Drosophila blood cell chemotaxis

Highlights • Integrin-mediated adhesion used by Drosophila blood cells to migrate in vivo.• SCAR/WAVE is required for lamellipodia but also for clearance of apoptotic cells.• The formins Fhos and Diaphanous regulate Drosophila macrophage migration and morphology.• Calcium waves drive hydrogen peroxide production to regulate inflammatory migrations.• The steroid hormone Ecdysone controls the onset of immune competence.


Introduction
Chemotaxis is the directed movement of cells (or an organism) towards or away from a chemical source. A classical example of chemotaxis is the movement of immune cells, such as neutrophils or macrophages, towards chemoattractants released at sites of infection or injury (e.g. fMLP and CSF-1) [1]. This process has been studied intensively in vitro, while the slime mould Dictyostelium discoideum has also proven vital in dissecting out the migration machinery and its regulation [2]. Whilst understanding regulation of cell migration represents a key biological problem, the fact that so many studies focus on immune cell motility reflects the diverse range of human diseases driven or exacerbated by inflammation.
Insects contain a population of blood cells, called hemocytes (Box 1), which make up the cellular component of their innate immune system [3,4]. Given the genetic tractability and imaging capabilities of Drosophila melanogaster, the hemocytes of this organism have emerged as a prime cell type with which to study the regulation of migration and inflammation in vivo. Hemocytes are functionally equivalent to vertebrate macrophages and undergo chemotaxis to undergo developmental migrations and reach sites of tissue damage, while also detecting and removing apoptotic cells, debris and pathogens [4]. In this review we will discuss recent developments in our understanding of the machinery used by Drosophila hemocytes to chemotax during both developmental and pathological events occurring over the lifespan of a fruit fly. We will also focus on the latest work elucidating how damage signals are triggered and immune cell activation controlled.

Box 1 Blood cell lineages in Drosophila
Drosophila fruit flies contain a population of blood cells called hemocytes that consists of at least three cell types: plasmatocytes, lamellocytes and crystal cells. Plasmatocytes are migratory, phagocytic and resemble vertebrate macrophages; lamellocytes are induced during immune responses to encapsulate invading parasites with their large lamellar processes [65]; crystal cells are non-motile and rupture during immune responses to activate the phenoloxidase pathway and the melanization cascade [66], a humoral form of host defense. Insect blood cells have been used extensively as a model for blood cell specification and proliferation, since many of the signaling pathways used during vertebrate hematopoiesis are conserved and related transcription factors employed [67,68], such as the GATA factor Serpent [69] and the RUNX homologue Lozenge, which is specifically required for the production of crystal cells [70]. Embryonic hemocytes are derived from the head mesoderm [71], while a second wave of hematopoiesis occurs in the lymph gland, with cells released from this organ during larval stages [72]. Migration studies typically focus on the highly motile plasmatocytes, which disperse over the entire embryo during the course of development [71]. Plasmatocytes persist through to adult stages [72] and are often referred to simply as hemocytes (as we have done in this review) or macrophages. developmental functions are necessary and facilitates surveillance against potential pathogens. Consequently, dispersal is a carefully orchestrated and hard-wired process and its stereotyped nature provides numerous opportunities at which to determine the genetic requirements for chemotaxis. After dispersal, hemocytes become responsive to wound stimuli owing to downregulation of developmental cues [12], suggesting a prioritization of developmental cues over wound cues; a large overlap exists in the machinery used to respond to either cue. Migrating hemocytes possess large actin-rich lamellipodia into which microtubules protrude from the cell body. These microtubules are often bundled into an 'arm-like' structure ( Figure 1c), which facilitates persistent motility [13]. A number of classic cytoskeletal regulators are autonomously required within hemocytes for dispersal or normal motility, including the GTPases Rho,  Embryonic migration routes and chemoattractant expression. Schematics showing expression of Pvf2 and Pvf3 chemoattractants (pink shading) in the developing Drosophila embryo at stages 11 (a) and 12 (b). Cartoons below embryos correspond to boxed regions and show RhoL-dependent invasion of the germband (gb) towards a source of Pvfs, some of which is expressed by the developing malphigian tubules (mp) (a) and movement along the developing ventral nerve cord (VNC; grey) (b); arrows indicate hemocyte movements at these stages of development. During progression along the VNC hemocytes are tightly confined between the ventral side of the VNC and epithelium (ep) and as they migrate along the VNC in response to the Pvf ligands that are expressed there, the epithelium and VNC separate, creating a channel for hemocyte progression. Hemocytes also migrate along the developing dorsal vessel at this stage (dv); a = anterior, p = posterior, d = dorsal, v = ventral, lat = lateral. Later in development cell-cell repulsion begins to occur and this depends upon the microtubules, which are frequently bundled into an arm-like structure (arrow) that facilitates persistent migration (c). Microtubules labeled via Clip-GFP expression in hemocytes; white line indicates edges of hemocytes, drawn according to mCherrymoesin localization (not shown). After initial dispersal hemocytes migrate at right angles from the ventral midline to the edges of the VNC (purple arrows) to form three lines (white arrows) on the ventral side of the embryo, immediately beneath the epithelium (d). Maximum projection images show GFP and nls-red stinger localization in hemocytes from the ventral side of the embryo; scale bars represent 50 mm; ant = anterior, post = posterior.
A family of PDGF/Vegf-related ligands called the Pvfs is expressed along the routes hemocytes take through the embryo (Figure 1a, , presumably explaining the defects in contact inhibition of motility (cell-cell repulsion -a phenomenon that depends on microtubules [13]) observed in myospheroid mutants. Collapse events may occur via uncoupling of the actin and microtubule cytoskeletons or increased actin retrograde flow forcing microtubules rearwards when integrinmediated anchoring of actin to ECM is absent.

