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

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that 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, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster 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.     

Developmental control of blood cell migration by the Drosophila VEGF pathway.

We show that 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 or Vegf27Cb have no effect, but simultaneous inactivation of all three Vegf genes phenocopied the receptor mutant, and ectopic expression of Vegf27Cb redirected 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 we speculate that the ancestral function of VEGF pathways was to guide blood cell movement.     

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.     

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

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. We now demonstrate 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 carrying multiple apoptotic corpses form. 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, we demonstrate that PVR directly controls 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.     

Guidance of cell migration by the Drosophila PDGF/VEGF receptor

Directed cell migrations are important for development, but the signaling pathways and mechanisms responsible for guiding cell migration in vivo are poorly understood. Migration of border cells during Drosophila oogenesis is a simple and attractive model system in which to address these questions. We demonstrate that 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. We present evidence implicating Rac and the Rac activator Mbc/DOCK180/CED-5 as mediators of the guidance signal.     

A role for the chaperone Hsp70 in the regulation of border cell migration in the Drosophila ovary

Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. Here, we show that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, we provide biochemical and genetic evidence for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, our results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function.     

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, we raised antibodies against a tumorous blood cell line and identified an antigenic determinant that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, we induced a dramatic increase in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis.     

Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity

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. Here, we investigate 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. We find that 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. We find that disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. Our data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and allow us to propose a mechanism whereby these processes work together to shape the developing central nervous system.     

Two splice variants derived from a Drosophila melanogaster candidate ClC gene generate ClC-2-type Cl- channels

Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.     

The role of megalin (LRP-2/Gp330) during development

Megalin (LRP-2/GP330), a member of the LDL receptor family, is an endocytic receptor expressed mainly in polarised epithelial cells. Identified as the pathogenic autoantigen of Heymann nephritis in rats, its functions have been studied in greatest detail in adult mammalian kidney, but there is increasing recognition of its involvement in embryonic development. The megalin homologue LRP-1 is essential for growth and development in Caenorhabditis elegans and megalin plays a role in CNS development in zebrafish. There is now also evidence for a homologue in Drosophila. However, most research concerns mammalian embryogenesis; it is widely accepted to be important during forebrain development and the developing renal proximal tubule. Megalin is also expressed in lung, eye, intestine, uterus, oviduct, and male reproductive tract. It is found in yolk sacs and the outer cells of pre-implantation mouse embryos, where interactions with cubilin result in nutrient endocytosis, and it may be important during implantation. Models for megalin interaction(s) with Sonic Hedgehog (Shh) have been proposed. The importance of Shh signalling during embryogenesis is well established; how and when megalin interacts with Shh is becoming a pertinent question in developmental biology.     

PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis

PVR, the Drosophila homolog of the PDGF/VEGF receptor, has been implicated in border cell migration during oogenesis and hemocyte migration during embryogenesis. It was earlier shown that Mbc, a CDM family protein, and its effector, Rac, transduced the guidance signal from PVR during border cell migration. Here we demonstrate that PVR is also required for the morphogenetic process, thorax closure, during metamorphosis. The results of genetic and biochemical experiments indicate that PVR activates the JNK pathway. We present evidence showing Crk (an adaptor molecule), Mbc, ELMO (a homolog of Caenorhabditis elegans CED-12 and mammalian ELMO), and Rac to be mediators of JNK activation by PVR. In addition, we suppose that not only Rac but also Cdc42 is activated and involved in JNK activation downstream of PVR.     

Adenoviral expression of vascular endothelial growth factor splice variants differentially regulate bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) are being explored for clinical applications, and genetic engineering represents a useful strategy for boosting the therapeutic potency of MSCs. Vascular endothelial growth factor (VEGF)-based gene therapy protocols have been used to treat tissue ischemia, and a combined VEGF/MSC therapeutics is appealing due to their synergistic paracrine actions. However, multiple VEGF splice variants exhibit differences in their mitogenicity, chemotactic efficacy, receptor interaction, and tissue distribution, and the differential regulatory effects of multiple VEGF isoforms on the function of MSCs have not been characterized. We expressed three rat VEGF-A splice variants VEGF120, 164, and 188 in MSCs using adenoviral vectors, and analyzed their effects on MSC proliferation, differentiation, survival, and trophic factor production. The three VEGF splice variants exert common and differential effects on MSCs. All three expressed VEGFs are potent in promoting MSC proliferation. VEGF120 and 188 are more effective in amplifying expression of multiple growth factor and cytokine genes. VEGF164 on the other hand is more potent in promoting expression of genes associated with MSC remodeling and endothelial differentiation. The longer isoform VEGF188, which is preferentially retained by proteoglycans, facilitates bone morphogenetic protein-7 (BMP7)-mediated MSC osteogenesis. Under serum starvation condition, virally expressed VEGF188 preferentially enhances serum withdrawal-mediated cell death involving nitric oxide production. This work indicates that seeking the best possible match of an optimal VEGF isoform to a given disease setting can generate maximum therapeutic benefits and minimize unwanted side effects in combined stem cell and gene therapy.     

Involvement of insect-derived growth factor (IDGF) in the cell growth of an embryonic cell line of flesh fly

Insect-derived growth factor (IDGF) is the first adenosine deaminase-related growth factor (ADGF) purified from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Here we show the requirement of IDGF for the growth of NIH-Sape-4 cells. Growth factor activity was abolished by adsorption of IDGF from the conditioned medium of NIH-Sape-4 cells. In addition, knockdown of IDGF gene expression by RNA interference (RNAi) significantly reduced IDGF secretion from the cells following cell growth inhibition. The IDGF gene was strongly expressed in the hemocytes, and IDGF increased the viability of the larval hemocytes. These data provide evidence that IDGF is required for the growth of NIH-Sape-4 cells and possibly for hemocyte viability.     

Sulf2 gene is alternatively spliced in mammalian developing and tumour tissues with functional implications

SULF2 enzyme regulates the activities of a number of signalling pathways that in many tissues are up-regulated during development and disease. As we recently showed for avian Sulf1, the present study demonstrates that mammalian Sulf2 gene can also generate functionally distinct splice variants that would regulate normal development and tumour growth differentially. It is thus important to distinguish SULF1/SULF2 isoforms in mammalian tissues to understand their functional and clinical relevance to disease. This study demonstrates that unlike normal adult lung with little or no SULF2 expression, this enzyme is expressed at high levels in most lung tumours showing differential cellular distribution of full length and shorter SULF2 variants in such tumours. Furthermore, we show that the short SULF2 splice variants are associated with those signalling pathways that are inhibited by full length SULF1/SULF2 variants and therefore could promote growth in such lung tumours.     

Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors

There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism.     

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound beta-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophila galectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophila galectin had a unique and specific tissue distribution. Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.     

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.