No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Macrophages and glia participate in the removal of apoptotic neurons from the Drosophila embryonic nervous system
Abstract
Cell death in the Drosophila embryonic central nervous system (CNS) proceeds by apoptosis, which is revealed ultrastructurally by nuclear condensation, shrinkage of cytoplasmic volume, and preservation of intracellular organelles. Apoptotic cells do not accumulate in the CNS but are continuously removed and engulfed by phagocytic haemocytes. To determine whether embryonic glia can function as phagocytes, we studied serial electronic microscopic sections of the Drosophila CNS. Apoptotic cells in the nervous system are engulfed by a variety of glia including midline glia, interface (or longitudinal tract) glia, and nerve root glia. However, the majority of apoptotic cells in the CNS are engulfed by subperineurial glia in a fashion similar to the microglia of the vertebrate CNS. A close proximity between macrophages and subperineurial glia suggests that glia may transfer apoptotic profiles to the macrophages. Embryos affected by the maternal-effect mutation Bicaudal-D have no macrophages. In the absence of macrophages, most apoptotic cells are retained at the outer surfaces of the CNS, and subperineurial glia contain an abundance of apoptotic cells. Some apoptotic cells are expelled from the CNS, which suggests that the removal of apoptotic cells can occur in the absence of macrophages. The number of subperineurial glia is unaffected by changes in the rate of neuronal apoptosis.
... However, these bone marrowderived professional phagocytes are excluded from the healthy CNS (Galea et al., 2007). Instead, glial cells in many different animal species take on the role of phagocytosing dead cells, defending the brain from invading pathogens, and maintaining tissue homeostasis (Awasaki and Ito, 2004;Block et al., 2004;Gomez et al., 2004;Kaur et al., 2004;Koenigsknecht and Landreth, 2004;Laporte et al., 2004;MacDonald et al., 2006;Sonnenfeld and Jacobs, 1995b;Watts et al., 2004). ...
... As is the case in mammals, the Drosophila CNS is immune privileged and inaccessible to hematopoietically derived immune cell types, such as peripheral macrophages, and glia are the primary endogenous phagocytic cell type within the nervous system. As outlined below, Drosophila glia, like their mammalian counterparts, act to clear a range of engulfment targets from the developing and mature nervous system, including apoptotic neurons and degenerating axon debris (Awasaki and Ito, 2004;Awasaki et al., 2006;Freeman et al., 2003;MacDonald et al., 2006;Sonnenfeld and Jacobs, 1995b). ...
... This amounts to a daunting number of embryonic neurons in the CNS that are destined to die, and must be removed from the developing brain. Glial cells (of which there are only ∼32 per hemisegment) are responsible for clearing this large population of neuronal cell corpses from the CNS within the short 20-hour period of embryonic nervous system development (Bangs and White, 2000;Buss et al., 2006;Freeman et al., 2003;Kurant et al., 2008;Marin-Teva et al., 2004;Sonnenfeld and Jacobs, 1995b). The first evidence that glia are capable of engulfing apoptotic neurons in the Drosophila embryo was discovered by using electron microscopy (EM) to visualize glia in the CNS and PNS during embryogenesis. ...
Glia, whose name derives from the original Greek word, meaning “glue,” have long been understood to be cells that play an important functional role in the nutritive and structural support of the central nervous system, yet their full involvement has been historically undervalued. Despite the strong evidence that glial reactions to cellular debris govern the health of the nervous system, the specific properties of damaged axonal debris and the mechanisms by which glia sense them, morphologically adapt to their presence, and initiate phagocytosis for clearance, have remained poorly understood. The work presented in this thesis was aimed at addressing this fundamental gap in our understanding of the role for glia in neurodegenerative processes.
I demonstrate that the cellular machinery responsible for the phagocytosis of apoptotic cell corpses is well conserved from worms to mammals. Draper is a key component of the glial response machinery and I am able to show here, for the first time, that it signals through Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70. Shark binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. I show that Shark activity is essential for Draper-mediated signaling events in vivo, including the recruitment of glial membranes to axons undergoing Wallerian degeneration. I further show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. Therefore I propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the subsequent downstream activation of the Draper pathway. I observed that these Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signaling events in myeloid and lymphoid cells. Thus, Draper appears to be an ancient immunoreceptor with an extracellular domain tuned to modified-self antigens and an intracellular domain that promotes phagocytosis through an ITAM domain-SFK-Syk-mediated signaling cascade.
I have further identified the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. I am able to demonstrate that Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway to promote a distinct step in the clearance of axonal debris. Whereas Draper signaling is required early during glial responses, promoting glial activation and extension of glial membranes to degenerating axons, the Crk/Mbc/dCed-12 complex functions at later stages of glial response, promoting the actual phagocytosis of axonal debris.
Finally, many interesting mutants have been identified in primary screens for genes active in neurons that are required for axon fragmentation or clearance by glia, and genes potentially active in glia that orchestrate clearance of fragmented axons. The further characterization of these genes will likely unlock the mystery surrounding “eat me” and “find me” cues hypothesized to be released or exposed by neurons undergoing degeneration. Illuminating these important glial pathways could lead to a novel therapeutic approach to brain trauma or other neurodegenerative conditions by providing a druggable means of inducing early attenuation of the glial response to injury down to levels less damaging to the brain.
Taken together, my combined work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and the termination of glial responses to injury are genetically separable events mediated by distinct signaling pathways.
... In addition to morphological similarities, Drosophila astrocyte-like glia have been shown to be functionally very similar to their mammalian counterpart as they are able to tightly control neurotransmitter levels at synapses by expressing specific transporters on their membranes (Kucukdereli et al., 2011;Rival et al., 2004;Stacey et al., 2010)( Dr. Tobias Stork, personal communication). A CNS glial subtype specifically assigned to immune functions, like mammalian microglia, does not appear to be present in the Drosophila embryo; rather, all glia seem competent to perform immune-like functions such as engulfment of neuronal corpses during development (Freeman et al., 2003;Sonnenfeld and Jacobs, 1995). The ectoderm derived surface glia form a flat sheath around the Drosophila CNS blood-brain barrier, isolating neural elements from surrounding tissues and hemolymph (Edwards et al., 1993;Ito et al., 1995). ...
... The ectoderm derived surface glia form a flat sheath around the Drosophila CNS blood-brain barrier, isolating neural elements from surrounding tissues and hemolymph (Edwards et al., 1993;Ito et al., 1995). Finally, in the PNS, CNS-derived peripheral glia ensheath and support peripheral nerves containing motor and sensory axons (Auld et al., 1995;Freeman et al., 2003;Leiserson et al., 2000;Sonnenfeld and Jacobs, 1995), much like mammalian Schwann cells. ...
... During Drosophila embryonic development, approximately 500 neurons are produced per hemisegment of the ventral nerve cord and roughly 30% of these will undergo PCD (Rogulja-Ortmann et al., 2007). These cell corpses are rapidly cleared from the CNS primarily by surface glial cells which ensheath the entire CNS and send projections into the cortex regions (Sonnenfeld and Jacobs, 1995) (Fig. 1-1A). It is unclear whether the glial cells then degrade and process the dead cells or whether they transfer the corpses to macrophages in close contact with the CNS for further processing. ...
