Wound Healing and Inflammation: Embryos Reveal the Way to Perfect Repair

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. May, 2004  |  Pubmed ID: 15293805

Tissue repair in embryos is rapid, efficient and perfect and does not leave a scar, an ability that is lost as development proceeds. Whereas adult wound keratinocytes crawl forwards over the exposed substratum to close the gap, a wound in the embryonic epidermis is closed by contraction of a rapidly assembled actin purse string. Blocking assembly of this cable in chick and mouse embryos, by drugs or by inactivation of the small GTPase Rho, severely hinders the re-epithelialization process. Live studies of epithelial repair in GFP-actin-expressing Drosophila embryos reveal actin-rich filopodia associated with the cable, and although these protrusions from leading edge cells appear to play little role in epithelial migration, they are essential for final zippering of the wound edges together-inactivation of Cdc42 prevents their assembly and blocks the final adhesion step. This wound re-epithelialization machinery appears to recapitulate that used during naturally occurring morphogenetic episodes as typified by Drosophila dorsal closure. One key difference between embryonic and adult repair, which may explain why one heals perfectly and the other scars, is the presence of an inflammatory response at sites of adult repair where there is none in the embryo. Our studies of repair in the PU. 1 null mouse, which is genetically incapable of raising an inflammatory response, show that inflammation may indeed be partly responsible for scarring, and our genetic studies of inflammation in zebrafish (Danio rerio) larvae suggest routes to identifying gene targets for therapeutically modulating the recruitment of inflammatory cells and thus improving adult healing.

Live Imaging of Wound Inflammation in Drosophila Embryos Reveals Key Roles for Small GTPases During in Vivo Cell Migration

The Journal of Cell Biology. Feb, 2005  |  Pubmed ID: 15699212

Aa robust inflammatory response to tissue damage and infection is conserved across almost all animal phyla. Neutrophils and macrophages, or their equivalents, are drawn to the wound site where they engulf cell and matrix debris and release signals that direct components of the repair process. This orchestrated cell migration is clinically important, and yet, to date, leukocyte chemotaxis has largely been studied in vitro. Here, we describe a genetically tractable in vivo wound model of inflammation in the Drosophila melanogaster embryo that is amenable to cinemicroscopy. For the first time, we are able to examine the roles of Rho-family small GTPases during inflammation in vivo and show that Rac-mediated lamellae are essential for hemocyte motility and Rho signaling is necessary for cells to retract from sites of matrix- and cell-cell contacts. Cdc42 is necessary for maintaining cellular polarity and yet, despite in vitro evidence, is dispensable for sensing and crawling toward wound cues.

Distinct Mechanisms Regulate Hemocyte Chemotaxis During Development and Wound Healing in Drosophila Melanogaster

The Journal of Cell Biology. May, 2006  |  Pubmed ID: 16651377

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.

Drosophila Melanogaster Embryonic Haemocytes: Masters of Multitasking

Nature Reviews. Molecular Cell Biology. Jul, 2007  |  Pubmed ID: 17565363

Drosophila melanogaster haemocytes constitute the cellular arm of a robust innate immune system in flies. In the adult and larva, these cells operate as the first line of defence against invading microorganisms: they phagocytose pathogens and produce antimicrobial peptides. However, in the sterile environment of the embryo, these important immune functions are largely redundant. Instead, throughout development, embryonic haemocytes are occupied with other tasks: they undergo complex migrations and carry out several non-immune functions that are crucial for successful embryogenesis.

Drosophila Embryos As Model Systems for Monitoring Bacterial Infection in Real Time

PLoS Pathogens. Jul, 2009  |  Pubmed ID: 19609447

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid 'freezing' phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors.

Inflammation and Wound Healing in Drosophila

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19763964

Cell motility is a widely researched and clinically relevant process that has primarily been investigated using cell culture models. While these in vitro assays are useful in allowing for high-resolution analysis of cell movement, there will always be questions surrounding the physiological relevance of studying cell migration on artificial 2-dimensional substrates. Therefore, a number of groups in recent years have started developing alternative systems, either ex vivo or in vivo, to begin extrapolating our knowledge surrounding cell motility to actual developmental and disease processes. One such example exploits the translucence of Drosophila embryos, and the genetic tractability of this well-characterized model organism, to understand the cellular and molecular events surrounding inflammation and wound healing. Laser ablation of a small patch of embryonic epithelium in the Drosophila embryo results in a repair process that can be timelapse imaged in its entirety as the epithelial hole is sealed shut. Additionally, Drosophila macrophages can be imaged as they rapidly respond and chemotax to these sites of damage in a process reminiscent of the vertebrate inflammatory response. In both cases the imaging is of a spatial and temporal resolution approaching that which can be obtained from in vitro systems, making the Drosophila embryo an ideal model to begin dissecting the genetic control of cell migration during wound healing and inflammation in an in vivo setting.

