PVF2, a PDGF/VEGF-like growth factor, induces hemocyte proliferation in Drosophila larvae.
ABSTRACT: 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.
Project description: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.
Project description: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.
Project description:Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. Here we show that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1-GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. We propose that the conserved ARF1-Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic.
Project description:Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. Here we develop a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. We fabricated von Frey filaments that span the subthreshold to high noxious range for Drosophila larvae. Using these, we discovered that pressure (force/area), rather than force per se, is the main determinant of aversive rolling responses to noxious mechanical stimuli. We demonstrated that the RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, whereas Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. PDGF, but not VEGF, peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of VEGF receptor Type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR-2 inhibition also attenuated morphine analgesic tolerance in rats. Our results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.SIGNIFICANCE STATEMENT Hypersensitivity to touch is poorly understood and extremely difficult to treat. Using a refined Drosophila model of mechanical nociception, we discovered a conserved VEGF-related receptor tyrosine kinase signaling pathway that regulates mechanical nociception in flies. Importantly, pharmacological inhibition of VEGF receptor Type 2 signaling in rats causes analgesia and blocks opioid tolerance. We have thus established a robust, genetically tractable system for the rapid identification and functional analysis of conserved genes underlying mechanical pain sensitivity.
Project description:Age-associated changes in stem cell populations have been implicated in age-related diseases, including cancer. However, little is known about the underlying molecular mechanisms that link aging to the modulation of adult stem cell populations. Drosophila midgut is an excellent model system for the study of stem cell renewal and aging. Here we describe an age-related increase in the number and activity of intestinal stem cells (ISCs) and progenitor cells in Drosophila midgut. We determined that oxidative stress, induced by paraquat treatment or loss of catalase function, mimicked the changes associated with aging in the midgut. Furthermore, we discovered an age-related increase in the expression of PVF2, a Drosophila homologue of human PDGF/VEGF, which was associated with and required for the age-related changes in midgut ISCs and progenitor cell populations. Taken together, our findings suggest that PDGF/VEGF may play a central role in age-related changes in ISCs and progenitor cell populations, which may contribute to aging and the development of cancer stem cells.
Project description:Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. Here, we use insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. We demonstrate that a Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, we describe mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.
Project description:In this study, more than 1,000 cricket (Gryllus bimaculatus) hemocytes were classified based on their size and morphology. These hemocytes were classified into six types: granulocytes, plasmatocytes, prohemocytes, spherulocytes, coagulocytes, and oenocytoids. Hemocyte cultures was observed in real time to determine which hemocytes were associated with cellular immune responses against potential pathogens. Granulocytes were identified as the professional immune cell that mediates nodulation, encapsulation, and phagocytosis of pathogens. Granulocytes have been shown to actively produce various sticky nets (amoeba-like hairs and extracellular traps) from their plasma membranes that they use to gather other hemocytes and to implement cellular immune responses. The activation of lysosomes in granulocytes started at 4?h, peaked at 12?h, and returned to baseline by 24?h post-infection. At 48?h post-infection, cells could be found within the cytoplasm of granulocytes and reactivated lysosomes surrounding these cells were visible. This result seems to reflect a phenomenon in which necrotic granulocytes are removed by other healthy granulocytes. This unique mechanism of cellular immunity is therefore a way to efficiently and effectively remove pathogens and simultaneously maintain healthy hemocytes.
Project description:The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.
Project description:Studies using Drosophila have contributed significantly to our understanding of regulatory mechanisms that control stem cell fate choice. The Drosophila blood cell progenitor or prohemocyte shares important characteristics with mammalian hematopoietic stem cells, including quiescence, niche dependence, and the capacity to form all three fly blood cell types. This report extends our understanding of prohemocyte fate choice by showing that the zinc-finger protein Odd-skipped promotes multipotency and blocks differentiation. Odd-skipped was expressed in prohemocytes and downregulated in terminally differentiated plasmatocytes. Furthermore, Odd-skipped maintained the prohemocyte population and blocked differentiation of plasmatocytes and lamellocytes but not crystal cells. A previous study showed that Odd-skipped expression is downregulated by Decapentaplegic signaling. This report provides a functional basis for this regulator/target pair by suggesting that Decapentaplegic signaling limits Odd-skipped expression to promote prohemocyte differentiation. Overall, these studies are the basis for a gene regulatory model of prohemocyte cell fate choice.
Project description:<h4>Background</h4>Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster.<h4>Methodology/principal findings</h4>To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids.<h4>Conclusions/significance</h4>From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.