The nervous system is made up of two major cell types, neurons and glia. The major distinguishing feature between neuronal cells and glial cells is that neurons are capable of transmitting action potentials while glial cells are electrically incompetent. For over a century glial cells were neglected and it was thought they existed merely to provide trophic and structural support to neurons. However, in the past few decades it has become increasingly clear that glial cell functions underlie almost all aspects of nervous system development, maintenance, and health. During development, glia act as permissive substrates for axons, provide guidance cues, regulate axon bundling, facilitate synapse formation, refine synaptic connections, and promote neuronal survival. In the mature nervous system glial cells regulate adult neurogenesis through phagocytosis, act as the primary immune cell, and contribute to complex processes such as learning and memory. In recent years, glial cells have also become a primary focus in the study of neurodegenerative diseases. Mounting evidence shows that glial cells exert both beneficial as well as detrimental effects in the pathology of several nervous system disorders, and modulation of glial activity is emerging as a viable therapeutic strategy for many diseases. Although glial cells are critical to the proper development and functioning of the nervous system, there is still relatively little known about the molecular mechanisms used by glial cells, how they exert their effects on neurons, and how glia and neurons communicate.
Despite the relative simplicity and small size of the Drosophila nervous system, glial cell organization and function in flies shows a remarkable complexity similar to vertebrate glial cells. In this study I use Drosophila as a model organism to study cellular and molecular mechanisms of glial clearance of axonal debris after acute axotomy. In chapter two of this thesis, I characterize three distinct subtypes of glial cells in the adult brain; cell body glia which ensheath neuronal cell bodies in the cortex region of the brain, astrocyte like glial cells which bear striking morphological similarity to mammalian astrocytes and share common molecular components, and ensheathing glial cells which I show act as the primary phagocytic cell type in the neuropil region of the brain. In addition, I identify dCed-6, the ortholog of mammalian GULP, as a necessary component of the glial phagocytic machinery.
In chapter three of this thesis, I perform a candidate based, in vivo, RNAi screen to identify novel genes involved in the glial engulfment of degenerating axon material. The Gal4/UAS system was used to drive UAS-RNAi for approximately 300 candidate genes with the glial specific repo-Gal4 driver. Two assays were used as a readout in this screen, clearance of axon material five days after injury, and Draper upregulation one day after maxillary palp or antennal injury. Overall, I identified 20 genes which, when knocked down specifically in glial cells, result in axon clearance defects after injury. Finally, in chapter four I identify Stat92E as a novel glial gene required for glial phagocytic function. I show that Stat92E regulates both basal and injury induced Draper expression. Injury-induced Draper expression is transcriptionally regulated through a Stat92E dependent non-canonical signaling mechanism whereby signaling through the Draper receptor activates Stat92E which in turn transcriptionally activates draper through a binding site located in the first intron of Draper. Draper represents only the second receptor known to positively regulate Stat92E transcriptional activity under normal physiological conditions.
... In the CNS of Drosophila melanogaster embryos, programmed cell death eliminates many neurons and glia (Jacobs, 2000;Sonnenfeld and Jacobs, 1995b). In the case of the midline glia, approximately ten midline glial cells are generated in each segment by stage 13 of embryonic development. ...
... Recent work indicates that midline glial cell survival is mediated through activation of MAP kinase signaling in the midline glia via the reception of the EGFR ligand Spitz, which is provided by the developing neurons (Bergmann et al., 2002). In addition to midline glia, a subset of developing neurons and longitudinal glia (which flank the midline) are also removed through cell death (Hidalgo et al., 2001;Sonnenfeld and Jacobs, 1995b). Electron microscopic studies by Sonnenfeld and Jacobs demonstrated that the majority of cell corpses are expelled from the CNS and engulfed by macrophages (Sonnenfeld and Jacobs, 1995b). ...
... In addition to midline glia, a subset of developing neurons and longitudinal glia (which flank the midline) are also removed through cell death (Hidalgo et al., 2001;Sonnenfeld and Jacobs, 1995b). Electron microscopic studies by Sonnenfeld and Jacobs demonstrated that the majority of cell corpses are expelled from the CNS and engulfed by macrophages (Sonnenfeld and Jacobs, 1995b). Cell corpses can also be detected in glial cells both within and at the surface of the CNS, indicating that glial cells also contribute to removal of dead cells. ...
Cell death plays an essential role in development, and the removal of cell corpses presents an important challenge for the developing organism. Macrophages are largely responsible for the clearance of cell corpses in Drosophila melanogaster and mammalian systems. We have examined the developmental requirement for macrophages in Drosophila and find that macrophage function is essential for central nervous system (CNS) morphogenesis. We generate and analyze mutations in the Pvr locus, which encodes a receptor tyrosine kinase of the PDGF/VEGF family that is required for hemocyte migration. We find that loss of Pvr function causes the mispositioning of glia within the CNS and the disruption of the CNS axon scaffold. We further find that inhibition of hemocyte development or of Croquemort, a receptor required for macrophage-mediated corpse engulfment, causes similar CNS defects. These data indicate that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS.
... Some aspects of this migration, in particular invasion of the posterior end and migration along the VNC are mediated by PVR (Brückner et al., 2004;Wood et al., 2006), although PVR is primarily required for anti-apoptotic survival of hemocytes (Brückner et al., 2004). In the VNC, a significant amount of programmed cell death takes place in various cell types from the early stages of CNS formation to the end of embryogenesis (Abrams et al., 1993;White et al., 1994;Sonnenfeld and Jacobs, 1995;Zhou et al., 1995;Hidalgo et al., 2001;Peterson et al., 2002;Lundell, 2003;Miguel-Aliaga, 2004;Karcavich and Doe, 2005;Rogulja-Ortmann et al., 2007). Hemocytes phagocytose apoptotic bodies, opening up spaces and allowing for condensation of the nervous system (Olofsson and Page, 2005;Evans et al., 2010). ...
... As development proceeds, in the larva, and especially during metamorphosis and in the adult, functions of hemocytes are more predominantly adopted by glia. In particular, glia mediate phagocytosis of dead cells and neuron fragments during axonal and dendrite pruning, and following injury (Sonnenfeld and Jacobs, 1995;Watts et al., 2004;Kurant, 2011;Bittern et al., 2020;Furusawa and Emoto, 2020;Hilu-Dadia and Kurant, 2020). Hemocytes and glia show molecular parallels regarding their phagocytic receptors such as Simu and Drpr (MacDonald et al., 2006;Kurant et al., 2008;Shklyar et al., 2014;Evans et al., 2015;Weavers et al., 2016;Shlyakhover et al., 2018;Davidson and Wood, 2020), and their mutual dependence on the transcription factors glial cells missing (gcm) and glial cells missing 2 (gcm2), during embryonic development (Bernardoni et al., 1997;Alfonso and Jones, 2002;Trébuchet et al., 2019). ...
Across the animal kingdom, macrophages are known for their functions in innate immunity, but they also play key roles in development and homeostasis. Recent insights from single cell profiling and other approaches in the invertebrate model organism Drosophila melanogaster reveal substantial diversity among Drosophila macrophages (plasmatocytes). Together with vertebrate studies that show genuine expression signatures of macrophages based on their organ microenvironments, it is expected that Drosophila macrophage functional diversity is shaped by their anatomical locations and systemic conditions. In vivo evidence for diverse macrophage functions has already been well established by Drosophila genetics: Drosophila macrophages play key roles in various aspects of development and organogenesis, including embryogenesis and development of the nervous, digestive, and reproductive systems. Macrophages further maintain homeostasis in various organ systems and promote regeneration following organ damage and injury. The interdependence and interplay of tissues and their local macrophage populations in Drosophila have implications for understanding principles of organ development and homeostasis in a wide range of species.