Genetic Ablation of Drosophila Phagocytes Reveals Their Contribution to Both Development and Resistance to Bacterial Infection

Journal of Innate Immunity. 2009  |  Pubmed ID: 20375589

Drosophila phagocytes participate in development and immune responses through their abilities to perform phagocytosis and/or secrete extra-cellular matrix components, antimicrobial peptides, clotting factors and signalling molecules. However, our knowledge of their functional impact on development and host resistance to infection is limited. To address this, we have used a genetic cell ablation strategy to generate Drosophila individuals lacking functional phagocytes. Our results highlight the essential contribution of phagocytes to embryonic development including central nervous system morphogenesis. Phagocytes also ensure optimal viability during post-embryonic development through immune functions. The use of phagocyte-depleted flies reveals the contribution of phagocytes in the resistance of Drosophila adults upon systemic infections with specific bacteria. Phagocytes were not involved in the expression of antimicrobial peptides by the fat body indicating a clear separation between cellular and humoral immune responses at this stage. Finally, we confirm that phagocytosis is a critical effector mechanism of the cellular arm by demonstrating that phagocytosis contributes to resistance to infection with Staphylococcus aureus in adults. Our results highlight the power of this cell ablation strategy to reveal the contribution of phagocytes to specific biological processes. We now provide a blueprint of phagocyte importance during both development and innate immune responses in Drosophila.

Interdependence of Macrophage Migration and Ventral Nerve Cord Development in Drosophila Embryos

Development (Cambridge, England). May, 2010  |  Pubmed ID: 20392742

During embryonic development, Drosophila macrophages (haemocytes) undergo a series of stereotypical migrations to disperse throughout the embryo. One major migratory route is along the ventral nerve cord (VNC), where haemocytes are required for the correct development of this tissue. We show, for the first time, that a reciprocal relationship exists between haemocytes and the VNC and that defects in nerve cord development prevent haemocyte migration along this structure. Using live imaging, we demonstrate that the axonal guidance cue Slit and its receptor Robo are both required for haemocyte migration, but signalling is not autonomously required in haemocytes. We show that the failure of haemocyte migration along the VNC in slit mutants is not due to a lack of chemotactic signals within this structure, but rather to a failure in its detachment from the overlying epithelium, creating a physical barrier to haemocyte migration. This block of haemocyte migration in turn disrupts the formation of the dorsoventral channels within the VNC, further highlighting the importance of haemocyte migration for correct neural development. This study illustrates the important role played by the three-dimensional environment in directing cell migration in vivo and reveals an intriguing interplay between the developing nervous system and the blood cells within the fly, demonstrating that their development is both closely coupled and interdependent.

Prioritization of Competing Damage and Developmental Signals by Migrating Macrophages in the Drosophila Embryo

Current Biology : CB. Mar, 2010  |  Pubmed ID: 20188558

The function of immune cells is critically dependent on their capacity to respond to a complex series of navigational cues that enable them to home to various organ sites in the body or to respond to inflammatory cues such as those released at sites of tissue damage. From early embryonic stages, immune cells are faced with a barrage of signals that will not all be directing the cell to do the same thing. Here we use the Drosophila embryo to investigate how hemocytes (Drosophila macrophages), are able to prioritize key guidance signals and ignore others so that they are not pulled every which way. We identify the immediate wound attractant signal as H(2)O(2) and investigate how Drosophila macrophages respond to competing guidance cues-those emanating from a wound-versus standard developmental guidance cues, as well as those signals drawing cells toward neighboring dying cells. We reveal a hierarchy of responsiveness to attractant cues that varies over time and we identify why there is a wound refractile period early in embryonic development when macrophages cannot be distracted from their developmental migratory pathway to a site of tissue damage.

Clasp-mediated Microtubule Bundling Regulates Persistent Motility and Contact Repulsion in Drosophila Macrophages in Vivo

The Journal of Cell Biology. May, 2010  |  Pubmed ID: 20457764

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell-cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.

