Fernandez-Espartero, 2013).

In summary, this work demonstrates that Vav functions downstream of RTKs to control directed cell migration during development. Furthermore, this study has unravelled the cellular and molecular mechanism by which Vav regulates cell migration in the developing Drosophila egg chamber: binding of PDGF/EGF to their receptors would induce Vav activation through tyrosine phosphorylation and its association with the activated receptors. This would lead to an increase in Rac activity at the leading edge of migrating cells, which promotes the stabilization and growth of the cellular front extensions, thus controlling directed cell migration (Fernandez-Espartero, 2013).

Regulation of Vav signalling downstream of RTKs can participate not only in development or normal physiology but also in tumorigenesis. Vav1 is mis-expressed in a high percentage of pancreatic ductular adenocarcinomas and lung cancer patients. Thus, understanding the mechanisms by which Vav controls cellular processes downstream of RTKs is likely to be relevant for both developmental and tumor biology (Fernandez-Espartero, 2013).

DEVELOPMENTAL BIOLOGY

Fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor have been identified and characterized. The gene encoding the ligand PDGF/VEGF factor 1 (Pvf1) has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The gene encoding the receptor, PDGF- and VEGF-receptor related (Pvr), has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic hematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the Pvr gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signaling system may regulate the migration of the Drosophila embryonic hemocyte precursor cells (Heino, 2001).

Northern blot analyses of total RNA from various developmental stages using a probe recognizing the intracellular part of all splice variants of Pvr has revealed a single major mRNA of about 5.5 kb. The transcript occurs in all embryonic stages and is also maternally supplied to the earliest embryonic stage. Zygotic Pvr mRNA is most abundant during later embryonic stages and nearly absent during larval development in the first and second instars, appearing again during the third instar. This expression persists during the pupal as well as adult stages (Heino, 2001).

In order to discriminate the various splice variants from each other, it was necessary to use isoform-specific probes, since the resolution of the agarose gel was not sufficient to clearly separate them. The major part of the RNA hybridization signal corresponds to the splice variant #1 as shown by comparison of the hybridizations with exon 6- and 7-specific probes. Small amounts of the SPV-2 and SPV-4 could be detected with the exon 7-specific and exon 5-specific probes, respectively. Both variants occurred with the same dynamic pattern as the dominant SPV-1. SPV-3 could not be assessed directly, but comparison of the hybridization signals obtained with the various probes shows that this variant is not expressed to any significant degree (Heino, 2001).

Pvr shows a very restricted temporal and spatial expression pattern during embryogenesis. At early stages (prior to stage 10), no signal is detected. Thereafter the gene is strongly expressed anteriorly in the presumptive head region. Later the expression becomes restricted to cells displaying a scattered distribution throughout the embryo. During stage 16, a sub-population of the cells expressing Pvr is clearly concentrated along the ventral midline. This pattern corresponds to the origin, migration and final distribution of the embryonic hemocytes (Heino, 2001).

Pvr transcripts are detected in mRNA from ovaries and from embryos. Pvr mRNA is detected in embryonic hemocytes and in the related tissue culture cells, Schneider cells. Endogenous Pvr protein was detected in Schneider cell extracts as an approximately 180 kDa protein, corresponding well to the predicted molecular weight of 170 kDa (Duchek, 2001b).

The embryonic expression pattern of Vegfr was determined by RNA in situ hybridization. Transcript is first detected in two bilaterally symmetric clusters of mesodermal cells in the head region of early stage 8 embryos (~4 hr after egg lay [AEL]). During the next 11 hr of development, these cells undergo a stereotyped series of migrations that disperse them throughout the embryo. The cells migrate out from the clusters in three directions. Some cells migrate anteriorly into the clypeolabrum, while others migrate ventrally toward the gnathal buds. The majority migrate posteriorly toward the caudal end (tail) of the germband-extended embryo, coursing between the amnioserosa and the yolk sac to reach its posterior margin. Once cells move past the posterior margin into the tail, they surround the hindgut opening and migrate along the ventral midline. By the beginning of stage 12 (~9 hr AEL), these migrations produce two populations of cells, one in the anterior scattered around their site of origin, the other in the posterior clustered around the hindgut and ventral midline. During stages 12-14 (~10-12 hr AEL), both populations migrate toward the middle of the embryo along three major routes: (1) the ventral midline (dorsal and ventral to the developing nerve cord); (2) the gut, and (3) the dorsal epidermis. Later, cells leave these paths and become uniformly distributed (Cho, 2002).

The positions and movements of most Vegfr-expressing cells resemble those of developing blood cells. Indeed, the Vegfr expression pattern is difficult to distinguish from that of the blood cell (plasmatocyte) markers Peroxidasin and Croquemort except that Vegfr turns on ~2 hr earlier. (It is not known if Vegfr is expressed in crystal cells, a minor subpopulation of hemocytes.) To confirm that Vegfr-expressing cells are hemocytes, serpent^AS mutants, which lack blood cells, were examined. With the exception or three groups of cells, Vegfr-expressing cells are absent in srp^AS embryos. The three exceptions are (1) tracheal cells from stages 11-13; (2) a small group of cells near each tracheal visceral branch; and (3) a small group of ventral midline cells in each segment, possibly midline glia. In situ hybridization of fixed larvae indicated that larval blood cells also express Vegfr. Thus, Vegfr is expressed in most or all embryonic and larval blood cells throughout their development (Cho, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn⁰⁶⁹⁴⁶). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca²⁺-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S⁺O⁺) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S⁺O⁺ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S⁺O⁺H⁺Pv⁺) pre-prohemocytes initiate the expression of dome-gal4 (d_g4), thereby maturing into prohemocytes. The prohemocyte population (S⁺O⁺H⁺Pv⁺d_g4⁺) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S⁺O⁺H⁺Pv⁺d_g4^lowM⁺). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S⁺O⁺H⁺Pv⁺d_g4^-M⁺), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

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

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

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

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

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

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

Three genes in the Drosophila genome code for Pvr ligands: Pvf1, Pvf2, and Pvf3. Both Pvf2 and Pvf3 are expressed in the ventral midline, where they are thought to act in a partially redundant manner as attractive cues for hemocytes migrating out of the head. 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. 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 (Harris, 2007 and references therein).