... Migration along the developing ventral nerve cord (VNC) is an essential route for macrophage dispersal, with macrophages contacting the overlying epithelium and glial cells on the surface of the nerve cord ( Fig. 1a, b). During dispersal, macrophages encounter and clear apoptotic cells (Fig. 1c), while VNC glia also phagocytose dying cells 47 . The interaction of macrophages and glia suggested to us that impairing glialmediated apoptotic cell clearance could increase exposure of macrophages to apoptotic cell death in vivo, thus providing a model with which apoptotic cell-macrophage interactions and their effects on macrophage behaviour could be studied in detail. ...
... Therefore, a similar process may occur in flies facilitating contact between macrophages and apoptotic cells in repo mutants. In the absence of macrophages, glial cells become engorged with apoptotic cells, highlighting the interplay and competition for apoptotic cells between these phagocytes 47 . ...
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.
... Neurotrophic factors are also present in the CNS, and play a role in controlling neuronal and glia survival (Hidalgo et al. 2011). Apoptotic CNS cells are phagocytosed by CNS glia and removed from the CNS (Sonnenfeld and Jacobs 1995). ...
... The CNS is ensheathed by surface glia, which are impermeable to circulating hemocytes. Consequently, it is macrophage-like CNS glia, and not hemocytes, that undertake phagocytosis of dying CNS cells (Sonnenfeld and Jacobs 1995;Kurant et al. 2008). These phagocytic cells are cell body glia that morphologically resemble astrocytes. ...
The Drosophila embryonic central nervous system (CNS) is a complex organ consisting of ∼15,000 neurons and glia that is generated in ∼1 day of development. For the past 40 years, Drosophila developmental neuroscientists have described each step of CNS development in precise molecular genetic detail. This has led to an understanding of how an intricate nervous system emerges from a single cell. These studies have also provided important, new concepts in developmental biology, and provided an essential model for understanding similar processes in other organisms. In this article, the key genes that guide Drosophila CNS development and how they function is reviewed. Features of CNS development covered in this review are neurogenesis, gliogenesis, cell fate specification, and differentiation.
... Haemocytes perform vital roles during this stage, being involved in the functional restructuring itself (Kurata, Saito & Natori, 1992), and in cleaning up the remnants of these apoptotic cells (Sonnenfeld & Jacobs, 1995). Their failure to do so could result in a build-up of decaying cellular material in the pupae (Sonnenfeld & Jacobs, 1995). ...
... Haemocytes perform vital roles during this stage, being involved in the functional restructuring itself (Kurata, Saito & Natori, 1992), and in cleaning up the remnants of these apoptotic cells (Sonnenfeld & Jacobs, 1995). Their failure to do so could result in a build-up of decaying cellular material in the pupae (Sonnenfeld & Jacobs, 1995). Consequently, disruption of haemocyte proliferation, maturation and migration by pesticides during early developmental stages might not only directly impair immune functions, but also have delayed long-term effects during metamorphosis (Figure 1). ...
There is growing concern that declines in some managed and wild bee pollinator populations threaten biodiversity, the functioning of vital ecological processes and sustainable food production on a global scale.
In recent years, there has been increasing evidence that sublethal exposure to the neurotoxic class of insecticides (neonicotinoids) can undermine pollinator immunocompetence and amplify the effects of diseases, which have been suspected to be one of the drivers of pollinator declines. However, exactly how neonicotinoids might inhibit pollinator immunity remains elusive.
Here, we put forward a mechanistic framework to explain the effects of neurotoxic pesticides on insect immunocompetence. We propose that there is a close ontogenetic connection between the cellular arm (haemocytes) of the insect immune and nervous systems and that this connection makes the immune system of pollinators and other insects inherently susceptible to interference by neurotoxins such as neonicotinoids at sublethal doses.
Investigation of this connection is urgently needed to confirm the validity of this framework and develop a clear, mechanistically informed understanding of the interplay between neonicotinoids and disease ecology in pollinators. This in turn may enable us to develop strategies to mitigate impacts of neurotoxins on pollinators and/or enhance their impacts on pests.
A plain language summary is available for this article.
... The plasticity of Drosophila plasmatocytes can also be observed by increasing the apoptotic challenge they face in vivo. During embryogenesis, the other major cell-type involved in efferocytosis are glial cells, specified via the transcription factor repo (14,34,35). Loss of glial specification in repo null embryos results in increased levels of uncleared apoptotic cellsa change previously shown to impair plasmatocyte migration and wound responses (36). ...
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.
... We then visualised single glial cell morphology at 0 hours after larval hatching (0h ALH) to assess morphological diversity both within and between classes (Fig 1). Note that in addition to the 5 major glial classes described above, the VNC contains a distinct class called the midline glia, which are a transient population found only during embryonic and larval stages [28][29][30][31]. Although midline glia express wrapper, otherwise known as a cortex glia marker [32][33][34], they do not resemble cortex glia in form or function but instead ensheath commissural axons and play critical roles in axon guidance and VNC morphogenesis [35]. ...
Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system (CNS), where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single-cell RNA sequencing data of Drosophila glia in 2 well-characterised tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila , and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.
... The plasticity of Drosophila plasmatocytes can also be observed by increasing the apoptotic challenge they face in vivo. During embryogenesis, the other major cell-type involved in efferocytosis are glial cells, specified via the transcription factor repo (Halter et al., 1995;Kurant et al., 2008;Sonnenfeld & Jacobs, 1995). Loss of glial specification in repo null embryos results in increased levels of uncleared apoptotic cells -a change previously shown to impair plasmatocyte migration and wound responses (Armitage et al., 2020). ...
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.
... Flies have glia that cover the range of functions expected of mammalian glial types, including those for nerve insulation (wrapping glia), barrier function (subperineurial and perineurial glia), synaptic regulation (astrocyte-like glia) and neuronal cell body contacts (cortex glia) (Logan and Freeman, 2007;Yildirim et al., 2019). Drosophila subperineurial glia also have engulfment functions in both development and disease (Sonnenfeld and Jacobs, 1995). In the absence of injury, peripheral axons in Drosophila must be maintained via appropriate wrapping and metabolic signaling; these processes are controlled by the discoidin domain receptor (Ddr) and TGFβ signaling, respectively (Corty et al., 2022;Lassetter et al., 2023). ...
The degeneration of axons and their terminals occurs following traumatic, toxic, or genetically-induced insults. Common molecular mechanisms unite these disparate triggers to execute a conserved nerve degeneration cascade. In this review, we will discuss how models of peripheral nerve injury and neuropathy in Drosophila have led the way in advancing molecular understanding of axon degeneration and nerve injury pathways. Both neuron-intrinsic as well as glial responses to injury will be highlighted. Finally, we will offer perspective on what additional questions should be answered to advance these discoveries toward clinical interventions for patients with neuropathy.
... Glial cells carry out a number of vital functions in the Drosophila nervous system, many of which are analogous to those of mammalian glial cells [43][44][45][46] . To determine the type(s) of glial cells in which Vexed is required, we next knocked down expression in individual glial cell subtypes using specific Gal4 drivers. ...