Nucleation of actin filaments in migrating hemocytes
Although Regulation of migration to sites of pathology The embryonic wound response is perhaps the bestcharacterized example of hemocyte chemotaxis; here hemocytes rapidly repolarize and migrate to sites of damage (Figures 2 and 3). As is the case following tail fin wounds in zebrafish larvae [48], the NADPH oxidase Duox becomes activated, leading to the production of hydrogen peroxide at wound sites. Duox is both necessary and sufficient for hemocyte recruitment [12,49 ]. In worms, flies and fish wounding induces a rapid calcium flash through the epithelium [49 ,50,51 ], which, in flies at least, leads to Duox activation via a pair of calciumsensing EF hands in an intracellular loop (Figure 2) [49 ]. How hemocytes decode the hydrogen peroxide wound cue is not known, but the zebrafish Src family kinase Lyn contains a conserved cysteine residue, oxidation of which regulates Lyn activity and is necessary for neutrophil chemotaxis to hydrogen peroxide and wounds [52]. This cysteine is conserved in Src42A in flies [52], suggesting this mechanism may be conserved through evolution, although Src42A has an anti-inflammatory role limiting epithelial cell responses to damage in flies [53].
PI3K signaling is specifically required for hemocyte wound responses in embryos, leading to the hypothesis that inflammatory responses could be regulated via

Adhesive capture and hemocyte activation
During late embryogenesis, the primitive fly heart begins to beat and hemocytes are pumped around internal spaces as a constituent of the insect blood for the rest of the lifecycle, although some hemocytes remain attached to the epithelium in sessile patches. From larval stages onwards hemocytes are captured from the circulation via adhesion, with no contribution from the sessile population [42]. This 'adhesive capture' superficially resembles the rolling and tethering of vertebrate leukocytes ahead of their extravasation; embryonic migration more closely resembles chemotaxis of macrophages through connective tissue after extravasation (Figure 3). In pupae sessile patch hemocytes recommence motility and become wound responsive [43]. The signals driving inflammatory migration in larvae and pupae remain uncharacterized, but as wounding of the latter triggers integrin-dependent migration and epithelial calcium waves [26 ,55 ], a similar mechanism to that of the embryo may be employed.
In larvae and adults activation of adhesion may facilitate capture: the typical blood cell response to damage and infection in Lepidopteran insects (the order containing moths and butterflies) is adhesion, which can be mediated via cytokine-like molecules such as plasmatocyte spreading peptide (PSP) [56,57]. Injection of PSP into lepidopterans removes immune cells from circulation, presumably via adhesion to internal tissues [56]. Likewise, hemocyte chemotactic peptide (HCP) facilitates recruitment to wounds in moth larvae and directs chemotaxis of their blood cells in vitro [58]. Therefore systemic release of similar molecules may activate Drosophila hemocytes to enable capture at wounds. Recruitment to other sites of pathology (e.g. tumors) post-embryogenesis is also likely to occur via adhesive capture from circulation. Whether local infections can trigger focal recruitment of hemocytes remains unclear -chemotaxis towards pathogens is yet to be demonstrated. Homing of hemocytes to Drosophila blood cell chemotaxis Evans and Wood 5 tumors is associated with damage or degradation of the basement membrane [45], which might expose adhesive signals or activate hemocytes to become adherent. Indeed, activation may represent the key step controlling immune responses. The steroid hormone ecdysone has long been associated with control of Drosophila development [59] and two recent studies have confirmed ecdysone to stimulate hemocyte motility, and its crucial role activating clearance of apoptotic cells and immune surveillance during metamorphosis [60 ,61 ]. The transition back to a more classical migratory chemotaxis to wounds correlates with the beginning of metamorphosis and is prevented by expression of dominant negative ecdysone receptor in hemocytes [60 ]. Ecdysone also turns on immune responses in embryos, since treatment with ecdysone analogues is sufficient to activate immune competence ahead of schedule [62 ]. Rac1 and Basket/JNK signaling have also been previously implicated in hemocyte activation [63] and therefore represent potential downstream targets of signaling pathways to trigger recruitment of hemocytes from the circulation.