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

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

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

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

Previous studies in Drosophila have shown that the PVF ligands act as attractants. In the border cells, ectopic expression of PVF1 is sufficient to redirect border cells towards the site of expression. 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. The ventral midline expression of PVF2, along with its ventral curving, mutant phenotype, suggests that PVF2 might be acting as a repellent for the salivary gland, despite the fact that there is no previous indication of PVF ligands acting as repellents. However, ectopic expression of PVF2 in the visceral mesoderm was not sufficient to redirect the salivary glands away from the visceral mesoderm (Harris, 2007).

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

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

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).

EFFECTS OF MUTATION

Embryonic hemocytes can be visualized with an anti-Peroxidasin antibody. Since no mutant was readily available for the Pvr gene, the deletion strain Df(2L)TE29Aa-14 was used to analyze any possible effect of a lack of the gene. Hemocytes were observed in the mutant as well as in the wild type, showing that Pvr is not essential for hemocyte development. However, while the number was, at the early developmental time points, about the same as in the control, the distribution of the hemocytes was hampered, especially on the dorsal side of the embryo in the deletion strain. At later stages, the antibody did not detect any hemocytes in the deficiency line. Since the deletion used uncovers ~25 genes predicted by the Genome Annotation Database of Drosophila, the defective migration of hemocytes might be due to deletion of other genes close to the Pvr gene. However, other aspects of embryonic development seem unaffected in the mutant embryos carrying the deletion, i.e. segmentation and the positioning of the tracheal system, indicating that the phenotype observed cannot be an indirect consequence of the disorganization of the embryonic development (Heino, 2001).

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).

A study of Pvr mutants suggests that engulfment of dead cells by hemocyte-derived macrophages is essential for CNS development

Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. The developmental requirement for macrophages in Drosophila was examined and macrophage function was found to be essential for central nervous system (CNS) morphogenesis. Mutations were generated and analyzed in the Pvr locus, which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration. Loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. Inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment, causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS (Sears, 2003).

The Pvr locus was disrupted using homologous recombination. Ten independent homologous recombination events were recovered from the progeny of 3000 females, and the products of the targeting events were examined by a combination of long-range PCR and Southern blotting. Nine out of ten targeted insertions, including Pvr^KO2, yield the products expected for a successful targeting event in both PCR and Southern blot analysis. These alleles all failed to complement the lethality of Df(2L)TE128x11, a chromosomal deficiency that removes Pvr: the embryos complete embryogenesis but fail to hatch (Sears, 2003).

In addition to creating targeted insertions at the Pvr locus, multiple Pvr alleles were identified though a genetic non-complementation screen. Nine-thousand eight-hundred and ten lines of EMS-mutagenized flies were screened for failure to complement the lethality of Df(2L)TE128x11. One-hundred and eighty-five lethal or semi-lethal lines were recovered, of which 20 failed to complement Pvr^KO lethality. Among these putative Pvr alleles, four that were identified carry missense mutations in the extracellular domain, four that create stop codons in the extracellular domain and three that carry missense mutations in the kinase domain. The mutations in the kinase domain disrupt residues highly conserved among protein kinases. In Pvr⁷²⁹ an alanine residue in the catalytic loop, highly conserved among tyrosine kinases, is changed to a threonine. In Pvr⁴¹⁸⁷ a glutamate residue in the activation loop, highly conserved among all protein kinases, is changed to a lysine. Finally, in Pvr⁷⁵⁰⁸ an aspartate residue in the DFG motif at the base of the activation loop is changed to a valine. Crystallographic data indicates this residue is involved in the divalent cation binding that contributes to nucleotide triphosphate recognition. The recovery of these changes in the kinase region are consistent with the importance of catalytic activity for Pvr function. All 11 of these EMS-induced Pvr alleles behave as embryonic lethal mutations (Sears, 2003).

Analysis was focused on three alleles predicted to yield severely truncated Pvr proteins. Pvr^KO2 contains two truncated copies of Pvr: one copy encodes a Pvr protein truncated after the fourth Ig domain; the other copy lacks promoter sequences and predicted start codons. Pvr⁹⁷⁴² contains a stop codon immediately after the first Ig domain, while Pvr⁵³⁶³ has a 61-base deletion that removes amino acids 114-134 from the first Ig domain and replaces them with three new residues and a stop codon. Stage 16/17 Pvr^KO2, Pvr⁹⁷⁴² and Pvr⁵³⁶³ embryos were examined for Pvr protein expression by Western blot using antisera raised against the C-terminal 275 amino acids of Pvr. Pvr expression was not detected in any of these mutant animals. Pvr^KO2, Pvr⁹⁷⁴² and Pvr⁵³⁶³ give equivalent results in the studies described below (Sears, 2003).

CNS patterning was examined in Pvr mutants. CNS axons in the Drosophila embryo establish a precise pattern reiterated in each segment. CNS axons establish two longitudinal tracts that run the length of the embryo on either side of the midline, with a subset of these axons crossing the midline of the embryo, forming two commissural axon bundles per segment. CNS axons are guided in part by signals from glia precisely positioned at the midline and along the longitudinal tracts. Although CNS axon architecture is grossly normal in Pvr mutants, the precise ladder-like axon scaffold seen in wild-type embryos is disrupted. The scaffold in each segment has a rounded appearance, owing to changes in the separation between the anterior and posterior commissures and the longitudinal tracts (Sears, 2003).

As a metric of CNS shape change in Pvr mutants, the ratio of the ratio between the longitudinal tracts in each segment of late stage 16 embryos to the distance between anterior and posterior commissures was calculated. This ratio was significantly smaller in Pvr mutants than in wild-type or Pvr heterozygote controls (Sears, 2003).