The hallmark of Parkinson’s disease (PD) is the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. We previously completed a screen identifying genes associated with the progressive degeneration of DA neurons. Here we describe the role of a previously uncharacterized gene, CG42339, in the loss of DA neurons using Drosophila Melanogaster. CG42339 mutants display a progressive loss of DA neurons and locomotor dysfunction, along with an accumulation of advanced glycation end products (AGEs) in the brain. Based on this phenotype, we refer to CG42339 as vexed. We demonstrate that vexed is specifically required within cortex glia to maintain neuronal viability. Loss of vexed function results in excessive activation of the innate immune response in the brain, leading to loss of DA neurons. We show that activation of the innate immune response leads to increased nitric oxide signaling and accumulation of AGEs, which ultimately result in neurodegeneration. These results provide further insight into the relationship between the role of the immune response in the central nervous system and how this impacts neuronal viability.
... Phagocytes in the fruit fly Drosophila melanogaster clear apoptotic corpses and axonal debris to maintain tissue homeostasis and repair the nervous system after injury. Engulfment in flies is carried out by an array of cell types including hemocytes (23), epithelial cells (24), and glia (2,25). The Draper receptor, expressed in each of these cell types, is comprised of an extracellular emlin (EMI) domain that facilitates protein-protein interactions (26) and nimrod (NIM) repeats, EGF-like domains found on multiple fruit fly innate immune receptors (27). ...
Transmembrane protein engulfment receptors expressed on the surface of phagocytes engage ligands on apoptotic cells and debris to initiate a sequence of events culminating in material internalization and immunologically beneficial outcomes. Engulfment receptors are modular, comprised of functionally independent extracellular ligation domains and cytosolic signaling motifs. Cognate kinases, adaptors, and phosphatases regulate engulfment by controlling the degree of receptor activation in phagocyte plasma membranes, thus acting as receptor-proximal signaling modules. Here, we review recent efforts to reprogram phagocytes using modular synthetic receptors composed of antibody-based extracellular domains fused to engulfment receptor signaling domains. To aid the development of new phagocyte reprogramming methods, we then define the kinases, adaptors, and phosphatases that regulate a conserved family of engulfment receptors. Finally, we discuss current challenges and opportunities for the field.
... Importantly, glia can remove entire neurons from neural circuits during developmental remodeling. For example, glia phagocytose neurons in the developing mammalian dorsal root ganglia 12 , and similar glial phagocytic activity occurs within the developing Drosophila nervous system [13][14][15][16][17] . It is through such glial elimination that the developing brain sculpts the neural circuitry, facilitating optimized function and behavioral output. ...
Glia engulf and phagocytose neurons during neural circuit developmental remodeling. Disrupting this pruning process contributes to Fragile X syndrome (FXS), a leading cause of intellectual disability and autism spectrum disorder in mammals. Utilizing a Drosophila FXS model central brain circuit, we identify two glial classes responsible for Draper-dependent elimination of developmentally transient PDF-Tri neurons. We find that neuronal Fragile X Mental Retardation Protein (FMRP) drives insulin receptor activation in glia, promotes glial Draper engulfment receptor expression, and negatively regulates membrane-molding ESCRT-III Shrub function during PDF-Tri neuron clearance during neurodevelopment in Drosophila. In this context, we demonstrate genetic interactions between FMRP and insulin receptor signaling, FMRP and Draper, and FMRP and Shrub in PDF-Tri neuron elimination. We show that FMRP is required within neurons, not glia, for glial engulfment, indicating FMRP-dependent neuron-to-glia signaling mediates neuronal clearance. We conclude neuronal FMRP drives glial insulin receptor activation to facilitate Draper- and Shrub-dependent neuronal clearance during neurodevelopment in Drosophila.
... However, the majority of apoptotic cells in the CNS are engulfed by subperineurial glia in a fashion similar to the microglia of the vertebrate CNS. In a Drosophila mutant, which lacks macrophages, subperineurial glia contain an abundance of apoptotic cells, indicating that there is a cross-talk between macrophages and subperineurial glia resulting in the removal of apoptotic cells (849). Inhibition of a receptor required for macrophage-mediated engulfment of dead cells causes CNS defects, indicating that macrophage-mediated clearance of cell corpses is required for proper morphogenesis of the Drosophila CNS (817). ...
Microglial cells derive from fetal macrophages which immigrate into and disseminate throughout the central nervous system (CNS) in early embryogenesis. After settling in the nerve tissue, microglial progenitors acquire an idiosyncratic morphological phenotype with small cell body and moving thin and highly ramified processes currently defined as “resting or surveillant microglia”. Physiology of microglia is manifested by second messenger-mediated cellular excitability, low resting membrane conductance, and expression of receptors to pathogen- or damage-associated molecular patterns (PAMPs and DAMPs), as well as receptors to classical neurotransmitters and neurohormones. This specific physiological profile reflects adaptive changes of myeloid cells to the CNS environment.
... We have characterized the time-course of these interactions in greater detail employing fluorescent markers of hemocyte identity in both living and dissected abdomens. The dynamic pattern of hemocyte migration during embryogenesis has been documented in detail, as their movement reflects the removal of apoptotic cells and the deposition of ECM (Moreira et al., 2010;Sonnenfeld and Jacobs, 1995). Embryonic hemocytes express Collagen IV (Viking), and can be tracked with both the hemolectin > Red-h-Stinger (hml-DsRed; Makhijani et al., 2011) and the Vkg-GFP gene trap. ...
Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. Here we characterize hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4 h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.
... In Drosophila, there are three cell types reported to function as phagocytic cells: professional phagocytes-macrophages/hemocytes, glial cells, and non-professional phagocytes-epithelial cells (30)(31)(32). Hemocytes are macrophage-like cells reported to engulf ACs or dendrite debris during pruning of Drosophila sensory dendrites (33) and embryogenesis (34). Drosophila glia act much similar role in engulfing dying cells or degenerating axons of the nervous system as their counterparts in mammals (35), degenerating dendrites are primarily cleared by the epidermal epithelia (36). ...
The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode Caenorhabditis elegans, which lacks “professional” phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In Drosophila melanogaster, ACs are engulfed by macrophages, glial, and epithelial cells. Drosophila macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in Drosophila has identified some key elements, like the receptors Croquemort and Draper, promoting Drosophila as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in Drosophila, and discuss the physiological outcomes and consequences of this process.
... Other glial cell types can also become phagocytic both in development and in response to injury. For instance, midline glia phagocytose apoptotic cells in normal development (Sonnenfeld and Jacobs, 1995). Injury to the larval ventral nerve cord (VNC) induces NG to become phagocytic and engulf cell debris and apoptotic cells (Kato, et al., 2011). ...
Although the central nervous system does not regenerate, injury induces repair and regenerative responses in glial cells. In mammals, activated microglia clear up apoptotic cells and debris resulting from the injury, astrocytes form a scar that contains the lesion, and NG2-glia elicit a prominent regenerative response. NG2-glia regenerate themselves and differentiate into oligodendrocytes, which remyelinate axons leading to some recovery of locomotion. The regenerative response of glial cells is evolutionarily conserved across the animals and Drosophila genetics revealed an underlying gene network. This involves the genes Notch, kon-tiki, eiger, dorsal and prospero, homologues of mammalian Notch1, ng2, TNF, NFκB and prox1, respectively. Feedback loops between these genes enable a surge in proliferation in response to injury and ensuing differentiation. Negative feedback sets a timer for proliferation, and prevents uncontrolled growth that could lead to glioma. Remarkable parallels are found in these genetic relationships between fruit-flies and mammals. Drosophila findings provide insights into gene functions that could be manipulated in stem cells and progenitors for therapeutic repair. This article is protected by copyright. All rights reserved.
