ASXL3-related disorder: Molecular phenotyping and comprehensive review providing insights into disease mechanism.

ASXL3-related disorder, sometimes referred to as Bainbridge-Ropers syndrome, was first identified as a distinct neurodevelopmental disorder by Bainbridge et al. in 2013. Since then, there have been a number of case series and single case reports published worldwide.

Macrophage subpopulation identity in is modulated by apoptotic cell clearance and related signalling pathways.

In blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established model with which to study macrophage function and behaviour. However, the use of plasmatocytes as a macrophage model has been limited by a historical perspective that plasmatocytes represent a homogenous population of cells, in contrast to the high levels of heterogeneity of vertebrate macrophages. Recently, a number of groups have reported transcriptomic approaches which suggest the existence of plasmatocyte heterogeneity, while we identified enhancer elements that identify subpopulations of plasmatocytes which exhibit potentially pro-inflammatory behaviours, suggesting conservation of plasmatocyte heterogeneity in .

The Epidermal Growth Factor Ligand Spitz Modulates Macrophage Efferocytosis, Wound Responses and Migration Dynamics During Embryogenesis.

How multifunctional cells such as macrophages interpret the different cues within their environment and undertake an appropriate response is a key question in developmental biology. Understanding how cues are prioritized is critical to answering this - both the clearance of apoptotic cells (efferocytosis) and the migration toward damaged tissue is dependent on macrophages being able to interpret and prioritize multiple chemoattractants, polarize, and then undertake an appropriate migratory response. Here, we investigate the role of Spitz, the cardinal epidermal growth factor (EGF) ligand, in regulation of macrophage behavior in the developing fly embryo, using activated variants with differential diffusion properties.

Identification of functionally distinct macrophage subpopulations in .

Vertebrate macrophages are a highly heterogeneous cell population, but while blood is dominated by a macrophage-like lineage (plasmatocytes), until very recently these cells were considered to represent a homogeneous population. Here, we present our identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development. These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults.

Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury.

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here, we seek to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells.

Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution.

Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage-apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, we have exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Six-microns-under (Simu) leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells.

Ena orchestrates remodelling within the actin cytoskeleton to drive robust macrophage chemotaxis.

The actin cytoskeleton is the engine that powers the inflammatory chemotaxis of immune cells to sites of tissue damage or infection. Here, we combine genetics with live imaging to investigate how cytoskeletal rearrangements drive macrophage recruitment to wounds in We find that the actin-regulatory protein Ena is a master regulator of lamellipodial dynamics in migrating macrophages, where it remodels the cytoskeleton to form linear filaments that can then be bundled together by the cross-linker Fascin (also known as Singed in flies). In contrast, the formin Dia generates rare, probing filopods for specialised functions that are not required for migration.

Corpse Engulfment Generates a Molecular Memory that Primes the Macrophage Inflammatory Response.

Macrophages are multifunctional cells that perform diverse roles in health and disease. Emerging evidence has suggested that these innate immune cells might also be capable of developing immunological memory, a trait previously associated with the adaptive system alone. While recent studies have focused on the dramatic macrophage reprogramming that follows infection and protects against secondary microbial attack, can macrophages also develop memory in response to other cues? Here, we show that apoptotic corpse engulfment by Drosophila macrophages is an essential primer for their inflammatory response to tissue damage and infection in vivo.

Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans.

Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles.

Draper/CED-1 mediates an ancient damage response to control inflammatory blood cell migration in vivo.

Tissue damage leads to a robust and rapid inflammatory response whereby leukocytes are actively drawn toward the wound. Hydrogen peroxide (H2O2) has been shown to be an immediate damage signal essential for the recruitment of these inflammatory blood cells to wound sites in both Drosophila and vertebrates [1, 2]. Recent studies in zebrafish have shown that wound-induced H2O2 is detected by the redox-sensitive Src family kinase (SFK) Lyn within the responding blood cells [3].

Drosophila blood cell chemotaxis.

Drosophila melanogaster contains a population of blood cells called hemocytes that represent the functional equivalent of vertebrate macrophages. These cells undergo directed migrations to disperse during development and reach sites of tissue damage or altered self. These chemotactic behaviors are controlled by the expression of PDGF/Vegf-related ligands in developing embryos and local production of hydrogen peroxide at wounds.

Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release.

A crucial early wound response is the recruitment of inflammatory cells drawn by danger cues released by the damaged tissue. Hydrogen peroxide (H2O2) has recently been identified as the earliest wound attractant in Drosophila embryos and zebrafish larvae. The H2O2 signal is generated by activation of an NADPH oxidase, DUOX, and as a consequence, the first inflammatory cells are recruited to the wound within minutes.

Understanding in vivo blood cell migration--Drosophila hemocytes lead the way.

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.

Ena drives invasive macrophage migration in Drosophila embryos.

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).

Interdependence of macrophage migration and ventral nerve cord development in Drosophila embryos.

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.

Live imaging of Drosophila melanogaster embryonic hemocyte migrations.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo.

Drosophila embryos as model systems for monitoring bacterial infection in real time.

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.

Ena/VASP proteins mediate repulsion from ephrin ligands.

Ena/VASP proteins negatively regulate cell motility and contribute to repulsion from several guidance cues; however, there is currently no evidence for a role downstream of Eph receptors. Eph receptors mediate repulsion from ephrins at sites of intercellular contact during several developmental migrations. For example, the expression of ephrin-Bs in posterior halves of somites restricts neural crest cell migration to the anterior halves.

Vav1 and Vav2 play different roles in macrophage migration and cytoskeletal organization.

Vav family proteins act as guanine nucleotide exchange factors for Rho family proteins, which are known to orchestrate cytoskeletal changes and cell migration in response to extracellular stimuli. Using mice deficient for Vav1, Vav2 and/or Vav3, overlapping and isoform-specific functions of the three Vav proteins have been described in various hematopoietic cell types, but their roles in regulating cell morphology and migration have not been studied in detail. To investigate whether Vav isoforms have redundant or unique functions in regulating adhesion and migration, we investigated the properties of Vav1-deficient and Vav2-deficient macrophages.