Since glial cells within the CNS are required for proper axon tract formation, the positioning of glial cells was examined in Pvr mutants. Many CNS glial cells express the homeobox protein Repo and require Repo for their proper development. Repo-expressing glia form a patterned array along the longitudinal axon tracts and are largely excluded from the midline, except for a thin line of glia that enter the midline in each embryonic segment. In Pvr mutants, however, large numbers of Repo-positive glia accumulate in the midline. Thus, Pvr mutants have defects in both CNS axon tract morphology and glial positioning (Sears, 2003).

Since Pvr mutants show disruptions in CNS axon tract shape and glial cell positioning near the CNS midline, the pathfinding of CNS axons near the midline in Pvr mutants was examined in greater detail. The monoclonal antibody 1D4 (mAb 1D4) recognizes the Fasciclin 2 protein and labels a subset of longitudinal bundles that grow adjacent to the CNS midline. Mab1D4 is a commonly used tool for assessing axon fasciculation patterns and detecting inappropriate axon crossing of the CNS midline. Despite the changes in CNS axon scaffold shape and longitudinal glial distribution observed in Pvr mutants, no inappropriate axon crossing of the midline was detected. In addition, in wild-type animals three major tracts of Fasciclin 2-positive axons are observed near the dorsal surface of the CNS on either side of the midline. Three major tracts of Fasciclin 2-positive axons are also observed in Pvr mutants (Sears, 2003).

Although these tracts are relatively normal, they show very mild defasciculation in some segments, with axons in Pvr mutants occasionally separating from one another by greater distances than normal. Because disruptions in longitudinal glial cell development disrupt the formation of these axon bundles, the minor axon tract defects observed in Pvr mutants were not unexpected given the glial cell mispositioning seen in Pvr mutants (Sears, 2003).

To investigate the source of the CNS defects in Pvr mutants, the cell populations expressing Pvr were identified. Pvr protein is detected on several cell populations during embryonic development. In stage 16 embryos, Pvr is prominently expressed by cells at the surface of the embryo, as well as by cells scattered throughout the embryo and by cells at the CNS midline. The large number of Pvr-expressing cells scattered throughout the embryo are hemocytes, since they co-express the hemocyte marker Peroxidasin. The Pvr-expressing cells at the CNS midline are midline glia (a population distinct from the Repo-positive glia mentioned above) and are intimately associated with the CNS commissures. Pvr expression could not be detected in Pvr mutant embryos, confirming the specificity of the antiserum (Sears, 2003).

Drosophila midline glia play important roles in separating and wrapping CNS axon commissures. Although midline glia express Pvr and Pvr mutants exhibit defects in commissure morphology, no role for Pvr in the midline cells could be detected. To test directly whether Pvr acted in midline glia, high-level expression of a dominant-negative form of Pvr was driven in all midline cells using the Sim:Gal4 driver or specifically in midline glia using the Slit:Gal4 driver. However, in neither case was a detectable CNS axon or Repo-positive glia phenotype generated (Sears, 2003).

Is the CNS phenotype related to Pvr expression in hemocytes? In Pvr mutants, hemocytes largely fail to migrate away from their birthplace in the head. To test whether Pvr function in hemocytes is important for CNS development, expression of dominant-negative Pvr was driven in the developing hemocytes. While no solely hemocyte-specific Gal4 driver is available, Gcm:Gal4 can be used to drive gene expression in embryonic hemocytes. Gcm:Gal4 drives gene expression specifically in hemocytes beginning at stage 11 and later, beginning at stage 15, in other cells that do not detectably express Pvr. Embryos expressing dominant-negative Pvr under the control of Gcm:Gal4 have hemocyte migration defects resembling those of Pvr mutants, consistent with Pvr acting cell-autonomously to control hemocyte migration. Most importantly, embryos expressing dominant-negative Pvr under Gcm:Gal4 control also exhibit rounding of CNS axon commissures and mispositioning of Repo-positive glial cells similar to Pvr mutants. These data are consistent with Pvr acting in hemocytes to control CNS patterning and suggest that hemocyte function is required during CNS development (Sears, 2003).

To further examine the potential contribution of hemocytes to CNS development, animals mutant for serpent (srp), which encodes a GATA-family transcription factor required for hemocyte development, were examined. srp^neo45 is a hemocyte-specific allele of serpent, and srp^neo45 animals lack all hemocytes. Examination of srp^neo45 embryos demonstrated that not only do srp^neo45 mutants lack macrophages, they also exhibit CNS axon scaffold defects similar to those in Pvr mutants, with characteristic rounding of commissures. Quantitative representation of CNS axon tract morphology in srp^neo45 mutants confirmed this observation. srp^neo45 animals also show longitudinal glia positioning defects similar to those seen in Pvr mutants. Thus, mutants that disrupt either hemocyte production or migration cause similar alterations in CNS morphogenesis (Sears, 2003).

One possible explanation for dependence of CNS morphogenesis on hemocytes is that hemocyte-derived macrophages are needed to engulf cell corpses generated during development. To test this possibility animals in which macrophages appear to develop normally, but fail to engulf cell corpses, were examined. This was achieved using animals with reduced function of crq, which encodes a CD36-related receptor required for Drosophila macrophages to engulf dead cells. crq loss-of-function was examined using RNAi. Embryos injected with dsRNA corresponding to either of two non-overlapping regions within the Crq transcript had CNS axon scaffold defects similar to those in Pvr and srp mutants. In crq RNAi embryos the ratio of distance between longitudinals to distance between commissures was significantly different from wild type, but not significantly different from Pvr or srp mutants. In addition, crq RNAi animals also showed defects in the positioning of Repo-positive glia similar to those seen in Pvr and srp mutants. These data further support the importance of hemocytes in CNS morphogenesis and specifically suggest that engulfment of dead cells by hemocyte-derived macrophages is essential for CNS development (Sears, 2003).

The PDGF/VEGF receptor controls blood cell survival in Drosophila

The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. It has been demonstrated that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages form carrying multiple apoptotic corpses. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, PVR has been demonstrated to directly control survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease (Brückner, 2004).