Ena Drives Invasive Macrophage Migration in Drosophila Embryos

Disease Models & Mechanisms. Jan, 2011  |  Pubmed ID: 21045209

It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

Understanding in Vivo Blood Cell Migration--Drosophila Hemocytes Lead the Way

Fly. Apr-Jun, 2011  |  Pubmed ID: 21150318

Drosophila embryonic hemocytes have emerged as a potent system to analyze the roles of key regulators of the actin and microtubule cytoskeletons live and in an in vivo context (see Table I and references therein). The relative ease with which live imaging can be used to visualize the invasive migrations of these highly motile macrophages and their responses to wound and chemoattractant signals make them a particularly appropriate and genetically tractable cell type to study in relation to pathological conditions such as cancer metastasis and inflammation. ( 1-3) In order to understand how signaling pathways are integrated for a coordinated response, a question with direct relevance to autoimmune dysfunction, we have sought to more fully characterize the inputs these cells receive in vivo over the course of their developmental dispersal. These studies have recently revealed that hemocyte migration is intimately associated with the development of the ventral nerve cord (VNC), a structure used by hemocytes to disperse over the embryo that itself requires this association for its correct morphogenesis. Crucially the VNC must separate from the epidermis to create a channel for hemocyte migration, revealing how constriction of extracellular space can be used to control cell migration in vivo. ( 4).

Drosophila Embryonic Hemocytes

Current Biology : CB. Mar, 2011  |  Pubmed ID: 21377090

Swatting Flies: Modelling Wound Healing and Inflammation in Drosophila

Disease Models & Mechanisms. Sep-Oct, 2011  |  Pubmed ID: 21810906

Aberrant wound healing can lead to a variety of human pathologies, from non-healing chronic wounds that can become dangerously infected, to exuberant fibrotic healing in which repair is accompanied by excessive inflammation. To guide therapeutic intervention, we need a better understanding of the fundamental mechanisms driving tissue repair; this will require complementary wound-healing studies in several model organisms. Drosophila has been used to model genetic aspects of numerous human pathologies, and is being used increasingly to gain insight into the molecular and genetic aspects of tissue repair and inflammation, which have classically been modelled in mice or cultured cells. This review discusses the advantages and disadvantages of Drosophila as a wound-healing model, as well as some exciting new research opportunities that will be enabled by its use.

Wound Healing: Calcium Flashes Illuminate Early Events

Current Biology : CB. Jan, 2012  |  Pubmed ID: 22240471

What are the earliest signals produced at a wound edge that mobilise epithelial cells to heal the wound? Live analysis of wound healing in the worm Caenorhabditis elegans shows that calcium may be the key early trigger.

Elucidating the in Vivo Targets of Photorhabdus Toxins in Real-time Using Drosophila Embryos

Advances in Experimental Medicine and Biology. 2012  |  Pubmed ID: 22127885

The outcome of any bacterial infection, whether it is clearance of the infecting pathogen, establishment of a persistent infection, or even death of the host, is as dependent on the host as on the pathogen (Finlay and Falkow 1989). To infect a susceptible host bacterial pathogens express virulence factors, which alter host cell physiology and allow the pathogen to establish a nutrient-rich niche for growth and avoid clearance by the host immune response. However survival within the host often results in tissue damage, which to some cases accounts for the disease-specific pathology. For many bacterial pathogens the principal determinants of virulence and elicitors of host tissue damage are soluble exotoxins, which allow bacteria to penetrate into deeper tissue or pass through a host epithelial or endothelial barrier. Therefore, exploring the complex interplay between host tissue and bacterial toxins can help us to understand infectious disease and define the contributions of the host immune system to bacterial virulence. In this chapter, we describe a new model, the Drosophila embryo, for addressing a fundamental issue in bacterial pathogenesis, the elucidation of the in vivo targets of bacterial toxins and the monitoring of the first moments of the infection process in real-time. To develop this model, we used the insect and emerging human pathogen Photorhabdus asymbiotica and more specifically we characterised the initial cross-talk between the secreted cytotoxin Mcf1 and the embryonic hemocytes. Mcf1 is a potent cytotoxin which has been detected in all Photorhabdus strains isolated so far, which can rapidly kill insects upon injection. Despite several in vitro tissue culture studies, the biology of Mcf1 in vivo is not well understood. Furthermore, despite the identification of many Photorhabdus toxins using recombinant expression in E. coli (Waterfield et al. 2008), very few studies address the molecular mechanism of action of these toxins in relation to specific immune responses in vivo in the insect model.