... In addition, during development about 50% of neurons undergo programmed cell death (PCD) while others require axonal, dendritic, or synaptic pruning. Clearance of apoptotic corpses and engulfment of pruned parts is mediated by microglia and astrocytes in vertebrates [1,[5][6][7][8][9] and by various types of glia in Drosophila [10][11][12][13][14][15][16][17]. ...
In the nervous system, glial cells provide crucial insulation and trophic support to neurons and are important for neuronal survival. In reaction to a wide variety of insults, glial cells respond with changes in cell morphology and metabolism to allow repair. Additionally, these cells can acquire migratory and proliferative potential. In particular, after axonal damage or pruning the clearance of axonal debris by glial cells is key for a healthy nervous system. Thus, bidirectional neuron-glial interactions are crucial in development, but little is known about the cellular sensors and signalling pathways involved. In here, we show that decreased cellular fitness in retinal progenitors caused by reduced Drosophila Myc expression triggers non cell-autonomous activation of retinal glia proliferation and overmigration. Glia migration occurs beyond its normal limit near the boundary between differentiated photoreceptors and precursor cells, extending into the progenitor domain and is stimulated by JNK activation (and the function of its target Mmp1), while proliferative responses are mediated by Dpp/TGF-β signalling activation.
... The removal of cellular debris in the CNS, which in vertebrates is carried out by microglia, is a function shared between hemocytes and (macro-)glia in Drosophila. In the fly embryo, primary neuropil glia and surface glia, in addition to hemocytes, act as macrophages (Sonnenfeld and Jacobs, 1995). Postembryonically, neuropil glia exhibits functions reminiscent of vertebrate microglia, depending on the context; astrocyte-like cells clear neuronal debris as a consequence of neuronal remodeling during metamorphosis, whereas ensheathing glia clears neuronal debris during neurite injury in the adult brain (Doherty et al., 2009, Tasdemir-Yilmaz and. ...
Glia of vertebrates and invertebrates alike represents a diverse population of cells in the nervous system, divided into numerous classes with different structural and functional characteristics. In insects, glia fall within three basic classes: surface, cell body, and neuropil glia. Due to the glial subclass-specific markers and genetic tools available in Drosophila, it is possible to establish the progenitor origin of these different populations and reconstruct their migration and differentiation during development. We review, and posit when appropriate, recently elucidated aspects of glial developmental dynamics. In particular, we focus on the relationships between mature glial subclasses of the larval nervous system (primary glia), born in the embryo, and glia of the adult (secondary glia), generated in the larva.
... Apart from their role in neuronal survival, glial cells act as the macrophages of the CNS by removing neuronal debris. They are activated after neuronal PCD (Sonnenfeld and Jacobs 1995, Freeman, Delrow et al. 2003, Mergliano and Minden 2003. A number of phagocytic receptors, such as Draper and Six-microns-under (Simu), are expressed in embryonic glia and participate in the phagocytosis of apoptotic neurons (Kurant, Axelrod et al. 2008, Kurant 2011. ...
Nervous system development is a process that integrates cell proliferation, differentiation and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerge while integrating this information. This article is protected by copyright. All rights reserved.
... Plasmatocytes phagocytose apoptotic neurons along the CNS midline, which ensures proper condensation of the nervous system [112,113] and is required for embryonic survival. This has been demonstrated in Bicaudal-D and srp mutants, which lack embryonic hemocytes [22,113,114], as well as crq mutants and mutants of the receptor tyrosine kinase Pvr (PDGF/VEGF Receptor) [113], in which embryonic hemocytes undergo premature apoptotic death [115]. In the embryonic tracheal system, macrophages have a similar role in the elimination of apoptotic cells during tissue remodeling [116]. ...
The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life.
... CED-1 engagement with an unidentified ligand on the engulfment target is thought to initiate downstream signaling through the PTB domain coiled-coil protein Ced-6 (Reddien and Horvitz, 2004) and subsequent corpse engulfment. Drosophila glial cells have long been known to be the primary engulfing cell type in the nervous system by EM studies (Sonnenfeld and Jacobs, 1995) . Previous work from the Freeman laboratory demonstrated that nearly all glia express Draper, and that neuronal cell corpses accumulate in draper mutant embryos (Freeman et al., 2003a). ...
Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ).
During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth.
My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis.
My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.
... This suggests that cortex glia could supply gases and nutrients to the neurons that they ensheath; this role is similar to mammalian astrocytic functions. In the Drosophila embryo, a glial subtype having specific immune functions like microglia does not exist; all glia seem to have the capability of immune functions, such as engulfment of cell corpses (Freeman et al., 2003;Sonnenfeld and Jacobs, 1995). ...
The nervous system is composed of neurons and glia. Glial cells have been neglected and thought to have only a supportive role in the nervous system, even though ~60% of the mammalian brain is composed of glia. Yet, in recent years, it has been shown that glial cells have several important functions during the development, maintenance and function of the nervous system. Glial cells regulate both pre and post mitotic neuronal survival during normal development and maintenance of the nervous system as well as after injury, are necessary for axon guidance, proper axon fasciculation, and myelination during development, promote synapse formation, regulate ion balance in the extracellular space, are required for normal synaptic function, and have immune functions in the brain. Although glia have crucial roles in nervous system development and function, there are still much unknown about the underlying molecular mechanisms in glial development, function and glial-neuronal communication.
Drosophila offers great opportunity to study glial biology, with its simple yet sophisticated and stereotypic nervous system. Glial cells in flies show great complexity similar to the mammalian nervous system, and many cellular and molecular functions are conserved between flies and mammals. In this study, I use Drosophila as a model organism to study the function of one subtype of glia: astrocytes. The role of astrocytes in synapse formation, function and maintenance has been a focus of study. However, their role in engulfment and clearance of neuronal debris during development remains unexplored.
I generated a driver line that enables the study of astrocytes in Drosophila.In chapter two of this thesis, I characterize astrocytes during metamorphosis, when extensive neuronal remodeling takes place. I found that astrocytes turn into phagocytes in a cell-autonomous, steroid-dependent manner, by upregulating the phagocytic receptor Draper and forming acidic phagolysosomal structures. I show that astrocytes clear neuronal debris during nervous system remodeling and that this is a novel function for astrocytes during the development of nervous system. I analyzed two different neuronal populations: MB γ neurons that prune their neurites and vCrz+ neurons that undergo apoptosis. I discovered that MB γ axons are engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 pathways in a partially redundant way. Interestingly, Draper is required for clearance of vCrz+ cell bodies, while Crk/Mbc/dCed-12, but not Draper, are required for clearance of vCrz+ neurites. Surprisingly, I also found that loss of Draper delayed vCrz+ neurite degeneration, suggesting that glia facilitate neurite destruction through engulfment signaling.
Taken together, my work identifies a novel function for astrocytes in the clearance of synaptic and neuronal debris during developmental remodeling of the nervous system. Additionally, I show that Crk/Mbc/dCed-12 act as a new glial signaling pathway required for pruning, and surprisingly, that glia use different engulfment pathways to clear neuronal debris generated by cell death versus local pruning.