These results demonstrate that PVR controls the trophic survival of embryonic blood cells in vivo and an embryonic hemocyte cell line in vitro. Inhibition of PVR signaling or loss of Pvr function results in caspase-dependent apoptotic hemocyte death and formation of large blood cell aggregates. Blood cell clustering in Pvr mutant embryos has previously been interpreted as a migration defect. Based on the current findings, a different scenario is proposed: Dying hemocytes lose their ability to migrate, which may happen in many cases before cells leave the anterior locations of their origin. The resulting apoptotic corpses pose a strong attractant for their still-viable peer macrophages, which migrate toward them to fulfil their phagocytic function. Subsequently, cannibalistic phagocytosis leads to engorgement of macrophages with multiple blood cell corpses and results in the overall appearance of large hemocyte aggregates (Brückner, 2004).

Blood cell death in Pvr mutants occurs progressively throughout embryonic development. While hemocyte clustering in Pvr¹ and Pvr⁴ becomes evident at different stages of embryonic development, blood cell aggregates persist until the end of embryogenesis and intensify over time. This is consistent with observations in the hemocyte cell line Kc 167, where silencing of Pvr results in apoptotic cell death over the course of days. Slow hemocyte death suggests activity of other partially redundant signaling pathways supporting cell survival. The Jak/Stat and Toll/Cactus pathways have been proposed to play roles in larval hemocyte survival, and it remains to be shown whether these pathways have a trophic function in the embryonic hematopoietic system. Further, it needs to be determined whether Pvr mutant blood cells die in a stochastic manner, or whether macrophages comprise different sublineages with variable dependence on trophic PVR signaling. While these studies indicate that both dying and actively phagocytosing blood cells express the same macrophage markers, identification of additional markers may reveal a distinction between sublineages (Brückner, 2004).

Defects in hemocyte function may indirectly account for the associated lethality of Pvr mutants. Pvr mutants suffer from secondary defects in the morphogenesis of the embryonic central nervous system. These defects are caused by a failure of hemocytes to remove dead cells; similar effects were seen in embryos that lack srp function, or in which phagocytosis is impaired. It is likely that additional secondary defects of Pvr mutants will be found in other organ systems that rely on reshaping by the phagocytic activity of macrophages (Brückner, 2004).

Trophic survival is well known in mammalian systems, where it serves as an important mechanism to control cell number -- extracellular factors produced by target tissues trigger survival pathways that suppress apoptotic cell death and thereby control cell number. In Drosophila, few rare described cases of antiapoptotic survival signaling are mediated through activation of the Drosophila EGFR (DER) by its ligands Vein or Spitz in the survival of glia cells, and by both ligands in the survival of smooth cuticle cells. Other signaling pathways may have similar roles during development, but detailed analysis is often limited. In accordance with the classical model of trophic cues, the PVR ligands PVF1-3 are expressed dynamically in multiple embryonic tissues throughout embryogenesis. Only simultaneous RNAi silencing of all three ligands leads to hemocyte aggregation, consistent with PVF1-3 providing the crucial stimuli for hemocyte survival by activation of PVR (Brückner, 2004).

Pvr mutant rescue experiments demonstrate that activated Ras is sufficient to restore hemocyte survival. This result resembles findings from survival signaling by DER, which was shown to inhibit action of the proapoptotic protein HID by phosphorylation through Ras-activated MAPK. Since PVR signaling triggers MAPK activation in Schneider cells and may have the same effect in embryonic hemocytes, it is likely that the Ras/MAPK pathway is a route of antiapoptotic PVR signaling. Expression of dominant-negative Ras^N17 did not lead to large hemocyte aggregates but induced mild enlargement of hemocytes at a low penetrance. This mild phenotype points to weak defects in blood cell survival. The incomplete effect of Ras^N17 may be due to a number of reasons. Ras^N17may be too weak to fully block endogenous Ras signaling, or Ras signaling may be redundant with other signaling pathways that are active in PVR-dependent cell survival. In Pvr¹ rescue experiments, activated Ras^V12 was used, which was shown to ectopically activate other signaling pathways such as the PI3K pathway. Therefore, rescue by Ras^V12 may involve a number of downstream pathways, but the Ras/MAPK pathway itself may still be central to the observed effect, consistent with a current model for apoptosis in Drosophila. Regardless of the upstream pathways involved, inhibition of caspases by the baculovirus inhibitor of apoptosis p35 was sufficient to rescue the Pvr mutant hemocyte death and aggregation phenotype. In these rescue experiments, p35 appeared slightly less potent than Ras^V12, which may be due to the inability of p35 to inhibit the upstream caspase Dronc, or partially insufficient expression levels, since p35 inhibits caspases by stoichiometric binding (Brückner, 2004).

While activated lambdaPVR and other activated rescue transgenes efficiently revert hemocyte clustering and cell death, embryos still retain residual defects in hemocyte distribution. In stage 11/12 embryos, blood cell entry into the posterior end of the elongated germ band is affected, and in stage 15/16 embryos, the posterior area of the ventral nervous system is lacking hemocytes. This is consistent with a role for PVR in the entry/migration of hemocytes into the posterior end of stage 11/12 embryos. Since the natural route of hemocytes is to distribute from the posterior end into ventral posterior areas, it is proposed that the observed ventral-posterior lack in hemocytes may reflect the initial inability of blood cells to invade, or migrate within, the posterior end. Alternatively, PVR activity may be required at multiple steps of embryonic hemocyte migration. In this case, defects at early steps of hemocyte migration (i.e., entry into the posterior end) may be more readily visualized than defects at later steps, which may be masked by additional independent mechanisms of hemocyte dispersal. To distinguish between these possibilities, more refined techniques to study blood cell migration will be required. Consistent with both models, uniform expression of activated lambdaPVR in hemocytes leads to a similar, yet milder, phenotype at a low penetrance, suggesting that correct hemocyte entry or migration requires tightly controlled or locally asymmetric activation of PVR. By analogy, migration of border cells in the egg chamber is dramatically affected by uniform activation of PVR, revealing a role for locally asymmetric PVR activation in the guidance of border cells (Brückner, 2004).