Conclusions
Hemocytes have long been investigated as part of the innate immune responses to systemic infection [64], but have recently received substantial interest as a model cell type to understand the regulation of cell migration in the context of an intact and immune competent organism. We are now beginning to have a more complete understanding of the molecular mechanisms used by these highly migratory cells to reach the locations necessary for their range of functions and needed for their responses to pathology. As researchers fill in the gaps in our knowledge, we anticipate hemocytes will become a prime cell type to probe regulation of signal integration in vivo and the challenge for Drosophila researchers will be to use the powerful genetics available in the fly to identify novel targets involved in these processes.

17.
Zanet J, Jayo A, Plaza S, Millard T, Parsons M, Stramer B: Fascin promotes filopodia formation independent of its role in actin bundling. J Cell Biol 2012, 197:477-486. This work revealed that phosphomimetic mutants of an evolutionarily conserved serine residue prevent both actin bundling and filopodia formation in Drosophila hemocytes, but non-phosphorylatable mutants can rescue filopodia and migration (but not bundling), suggesting that actin filament bundling by fascin might be less important for hemocyte migration than filopodia formation. The role of fascin in nurse cells was also addressed and the authors showed similar roles for fascin in carcinoma cells. Loss of myospheroid gene function plays two roles in the developing embryo -one in enabling separation of the VNC from epithelium, creating a channel for hemocytes to move into as they progress down the midline and another autonomous role in hemocytes in which they are needed for efficient migration in vivo. Live imaging studies using hemocytes with labeled microtubules showed that the b-integrin Myospheroid is also necessary to maintain the bundles of microtubules that enable persistent migration and contact inhibition. This paper indicates that Pico/Lamellipodin and SCAR/WAVE work together to drive cell migration in Xenopus neural crest cells and Drosophila border cells, while melanoblast dispersal is affected in Lamellipodin knock out mice, suggestive of neural crest migration defects. The authors show a direct interaction between Lamellipodin and Rac-GTP and that this association helps promote Lamellipodin-SCAR/WAVE interactions via Abi. This coupling helps to drive lamellipodia formation and enhances migration in vitro.

33.
Evans IR, Ghai PA, Urbancic V, Tan KL, Wood W: SCAR/WAVEmediated processing of engulfed apoptotic corpses is essential for effective macrophage migration in Drosophila. Cell Death Differ 2013, 20:709-720. The work was the first to show a genetic and cell autonomous requirement for SCAR/WAVE in hemocytes in vivo, where it is vital for normal hemocyte migration. In addition to driving lamellipodial protrusion SCAR seems important in normal degradation of apoptotic cells. Surprisingly removing apoptosis from the developing embryo rescued lamellipodia and partly restored migration in a SCAR mutant background.

47.
Kelsey EM, Luo X, Bruckner K, Jasper H: Schnurri regulates hemocyte function to promote tissue recovery after DNA damage. J Cell Sci 2012, 125:1393-1400. A transcriptional regulator called Schnurri helps promote regeneration after UV irradiation of Drosophila eye discs. Schnurri drives retinal expression of Pvf1, which activates hemocytes to become phagocytic. Removal of hemocytes or attenuation of this signaling network hampers regeneration.

49.
Razzell W, Evans IR, Martin P, Wood W: Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release. Curr Biol 2013, 23:424-429. This paper was the first to show a calcium wave through a multicellular epithelium in vivo and demonstrates that this calcium response is coupled to hemocyte inflammatory responses. The TRP channel TrpM and gap junctions are necessary for the spread of the calcium wave and dampening the wave using mutants in these genes or pharmacological means reduces inflammatory responses of hemocytes to laser-induced wounds in the embryo. The paper also shows that the maturation factor Nip/Mol is necessary for the role of Duox in triggering inflammatory migration.

51.
Yoo SK, Freisinger CM, LeBert DC, Huttenlocher A: Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish. J Cell Biol 2012, 199:225-234. In contrast to Razzell et al. this study failed to find a link between the calcium wave induced at sites of injury in zebrafish larvae and neutrophil responses (using the drug thapsigargin). However the calcium response was important (in concert with Src family kinases and reactive oxygen species) for normal regeneration after injury.