... Glial cells are the primary immunocompetent cells of the brain, and they perform many functions during the development of the nervous system. They modu late proliferation of the neural stem cells (Ebens et al., 1993), regulate differentiation of neuronal precursors, direct axon growth Sepp et al., 2001;Gilmour et al., 2002), form the environ ment of nerves and the individual axons (Barres, 2008;Nave and Trapp, 2008), divide and isolate lobes of the brain and their compartments (Oland and Tolbert, 2003;Awasaki et al., 2008), nourish neurons (Xiong and Montell, 1995;Booth et al., 2000), engulf neurons and various fragments of dead cells during the devel opment of the nervous system (Sonnenfeld and Jacobs, 1995;Awasaki, Ito, 2004;Watts et al., 2004), and are involved in synapse formation and develop ment (Barres, 2008). In adult animals, glia establishes the ion balance in the CNS, absorbs neurotransmitters after carrying the electrical signal across the synapse (Danbolt, 2001), insulates and protects neurons via the formation or regulation of the blood brain barrier (Abbott, 2005), and modulates the activity of synapses (Barres, 2008). ...
Nervous and immune systems have many general features in their organization and functioning in various animal species from insects to mammals. These systems are capable to regulate effectively each other by exchange of information through rather small molecules like oligopeptides, cytokines, and neuropeptides. For many such molecules, that function as transmitters or signaling peptides, their origin and receptors are common within nervous and immune systems. Development of nervous and immune systems during ontogenesis and their functions in various species are controlled by the ubiquitous HYPERLINK "http://slovari.yandex.ru/proteolytic/en-ru/Medical/" \1 "longvo/" proteolytic ubiquitin-proteasome system (UPS). UPS regulates key biochemical processes in both systems by providing formation of synaptic connections and synaptic plasticity, and governs immune responses. In the review, the molecular mechanisms of functioning and interaction between nervous and immune systems are considered in different species of invertebrats and vertebrats. The role of UPS in these processes in the main subject of this review.
... These cells exhibit many morphological and functional similarities to mammalian counterparts. However, the fruit fly is deprived of a CNS glia subtype specifically assigned to immune functions like microglia in mammals; rather, all glia seem competent to perform immune--like functions such as engulfment of neuronal corpses during development (Freeman et al., 2003;Freeman and Doherty, 2006; Sonnenfeld and Jacobs, 1995). Draper is a transmembrane protein that acts as a docking phagocytic receptor. ...
Alzheimer’s disease (AD) is characterized by the accumulation of amyloid β (Aβ) in the brain. Several lines of evidences point towards a strong link between AD and neuroinflammation. However, the exact molecular events of the innate immune reactions against Aβ need to be elucidated. We used Drosophila as a model organism to study the impact of innate immune reactions on AD. During my PhD I have been able to: (1) establish a Drosophila model to study the inflammatory responses inAD, (2) demonstrate that the Drosophila inflammatory IMD pathway plays a neuroprotective role in the development of AD-like phenotypes, (3) generate the IMD interactome dataset that could help elucidate the mechanisms linking AD to neuroinflammation, and (4) introduce a forward genetic screen for the identification of modifier genes of AD. We believe that the outcomes from our Drosophila studies could provide the basis for new therapeutic interventions against AD.
... Macrophages and glia share many similarities in corpse clearance, not only functionally, but also molecularly. Many known macrophage phagocytosis factors are upregulated in glia, pointing to these cells having a similar molecular repertoire as macrophages (Alfonso & Jones 2002;Sonnenfeld & Jacobs 1995). ...
Phagozyten entfernen apoptotische Zellen während der Entwicklung und beseitigen Pathogene im Immunsystem. Die zugrundeliegenden molekularen und zellulären Mechanismen, insbesondere die Unterschiede zwischen Makrophagen und nicht-professionellen Phagozyten wie Gliazellen, sind weitestgehend unklar. Wir haben neuartige Zellkultur-basierte Assays entwickelt, um 86 Kandidatengene zu testen, die wir aus der Literatur sowie unserem Expressions-Profiling in embryonalen Gliazellen von Drosophila melanogaster zusammengestellt haben. Die Genfunktion wurde durch RNAi herabgesenkt und die Phagozytoseeffizienz wurde mittels FACS untersucht; um die funktionelle Spezifität der Gene zu messen, haben wir nicht nur apoptotische Zellen, sondern auch Bakterien und Beads als „Essen“ angeboten. Mit Hilfe von Null-Mutanten und transgenem RNAi wurden die Ergebnis in vivo validiert. Um die Phagozytose apoptotischer Zellen testen, haben wir untersucht, wie Makrophagen und Gliazellen tote Zellen während der Embryonalentwicklung entfernen, während zur Untersuchung der bakteriellen Phagozytoze adulte Fliegen mit Bakterien infiziert wurden. Unser Screen liefert einen Querschnitt durch die verschiedenen Schritte der Phagozytose. In Bezug auf die Erkennung apoptotischer Zellen finden wir sowohl bekannte als auch neue Akteure für Makrophagen und Gliazellen. Außerdem zeigen wir, dass Vesikeltransport für die Phagozytose apoptotischer Zellen erforderlich ist. Überraschenderweise werden Rezeptoren zur Bakterienerkennung auch für apoptotische Zellen benötigt. Umgekehrt sind Apoptose- Rezeptoren auch für bakterielle Phagozytose notwendig, wodurch eine grundlegende Kreuz-Spezifität zutage tritt. Unsere Arbeit liefert die erste systematische und vergleichende Analyse der verschiedenen Phagozytosearten. Durch die Identifizierung vieler neuer Faktoren legt diese Arbeit den Grundstein für ein mechanistisches Verständnis der Phagozytose von apoptotischen Zellen und Bakterien durch Makrophagen und Gliazellen.
... In the past, however, most manipulations were performed on the entire glial population because of the availability of a very strong and glial-specific driver (repo-Gal4). Many very interesting glial functions have been described (too many to be summarized here), including the engulfment of neuronal cell corpses during embryonic development (Sonnenfeld and Jacobs 1995;Freeman et al. 2003;Kurant et al. 2008), and neurotrophic support through the generation of a neurotrophin-like family of molecules, includ-ing Drosophila neurotrophin 1 (DNT1), neurotrophin 2 (DNT2), Spatzle (Spz) (Zhu et al. 2008), and dmMANF (Palgi et al. 2009), which appear to act through the TLRs Toll6 and Toll7 receptors (McIlroy et al. 2013). Future studies should be aimed not only at further defining the molecular pathways mediating these neuronglia signaling events, but also the precise glial subtype(s) involved. ...
Molecular genetic approaches in small model organisms like Drosophila have helped to elucidate fundamental principles of neuronal cell biology. Much less is understood about glial cells, although interest in using invertebrate preparations to define their in vivo functions has increased significantly in recent years. This review focuses on our current understanding of the three major neuron-associated glial cell types found in the Drosophila central nervous system (CNS)-astrocytes, cortex glia, and ensheathing glia. Together, these cells act like mammalian astrocytes: they surround neuronal cell bodies and proximal neurites, are coupled to the vasculature, and associate closely with synapses. Exciting recent work has shown essential roles for these CNS glial cells in neural circuit formation, function, plasticity, and pathology. As we gain a more firm molecular and cellular understanding of how Drosophila CNS glial cells interact with neurons, it is becoming clear they share significant molecular and functional attributes with mammalian astrocytes.
Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
... Macrophages/hemocytes, or microglia-like cells have not been identified in the fly CNS. During Drosophila embryogenesis, apoptotic cell clearance is performed by professional phagocytes, called macrophages [55]. Macrophages, also designated as hemocytes, are found in the hemolymph either as sessile or as freely circulating moieties, being associated with various tissues. ...