In mammals, PDGF/VEGF receptors are known to mediate additional cellular responses, such as cell proliferation and differentiation. Drosophila PVF2 has been described to promote proliferation of larval hemocytes. The current experiments do not point to a role for PVR in the proliferation of embryonic blood cells, since Pvr RNAi-treated Kc cells did not show an obvious arrest in the cell cycle, and p35 was sufficient to rescue blood cell counts in mutant embryos. Since blood cell proliferation occurs very early during embryonic development, it cannot be excluded that an unnoticed maternal contribution of Pvr may obscure a role for the receptor in embryonic hemocyte proliferation in vivo. Likewise, with respect to blood cell differentiation, no effects of Pvr loss-of-function on macrophage differentiation were detected as judged by the marker Crq. Nevertheless, a role for PVR in hemocyte differentiation may become apparent once more specific lineage markers become available (Brückner, 2004).

Taken together, PVR has at least two functions in the embryonic hematopoietic system: (1) PVR mediates antiapoptotic survival of blood cells throughout embryonic development, and (2) PVR is required for invasion into/migration within the posterior end of the embryo. Rescue of cell viability reverts the dramatic blood cell aggregation phenotype and leads to dispersal of cells in anterior and dorsal areas of the embryo (Brückner, 2004).

The role of Drosophila PVR in trophic cell survival emphasizes the high degree of conservation between Drosophila and vertebrate PDGF/VEGF family receptor function. In vivo and cell culture work now provides the basis to study cell survival in a simple but highly conserved hematopoietic system. In vertebrates, control of cell survival is an important aspect of hematopoiesis and stem cell maintenance. Antiapoptotic cell survival and its aberrant prolongation are a major mechanism in the formation of human neoplasias. In many cases, connections to deregulated upstream signaling pathways remain unclear. Interestingly, in Acute Myeloid Leukemias (AML), more than one-third of cases are associated with specific activating mutations in the PDGF/VEGF receptors Flt3 and c-Kit, and activating fusions of PDGFßR replace the more common oncogenic BCR-ABL fusions in some cases of Chronic Myeloid Leukemia (CML). The contribution of these and other disease-associated genes to aspects of cell survival versus proliferation is still difficult to assess in vivo, yet their mechanism of action is important for the selection of molecularly targeted therapies. Drosophila embryonic hematopoiesis allows the in vivo study of blood cell survival independent of cell proliferation, and this work has demonstrated the antiapoptotic potential of the Drosophila PDGF/VEGF receptor and activated signaling components such as Ras^V12. It will be interesting to exploit the system further by testing disease-related genes of the same and particularly other families for their in vivo potential to rescue blood cell survival in Drosophila. Complementary to these in vivo findings, a Drosophila cell culture system was established for the study of PVR-dependent blood cell survival. Genome-wide RNAi screens will allow identification of modifiers of PVR-dependent blood cell survival (Brückner, 2004).

Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity: Removing Pvr or disrupting Rac1 function inhibits CNS condensation

Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. This study investigated condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. Preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. Disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. These data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and suggest a mechanism whereby these processes work together to shape the developing central nervous system (Olofsson, 2005).

Thus, disrupting hemocyte migration inhibits VNC condensation in the embryo. Lack of hemocyte migration is associated with a severe reduction of ECM components (Collagen IV and Peroxidasin) throughout the embryo and more particularly a loss of these components around the VNC. This leads to a proposal that correct assembly of the ECM depends on hemocytes, and that basement membrane is required for condensation. Supporting a role for ECM in VNC condensation, defects are observed in loss-of-function mutants of integrins, which are ECM receptors and appear themselves to be required for correct assembly of basement membranes. Mutants in integrins or the ECM component Laminin A share at least one other phenotype with embryos in which hemocyte migration has been inhibited: gut morphogenesis is impaired. Thus, a dysfunctional ECM may explain several of the morphogenetic defects seen in embryos with defective hemocyte migration (Olofsson, 2005).

How might basement membrane contribute to VNC condensation? Basement membrane may serve as a substrate for cellular movements involved in condensation and/or regulate signaling events relevant to condensation. Basement membrane is also required for normal neuromuscular junction development, and might be part of the functional blood-brain barrier in Drosophila. Hence, neural function may be disrupted when basement membrane formation is inhibited. However, condensation phenotypes in embryos with impeded hemocyte migration are more severe than in embryos in which neural activity has been blocked. This argues that the condensation phenotype seen in hemocyte migration-blocked embryos cannot be explained simply by a loss of neural activity (Olofsson, 2005).

Although animals in which hemocyte migration is blocked fail to deposit Collagen IV appropriately, it has not been demonstrated that Collagen IV function is required for condensation. However, embryos expressing a dominant negative form of Collagen IV under the control of a heatshock promoter fail to condense their nerve cord. While these data point towards a functional role of Collagen IV in condensation, further studies will be necessary to clarify the specific role of Collagen IV during condensation (Olofsson, 2005).

This study has not investigated whether phagocytosis of cells within the VNC contributes to condensation. pvr mutants show a perdurance of unengulfed cells at the ventral surface of the CNS at stage 14. The majority of these cells seem to disappear later, possibly engulfed by epidermal cells. pvr mutants also maintain some very restricted points of attachment between the epidermis and the VNC. This phenotype is not observed when hemocyte migration is blocked using mutant Rac1 expressed by crq-GAL4. This likely reflects failure of hemocyte migration at a later stage, after the two tissues have separated (Olofsson, 2005).

The major cell type that engulfs apoptotic corpses within the CNS is the subperineural glia. In the absence of macrophages (in the Bic-D mutants), apoptotic cells are still expelled from the CNS but accumulate at the ventral surface, similar to the observations in the pvr mutant. Hemocytes are required for normal CNS morphogenesis: at stage 16, pvr mutants and Crq RNAi treated embryos have mispositioned glia and minor axon scaffolding defects. These data were interpreted to reflect a failure of engulfment of cell corpses. In the context of these findings, an additional cause for glial mispositioning in pvr mutant embryos could be a loss of basement membrane components and the failure to condense (Olofsson, 2005).

