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. A comprehensive review of the literature was carried out. Abstracts were screened, relevant literature was analysed, and descriptions of common phenotypic features were quantified. ASXL3 variants were collated and categorised. Common phenotypic features comprised global developmental delay or intellectual disability (97%), feeding problems (76%), hypotonia (88%) and characteristic facial features (93%). The majority of genetic variants were de novo truncating variants in exon 11 or 12 of the ASXL3 gene. Several gaps in our knowledge of this disorder were identified, namely, underlying pathophysiology and disease mechanism, disease contribution of missense variants, relevance of variant location, prevalence and penetrance data. Clinical information is currently limited by patient numbers and lack of longitudinal data, which this review aims to address.

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

In Drosophila blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established in vivo 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 Drosophila. These plasmatocyte subpopulations exhibit enhanced responses to wounds and decreased rates of efferocytosis when compared to the overall plasmatocyte population. Interestingly, increasing the phagocytic requirement placed upon plasmatocytes is sufficient to decrease the size of these plasmatocyte subpopulations in the embryo. However, the mechanistic basis for this response was unclear. Here, we examine how plasmatocyte subpopulations are modulated by apoptotic cell clearance (efferocytosis) demands and associated signalling pathways. We show that loss of the phosphatidylserine receptor Simu prevents an increased phagocytic burden from modulating specific subpopulation cells, while blocking other apoptotic cell receptors revealed no such rescue. This suggests that Simu-dependent efferocytosis is specifically involved in determining fate of particular subpopulations. Supportive of our original finding, mutations in amo (the Drosophila homolog of PKD2), a calcium-permeable channel which operates downstream of Simu, phenocopy simu mutants. Furthermore, we show that Amo is involved in the acidification of the apoptotic cell-containing phagosomes, suggesting that this reduction in pH may be associated with macrophage reprogramming. Additionally, our results also identify Ecdysone receptor signalling, a pathway related to control of cell death during developmental transitions, as a controller of plasmatocyte subpopulation identity. Overall, these results identify fundamental pathways involved in the specification of plasmatocyte subpopulations and so further validate Drosophila plasmatocytes as a heterogeneous population of macrophage-like cells within this important developmental and immune model.

The Epidermal Growth Factor Ligand Spitz Modulates Macrophage Efferocytosis, Wound Responses and Migration Dynamics During Drosophila 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 Drosophila epidermal growth factor (EGF) ligand, in regulation of macrophage behavior in the developing fly embryo, using activated variants with differential diffusion properties. Our results show that misexpression of activated Spitz can impact macrophage polarity and lead to clustering of cells in a variant-specific manner, when expressed either in macrophages or the developing fly heart. Spitz can also alter macrophage distribution and perturb apoptotic cell clearance undertaken by these phagocytic cells without affecting the overall levels of apoptosis within the embryo. Expression of active Spitz, but not a membrane-bound variant, can also increase macrophage migration speeds and impair their inflammatory responses to injury. The fact that the presence of Spitz specifically undermines the recruitment of more distal cells to wound sites suggests that Spitz desensitizes macrophages to wounds or is able to compete for their attention where wound signals are weaker. Taken together these results suggest this molecule regulates macrophage migration and their ability to dispose of apoptotic cells. This work identifies a novel regulator of Drosophila macrophage function and provides insights into signal prioritization and integration in vivo. Given the importance of apoptotic cell clearance and inflammation in human disease, this work may help us to understand the role EGF ligands play in immune cell recruitment during development and at sites of disease pathology.

Identification of functionally distinct macrophage subpopulations in Drosophila.

Vertebrate macrophages are a highly heterogeneous cell population, but while Drosophila 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. Our enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene (Calnexin14D) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by the Calnexin14D-associated enhancer. Finally, we show that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers. Taken together this demonstrates macrophage heterogeneity in Drosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use of Drosophila to study macrophage heterogeneity in vivo.

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. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. We show that this leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage-apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be 'overtasked' to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo.

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. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and retention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player regulating macrophages during resolution of inflammation.

Ena orchestrates remodelling within the actin cytoskeleton to drive robust Drosophila 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 in vivo imaging to investigate how cytoskeletal rearrangements drive macrophage recruitment to wounds in Drosophila 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. The role of Ena in lamellipodial bundling is so fundamental that its overexpression increases bundling even in the absence of Fascin by marshalling the remaining cross-linking proteins to compensate. This reorganisation of the lamellipod generates cytoskeletal struts that push against the membrane to drive leading edge advancement and boost cell speed. Thus, Ena-mediated remodelling extracts the most from the cytoskeleton to power robust macrophage chemotaxis during their inflammatory recruitment to wounds.

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. Priming is triggered via calcium-induced JNK signaling, which leads to upregulation of the damage receptor Draper, thus providing a molecular memory that allows the cell to rapidly respond to subsequent injury or infection. This remarkable plasticity and capacity for memory places macrophages as key therapeutic targets for treatment of inflammatory disorders.

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. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease.

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]. Here, we show the same signaling occurs in Drosophila inflammatory cells in response to wound-induced H2O2 with mutants for the Lyn homolog Src42A displaying impaired inflammatory migration to wounds. We go on to show that activation of Src42A is necessary to trigger a signaling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (a member of the CED-1 family of apoptotic cell clearance receptors) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark-mediated signaling axis is homologous to the well-established SFK-ITAM-Syk-signaling pathway used in vertebrate adaptive immune responses. Consequently, our results suggest that adaptive immunoreceptor-signaling pathways important in distinguishing self from non-self appear to have evolved from a more-ancient damage response. Furthermore, this changes the role of H2O2 from an inflammatory chemoattractant to an activator signal that primes immune cells to respond to damage cues via the activation of damage receptors such as Draper.

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. Recent work reveals that many molecules important in vertebrate cell motility, including integrins, formins, Ena/VASP proteins and the SCAR/WAVE complex, have a conserved function in these innate immune cells. The use of this model organism has elucidated how damage signals are activated by calcium signaling during inflammation and that the steroid hormone ecdysone activates immune competence at key developmental stages.

Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo.

Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.

A dual role for the ßPS integrin myospheroid in mediating Drosophila embryonic macrophage migration.

Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main ßPS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards 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. To date, nothing is known about how wounding activates DUOX. Here, we show that laser wounding of the Drosophila embryo epidermis triggers an instantaneous calcium flash, which travels as a wave via gap junctions several cell rows back from the wound edge. Blocking this calcium flash inhibits H2O2 release at the wound site and leads to a reduction in the number of immune cells migrating to the wound. We suggest that the wound-induced calcium flash activates DUOX via an EF hand calcium-binding motif and thus triggers the production of the attractant damage cue H2O2. Therefore, calcium represents the earliest signal in the wound inflammatory response.

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. 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).

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). 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.

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo.

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.

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. 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.

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.