Glia are abundant cells in the brain of animals ranging from flies to humans. They perform conserved functions not only in neural development and wiring, but also in brain homeostasis. Here we show that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. We call these cells MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. Our findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. We suggest that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-014-1789-7) contains supplementary material, which is available to authorized users.
... Glial cells play a critical role in the normal brain development in Drosophila through axonal growth guidance and removal of apoptotic cells (97,224). Whereas some astroglia-like cells are present in the expanded forebrain parenchyma in the cartilaginous fish such as rays and sharks, most bony fish lack parenchymal astrocytes in the gray and white matter of the brain and spinal cord (10,103,108). ...
Astrocytes are the most abundant cells in the central nervous system (CNS) that provide nutrients, recycle neurotransmitters, as well as fulfill a wide range of other homeostasis maintaining functions. During the past two decades, astrocytes emerged also as increasingly important regulators of neuronal functions including the generation of new nerve cells and structural as well as functional synapse remodeling. Reactive gliosis or reactive astrogliosis is a term coined for the morphological and functional changes seen in astroglial cells/astrocytes responding to CNS injury and other neurological diseases. Whereas this defensive reaction of astrocytes is conceivably aimed at handling the acute stress, limiting tissue damage, and restoring homeostasis, it may also inhibit adaptive neural plasticity mechanisms underlying recovery of function. Understanding the multifaceted roles of astrocytes in the healthy and diseased CNS will undoubtedly contribute to the development of treatment strategies that will, in a context-dependent manner and at appropriate time points, modulate reactive astrogliosis to promote brain repair and reduce the neurological impairment.
The clearance of dead and dying cells, termed efferocytosis, is a rapid and efficient process and one that is critical for organismal health. The extraordinary speed and efficiency with which dead cells are detected and engulfed by immune cells within tissues presents a challenge to researchers who wish to unravel this fascinating process, since these fleeting moments of uptake are almost impossible to catch in vivo. In recent years, the fruit fly (Drosophila melanogaster) embryo has emerged as a powerful model to circumvent this problem. With its abundance of dying cells, specialist phagocytes and relative ease of live imaging, the humble fly embryo provides a unique opportunity to catch and study the moment of cell engulfment in real‐time within a living animal. In this review, we explore the recent advances that have come from studies in the fly, and how live imaging and genetics have revealed a previously unappreciated level of diversity in the efferocytic program. A variety of efferocytic strategies across the phagocytic cell population ensure efficient and rapid clearance of corpses wherever death is encountered within the varied and complex setting of a multicellular living organism.
Although glial cells have been typically regarded as support cells of the neurons, it is increasingly clear now that they play a critical role(s) in the development, function, and maintenance of the nervous system and are also required for the maintenance of ionic balance in the CNS, synaptic signaling, regulation of the blood-brain barrier, and brain immune response. However, several functional aspects of glial cells remain enigmatic; for instance, their presence and potential function(s) in glutamatergic, cholinergic or GABAergic synapses are largely unknown. Similarly, their precise contribution in the etiology of neuronal disorders is still elusive. In fact, mammalian glia are difficult to analyze in vivo, and our understanding of glia biology has largely emerged from the studies on the primary cultures. Nevertheless, the majority of such in vitro findings have not been confirmed or repeated in experiments with the living organism, which is important since glia and neurons exist in close association with each other soon after the differentiation. In view of the above, and also due to limitations attached with the studies on human genetics, Drosophila has emerged as one of the prime model systems for glial research. Intriguingly, despite the significant difference in size, the Drosophila adult brain exhibits structures and functions similar to the mammalian brains and shares a number of glial characteristics. In view of the availability of sophisticated genetic tools, diverse behavioral features, and evolutionarily conserved genome, the Drosophila glia seem well-positioned to provide exciting insights into glial biology, which will be relevant to the glial functions in higher organisms including humans. The present chapter summarizes the current knowledge and enduring contribution of Drosophila in glia research.Keywords
Drosophila
BrainGlia
Neurodegeneration
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.
The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells appeared to transiently accumulate in egr
3
mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr
3
mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr
3
mutant, its sister allele, egr
1
, and its parental strain, Regg1GS9830 As the egr
3
and egr
1
alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.
The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells persisted longer in egr ³ mutant wing discs than in wild type discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr ³ mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr ³ mutant, its sister allele, egr ¹ , and its parental strain, Regg1 GS9830 . As the egr ³ and egr ¹ alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.
Multicellular organisms are not created through cell proliferation alone. It is through cell death that an indefinite cellular mass is pared back to reveal its true form. Cells are also lost throughout life as part of homeostasis and through injury. This detritus represents a significant burden to the living organism and must be cleared, most notably through the use of specialized phagocytic cells. Our understanding of these phagocytes and how they engulf cell corpses has been greatly aided by studying the fruit fly, Drosophila melanogaster Here we review the contribution of Drosophila research to our understanding of how phagocytes respond to cell death. We focus on the best studied phagocytes in the fly: the glia of the central nervous system, the ovarian follicle cells, and the macrophage-like hemocytes. Each is explored in the context of the tissue they maintain as well as how they function during development and in response to injury.
The molecular and cellular mechanism for clearance of dead neurons was explored in the developing Drosophila optic lobe. During development of the optic lobe, many neural cells die through apoptosis, and corpses are immediately removed in the early pupal stage. Most of the cells that die in the optic lobe are young neurons that have not extended neurites. In this study, we showed that clearance was carried out by cortex glia via a phagocytosis receptor, Draper (Drpr). drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Drpr that was expressed in other subtypes of glia did not mediate clearance. Shark and Ced-6 mediated clearance of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe.
In traumatic brain injury (TBI) the initial injury phase is followed by a secondary phase that contributes to neurodegeneration. Yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI, which we term Drosophila Closed Head Injury (dCHI), where well-controlled, non-penetrating strikes are directly delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. To discover novel mediators of TBI, we used glial targeted translating ribosome affinity purification in combination with RNA sequencing. We detected significant changes in the transcriptome at various times after TBI including in genes involved in innate immunity within 24 hours after TBI. To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB and found that while lethality effects were still evident, changes in sleep and motor function were substantially reduced. These studies validate a new head-specific model for TBI in Drosophila and identify glial immune pathways as candidate in vivo mediators of TBI effects.
Traumatic brain injury (TBI) is one of the leading causes of death and disability in the developed world [1-3]. Yet the underlying mechanisms that lead to long term physical, emotional, and cognitive impairment remain unclear.
Unlike in most forms of trauma, a large percentage of people killed by traumatic brain injuries do not die immediately but rather days or weeks after the insult [4]. TBI consists of a primary and a secondary phase. The primary brain injury is the result of an external mechanical force, resulting in damaged blood vessels, axonal shearing [5], cell death, disruption of the blood– brain barrier, edema, and the release of damage associated molecular patterns (DAMPs) and excitotoxic agents [6]. In response, local glia and infiltrating immune cells upregulate cytokines (tumor necrosis factor α) and interleukins (IL-6 and IL-1β) that drive post-traumatic neuroinflammation [7-10]. This secondary injury develops over a much longer time course, ranging from hours to months after the initial injury and is the result of a complex cascade of metabolic, cellular and molecular processes [11-13]. Neuroinflammation is beneficial when it is promoting clearance of debris and regeneration [14] but can become harmful, mediating neuronal death, progressive neurodegeneration, and neurodegenerative disorders [15-18]. The mechanisms underlying these opposing outcomes are largely unknown, but are thought to depend of the location and timing of the neuroinflammatory response [19, 20]. It remains to be determined what the relative roles of TBI-induced neuroinflammation and other TBI-induced changes are in mediating short and long-term impairments in brain function in vivo.