VNC condensation correlates with the onset of neural activity in the CNS, and it is found that expressing tetanus toxin light chain or the inwardly rectifying K+ channel Kir2.1 pan-neuronally impairs condensation. This suggests that neural activity influences normal condensation. Neural activity could contribute to condensation in multiple ways. It could directly regulate cellular events relevant to condensation, such as adhesion or actin-based motility, or activity could influence the transcription of genes relevant to such events. Alternatively, neural activity could maintain synaptic connectivity among cells necessary for condensation, rather than directing changes in cellular behavior leading to condensation. Some condensation occurs before neural activity begins, and the condensation phenotypes resulting from impeding hemocyte migration are more severe than those resulting from blocking neural activity. This suggests that there may be multiple stages of condensation, including an earlier activity-independent stage and a later stage that is influenced by activity (Olofsson, 2005).

VNC condensation can be inhibited by expressing mutant Rac1 in lateral glia or neurons. In glia, migration and ensheathing behaviors require cytoskeletal integrity. When mutant Rac1 is expressed in peripheral glia, the formation of cellular extensions is disrupted, and this is accompanied by glia migration and axon ensheathment defects. Similarly, ensheathment of longitudinal axon tracts by longitudinal glia is disrupted in htl loss of function embryos. The VNC condensation phenotype in these embryos is interpreted as indication that glia need dynamic actin cytoskeleton to generate a condensing force. Two types of VNC glia are particularly well placed to generate such a force: longitudinal glia associated with VNC longitudinal connectives, and perineural glia, which ensheath the cortex of the VNC. Cell-cell contacts and cell-ECM contacts among these cells accompanied by remodeling of extracellular matrix could help generate a condensing force within and across neuromeres through changes in cell shape, adhesion or migration. A similar process occurs during mesenchymal condensation (Olofsson, 2005).

In neurons, neurite extension requires normal Rac GTPase activity. Expressing mutant Rac1 in these cells causes defects in axonal outgrowth. In wild type animals, VNC axons are arranged into longitudinal connectives that extend along the length of the nerve cord, and these are well placed to generate an anteroposterior condensing force. This could happen through differential cell adhesion of neurites within the longitudinal connectives or overall shortening of the axons. The observation that axons in VNC longitudinal connectives loop out during condensation in metamorphic insects is consistent with this idea. It is interesting to note that condensation is inhibited in embryos in which mutant Rac1 is expressed in glia, but longitudinal axon tracts appear normal in these animals. This suggests that if axons help generate a condensing force, they likely do this with the help of glia, possibly using these cells as a substrate (Olofsson, 2005).

It is also possible that at least part of the force required for condensation may come from outside the VNC. Somatic muscles connect to the VNC during embryogenesis, and embryonic muscle activity toward the end of embryogenesis is well timed for generating such a force. Also, the methods used to manipulate glia or neuron development in this study may affect neuromuscular activity by disrupting blood-brain barrier formation, or by affecting the Rac-dependent formation of synaptic structures. However, the observation that the CNS can condense in mutants in which muscles do not form normally argues against a major contribution from muscle activity (Olofsson, 2005).

These data identify several areas for further investigation. By following the behavior of small populations of cells in the VNC it may possible to analyze in vivo changes associated with the condensation process and get insight into how changes in organ shape are generated and coordinated. It will also be interesting to examine the contributions made by components of the ECM to normal blood-brain barrier function. Finally, it may be possible to use VNC condensation in embryonic Drosophila to investigate the molecular and cellular basis of how neural activity is translated into a morphogenetic event (Olofsson, 2005).

Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

DNA copy number evolution in Drosophila cell lines

Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, the genomes of 19 tissue-culture cell lines were sequenced and RNA-Seq profiles were generated. This work revealed dramatic duplications and deletions in all cell lines. Three lines of evidence were found indicating that copy number changes were due to selection during tissue culture. First, copy numbers were found to be correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, some copy number changes were identified that were shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). This study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis (Lee, 2014).

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. This study shows that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis; the mechanism for this protection was dissected. Upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, PDGF- and VEGF-related factor 1 (Pvf1), a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer (Xing, 2015).

A form of programmed cell death, apoptosis, is characterized as controlled, caspase-induced degradation of cellular compartments to terminate the activity of the cell. Apoptosis plays a vital role in various processes including normal cell turnover, proper development and function of the immune system and embryonic development. Apoptosis is also induced by upstream signals, such as DNA double-strand breaks (DSB), to destruct severely damaged cells. DSB activate ATM checkpoint kinase and Chk2 kinase-dependent p53 phosphorylation and induction of repair genes. However, if DSB are irreparable, p53 activation will result in pro-apoptotic gene expression and cell death. However, aggressive cancers contain cells that show inability to undergo apoptosis in response to stimuli that trigger apoptosis in sensitive cells. This feature is responsible for the resistance to anticancer therapies, as well as the relapse of tumours after treatment, yet the molecular mechanism of this resistance is poorly understood (Xing, 2015).

As the cell type that constantly regenerates and gives rise to differentiated cell types in a tissue, stem cells share high similarities with cancer stem cells, including unlimited regenerative capacity and resistance to genotoxic agents. Adult stem cells in model organisms such as Drosophila melanogaster, have been utilized to study stem cell biology and for conducting drug screens, thanks to their intrinsic niche, which provides authentic in vivo microenvironment. This study shows that Drosophila adult stem cells are resistant to radiation/chemical-induced apoptosis, and the mechanism for this protection was dissected. A previously reported cell survival gene with a human homologue, pineapple eye (pie) , acts in both stem cells and in differentiating cells to repress the transcription factor FOXO. Elevated FOXO levels in pie mutants lead to apoptosis in differentiating cells, but not in stem cells, indicating the presence of an additional anti-apoptotic mechanism(s) in the latter. We show that this mechanism requires Tie, encoding a homologue of human receptor tyrosine kinase Tie-2, and its target, bantam, encoding a microRNA. The downstream effector of FOXO, Tie and ban, is show to be Hid, encoding a Smac/DIABLO orthologue. Knocking down the ligand Pvf1/PDGF/VEGF/Ang in differentiating daughter cells made stem cells more sensitive to radiation-induced apoptosis, suggesting that Pvf1 from the apoptotic differentiating daughter cells protects stem cells (Xing, 2015).

This study shows that an anti-apoptotic gene, pie, is required for stem cell self-renewal but not for resistance to apoptosis, indicating a compensatory anti-apoptotic mechanism in stem cells. The cell cycle marker profile of pie GSCs resembles that of InR deficient GSCs, leading to the finding that pie controls GSC, as well as ISC self-renewal/division through FOXO protein levels. Surprisingly, pie targets FOXO as well in differentiating cells, failing to explain why the loss of pie does not induce apoptosis in stem cells. However, while the upregulation of FOXO leads to the upregulation of its apoptotic target Hid in differentiating cells, in adult stem cells Hid is not upregulated. Hence additional regulatory pathway is in place to repress Hid and thereby apoptosis in stem cells. This study identified Tie-receptor as the key gatekeeper for the process in the GSCs. The signal (Pvf1) from the dying daughter cells activates Tie in GSCs to upregulate bantam microRNA that represses Hid, thereby protecting the stem cells. Bantam is known to repress apoptosis and activate the cell cycle. However, while protected from apoptosis in this manner, the stem cells do not activate the cell cycle but rather stay in protective quiescence through FOXO activity. When the challenge is passed, stem cells repopulate the tissue (Xing, 2015).

The mammalian pie homologue, G2E3 was reported to be an ubiquitin ligase with amino terminal catalytic PHD/RING domains. G2E3 is essential for early embryonic development (Brooks, 2008). Importantly, microarray data show significant enrichment of G2E3 expression levels in human embryonic stem (ES) cell lines. These observations suggest a critical role of G2E3 in embryonic development, potentially in maintaining the pluripotent capacity. Since FOXO is shown to be an important ESC regulator, it will be interesting to test whether defects in G2E3 result in changes in FOXO levels. Furthermore, future studies are required to test whether human ES cells also are protected from apoptosis due to external signals from dying neighbouring cells (Xing, 2015).

The cell cycle defects of pie mutant stem cells, such as abnormal cell cycle marker profile, can be a consequence of elevated FOXO levels, since FOXO is a transcription factor with wide array of target genes, many of which are involved with cell cycle progress, such as the cyclin-dependent kinase inhibitor p21/p27 (Dacapo in Drosophila). This may be critical when bantam function is considered in the stem cells. Bantam is known to function as anti-apoptotic and cell cycle inducing microRNA. While in GSC bantam is critical through its anti-apoptotic function as a Hid repressor, it has no capacity to induce GSC cell cycle after irradiation. In a challenging situation, such as irradiation, an additional protection mechanism for the tissue is to keep the stem cell in a quiescent state during challenge. bantam's pro-cell division activity may be dampened by FOXO's capacity to upregulate p21/Dacapo (Xing, 2015).

The FOXO family is involved in diverse cellular processes such as tumor suppression, stress response and metabolism. The FOXO group of human Forkhead proteins contains four members: FOXO1, FOXO3a, FOXO4, and FOXO6. Studies to elucidate their function in various stem cell types in vivo using knockout mice have shown some potential redundancy of FOXO proteins. Recent publications have demonstrated a requirement for some of the FOXO family members in mouse hematopoietic stem cell proliferation, mouse neural stem cells, leukaemia stem cells and human and mouse ES cells in vitro. However, FOXO is shown to be dispensable in the early embryonic development in mouse. Drosophila genome has only one FOXO, allowing a definitive study of FOXO's function in stem cells. This study now demonstrates that tight regulation of FOXO protein levels is essential for in vivo GSC and ISC self-renewal in Drosophila. While the loss of FOXO function generates supernumerary stem cells, inappropriately high level of FOXO results in stem cell loss. Under challenge, such as exposure to irradiation, stem cells depleted of FOXO fail to stay quiescent and become more sensitive to the damage, leading to the loss of GSC population. These data demonstrate the importance of the balanced FOXO expression level for stem cell fate (Xing, 2015).

Previous studies have shown that multiple adult stem cell types manage to avoid cell death in response to severe DNA damage. This work has studied the mechanisms that stem cells utilize to avoid apoptosis in absence of pie and revealed that apoptosis is protected through a receptor, Tie and its target miRNA bantam that can repress the pro-apoptotic gene Hid. The ligand for Tie is likely secreted from the dying neighbours since Tie is essential in GSC only after irradiation challenge, IR induces Tie's potential ligand Pvf1 expression in cystoblasts and knockdown of Pvf1 in cystoblasts eliminates stem cells' protection against apoptosis. Further studies will reveal whether the same protective pathway is utilized in other stem cells. Community phenomenon have been described previously around dying cells: compensatory proliferation, Phoenix rising, bystander effect and Mahakali. While Bystander effect describes dying cells inducing death in the neighbours, compensatory proliferation, Phoenix rising and Mahakali describe positive effects in cells neighbouring the dying cells. The present work shows that adult stem cell can survive but show no immediate induction of proliferation when neighboured by dying cells. However, since adult stem cells can repopulate the tissue when death signals have passed, it is proposed that in adult stem cells these phenomenon merge. First, the GSCs survive by bantam repressing the apoptotic inducer, Hid, and later repopulate the tissue by activating cell cycle. Recent findings have suggested that p53 might play an important role in re-entry to cell cycle in stem cells51. The results from the current studies shed light on the general understanding of stem cell behaviour in response to surrounding tissue to ensure the normal tissue homeostasis. It is also plausible that cancer stem cells hijack these normal capacities of stem cells. (Xing, 2015).

EVOLUTIONARY HOMOLOGS

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

In vertebrates, PDGFA and its receptor, PDGFRalpha, are expressed in the early embryo. Impairing their function causes an array of developmental defects, but the underlying target processes that are directly controlled by these factors are not well known. In the Xenopus gastrula, PDGFA/PDGFRalpha signaling is required for the directional migration of mesodermal cells on the extracellular matrix of the blastocoel roof. Blocking PDGFRalpha function in the mesoderm does not inhibit migration per se, but results in movement that is randomized and no longer directed towards the animal pole. Likewise, compromising PDGFA function in the blastocoel roof substratum abolishes directionality of movement. Overexpression of wild-type PDGFA, or inhibition of PDGFA both lead to randomized migration, disorientation of polarized mesodermal cells, decreased movement towards the animal pole, and reduced head formation and axis elongation. This is consistent with an instructive role for PDGFA in the guidance of mesoderm migration (Nagel, 2004).

VEGF receptor and angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, two allelic zebrafish mutants were identifed in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression is severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis is unchanged. Overexpression of vegf_121+165 led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos, however, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis (Habeck, 2002).

Mammalian Ras, which is encoded by three independent genes, has been thought to be a versatile component of intracellular signalling. However, when, where and how Ras signalling plays essential roles in development and whether the three Ras genes have overlapping functions in particular cells remain unclear. This study shows that the three Ras proteins dose-dependently regulate lymphatic vessel growth in mice. Lymphatic vessel hypoplasia is a common phenotype in Ras compound knockout mice, and overexpressed normal Ras in an endothelial cell lineage selectively causes lymphatic vessel hyperplasia in vivo. Overexpression of normal Ras in lymphatic endothelial cells leads to sustained MAPK activation, cellular viability and enhanced endothelial network formation under serum-depleted culture conditions in vitro, and knockdown of endogenous Ras in lymphatic endothelial cells impairs cell proliferation, MAPK activation, cell migration and endothelial network formation. Ras overexpression and knockdown result in up- and downregulation of vascular endothelial growth factor receptor (VEGFR) 3 expression, respectively, in lymphatic endothelial cells in vitro. The close link between Ras and VEGFR3 in vitro is consistent with the result that Ras knockout and transgenic alleles are genetic modifiers in lymphatic vessel hypoplasia caused by Vegfr3 haploinsufficiency. These findings demonstrate a cooperative function of the three Ras proteins in normal development, and also provide a novel aspect of VEGFR3 signalling modulated by Ras in lymphangiogenesis (Ichise, 2010).

PDGF receptor in populations of cranial and cardiac neural crest cells

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, it has been shown that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFR NCC conditional embryos, suggesting that the PDGFR may play a role in a later stage of NCC development. These results demonstrate that the PDGFR plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development (Tallquist, 2003).

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. This study shows that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. Evidence is presented that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton (Duloquin, 2007).

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).

Hypoxia-selective macroautophagy and cell survival signaled by autocrine PDGFR activity

The selective regulation of macroautophagy remains poorly defined. This study reports that PDGFR signaling is an essential selective promoter of hypoxia-induced macroautophagy. Hypoxia-induced macroautophagy in tumor cells is also HIF1alpha-dependent, with HIF1alpha integrating signals from PDGFRs and oxygen tension. Inhibition of PDGFR signaling reduces HIF1alpha half-life, despite buffering of steady-state protein levels by a compensatory increase in HIF1alpha mRNA. This markedly changes HIF1alpha protein pool dynamics, and consequently reduces the HIF1alpha transcriptome. As autocrine growth factor signaling is a hallmark of many cancers, cell-autonomous enhancement of HIF1alpha-mediated macroautophagy may represent a mechanism for augmenting tumor cell survival under hypoxic conditions (Wilkinson, 2009).

Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. This study shows that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases (Calvo, 2011).

PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRalpha signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRalpha is unable to bind PI3K (Pdgfra(PI3K/PI3K)) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRalpha signaling, Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates were immunoprecipitated, and the peptides were analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). This analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. It was demonstrated that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra(PI3K/PI3K)-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. Ybox1 binds both the p53 promoter and the p53 protein and expression of p53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, this study demonstrated that introduction of a p53-null allele attenuates the vertebral defects found in Pdgfra(PI3K/PI3K) neonates. These findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRalpha signaling that regulates survival and proliferation during skeletal development in vivo (Fantauzzo, 2014).

Mutant p53 Drives Pancreatic Cancer Metastasis through Cell-Autonomous PDGF Receptor beta Signaling

Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. This study shows that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors (Weissmueller, 2014).

PDGFRalpha controls the balance of stromal and adipogenic cells during adipose tissue organogenesis

Adipose tissue is distributed in depots throughout the body with specialized roles in energy storage and thermogenesis. PDGFRα (see Drosophila PDGF- and VEGF-receptor related) is a marker of adipocyte precursors, and increased PDGFRα activity causes adipose tissue fibrosis in adult mice. However, the function of PDGFRα during adipose tissue organogenesis is unknown. By analyzing mice with juxtamembrane or kinase domain point mutations that increase PDGFRα activity (V561D or D842V), this study found that PDGFRα activation inhibits embryonic white adipose tissue organogenesis in a tissue-autonomous manner. By lineage tracing analysis, it was also found that collagen-expressing precursor fibroblasts differentiate into white adipocytes in the embryo. PDGFRα inhibited the formation of adipocytes from these precursors while favoring the formation of stromal fibroblasts. This imbalance between adipocytes and stromal cells was accompanied by overexpression of the cell fate regulator Zfp521. PDGFRα activation also inhibited the formation of juvenile beige adipocytes in the inguinal fat pad. These data highlight the importance of balancing stromal versus adipogenic cell expansion during white adipose tissue development, with PDGFRα activity coordinating this crucial process in the embryo (Sun, 2017).

REFERENCES

Search PubMed for articles about Drosophila PDGF- and VEGF-receptor related

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