To study the mechanisms that mediate TBI pathology in vivo over time, we employ the fruit fly Drosophila melanogaster , a model organism well suited to understanding the in vivo genetics of brain injury. Despite considerable morphological differences between flies and mammals, the fly brain operates on similar principles through a highly conserved repertoire of neuronal signaling proteins, including a large number of neuronal cell adhesion receptors, synapse-organizing proteins, ion channels and neurotransmitter receptors, and synaptic vesicle-trafficking proteins [21]. This homology makes Drosophila a fruitful model to study neurodegenerative disorders [22], including ALS [23], Alzheimer’s disease [24], Huntington’s disease [25] and Parkinson’s disease [26].
Trauma-induced changes in glial gene expression are a highly conserved feature of both mammalian [27, 28] and Drosophila glia [29-32] (reviewed in [33]). In Drosophila , glia are able to perform immune-related functions [32, 34]. Ensheathing glia can act as phagocytes and contribute to the clearance of degenerating axons from the fly brain [29, 31, 35]. The Drosophila innate immune system is highly conserved with that of mammals and consists primarily of the Toll, Immunodeficiency (Imd) and Janus Kinase protein and the Signal Transducer and Activator of Transcription (JAK-STAT) pathways, which together combat fungal and bacterial infections [36, 37]. Dysregulation of cerebral innate immune signaling in Drosophila glial cells can lead to neuronal dysfunction and degeneration [38, 39], suggesting that changes in glia cells could underlie secondary injury mechanisms in our Drosophila model of TBI.
Existing Drosophila TBI models [40, 41] deliver impacts to the entire body, not just the head, and thus, one cannot definitively attribute ensuing phenotypes to TBI. To remove the confound of bodily injury, we have developed a novel, head-specific Drosophila model for TBI, Drosophila Closed Head Injury (dCHI). Here we show that by delivering precisely controlled, non-penetrating strikes to an unanesthetized fly’s head, we can induce cell death and increased mortality in a dose-dependent manner. In addition, TBI results in impaired motor control and decreased, fragmented sleep. Impaired motor control persists for many days after TBI while the sleep phenotype disappears after three days. These TBI-induced behavioral phenotypes do not occur in mutants lacking the master immune regulator NF-κB Relish ( Rel ), even though TBI-induced mortality is greatly induced in these mutants. In wild type flies, TBI results in changes in glial gene expression, where many immune related genes are upregulated 24 hours after injury. Together, these results establish a platform where powerful Drosophila genetics can be utilized to study the complex cascade of secondary injury mechanisms that occur after TBI in order to genetically disentangle its beneficial and detrimental effects.
Monocyte/macrophage and natural killer (NK) cells are partners from a phylogenetic standpoint of innate immune system development and its evolutionary progressive interaction with adaptive immunity. The equally conservative ways of development and differentiation of both invertebrate hemocytes and vertebrate macrophages are reviewed. Evolutionary conserved molecules occurring in macrophage receptors and effectors have been inherited by vertebrates after their common ancestor with invertebrates. Cytolytic functions of mammalian NK cells, which are rooted in immune cells of invertebrates, although certain NK cell receptors (NKRs) are mammalian new events, are characterized. Broad heterogeneity of macrophage and NK cell phenotypes that depends on surrounding microenvironment conditions and expression profiles of specific receptors and activation mechanisms of both cell types are discussed. The particular tissue specificity of macrophages and NK cells, as well as their plasticity and mechanisms of their polarization to different functional subtypes have been underlined. The chapter summarized studies revealing the specific molecular mechanisms and regulation of NK cells and macrophages that enable their highly specific cross-cooperation. Attention is given to the evolving role of human monocyte/macrophage and NK cell interaction in pathogenesis of hypersensitivity reaction-based disorders, including autoimmunity, as well as in cancer surveillance and progression.
Master genes are known to induce the differentiation of a multipotent cell into a specific cell
type. These molecules are often transcription factors that switch on the regulatory cascade that
triggers cell specification. Gcm was first described as the master gene of the glial fate in
Drosophila as it induces the differentiation of neuroblasts into glia in the developing nervous
system. Later on, Gcm was also shown to regulate the differentiation of blood, tendon and
peritracheal cells as well as that of neuronal subsets. Thus, the glial master gene is used in at
least four additional systems to promote differentiation. To understand the numerous roles of
Gcm, we recently reported a genome-wide screen of Gcm direct targets in the Drosophila
embryo. This screen provided new insight into the role and mode of action of this powerful
transcription factor, notably on the interactions between Gcm and major differentiation pathways such as the Hedgehog, Notch and JAK/STAT. Here, we discuss the mode of action
of Gcm in the different systems, we present new tissues that require Gcm and we revise the
concept of ‘master gene’.
Holometabolous insects, such as the dipteran Drosophila, exhibit two distinct life forms and undergo a complete metamorphosis. The Drosophila embryo develops over the course of one day and hatches into a motile and feeding first instar larva, which grows and subsequently moults twice (Figure 1). The first and second larval instars each last about a day, while the third instar lasts approximately three days. At the end of the third larval instar, the animal ceases to feed, climbs up a suitable substrate, and initiates pupariation. The pupal stage lasts approximately four days, during which time the larva metamorphoses into the adult fly. The ease of rearing Drosophila, their fecundity, and short life cycle have all contributed to their use as a genetic model system (see Ashburner, 1989). In addition, the Drosophila genome is relatively small (∼1.6 X 108 base pairs per haploid genome), and is organised into only four chromosomes which are all linked by a common chromocentre. The large polytene chromosomes of the larval salivary gland greatly facilitate the cytological mapping of chromosomal rearrangements as well as the localisation of cloned genes via in situ hybridisation. In addition, the use of balancer chromosomes, which repress recombination and provide dominant phenotypic markers, greatly facilitates genetic screens and the stable maintenance of homozygous lethal chromosomes.
Microglia are the resident macrophages that colonize the central nervous system during embryonic development. In the developing brain, they exist as the round or amoeboid microglial cells (AMCs) which are localised preferentially in the white matter. In the postnatal period AMCs emit a variable number of processes to become the ramified microglia representing the mature form of microglia. Among the various hypotheses proposed for microglial ontogeny, the concept of the haemopoietic/monocytic origin of microglia is generally accepted. The phagocytic property of the AMCs is evidenced by their capability to ingest exogenous materials such as carbon particles, horseradish peroxidase etc. In addition to their phagocytic nature, demonstration of the role of microglia in vascularisation, neurogenesis and synaptogenesis has provided new insights into the varied functions of microglia. The immune functions of microglia are supported by the expression of major histocompatability complex class I and II antigens, cytokine and chemokine receptors. The cytokines and chemokines secreted by activated microglia have been implicated in augumenting neurotoxicity. This review focuses on the ontogeny, functions, and involvement of microglia in anomalies of the developing brain.
This resource is the long-awaited new revision of the most highly regarded reference volume on glial cells, and has been completely revised, greatly enlarged, and enhanced with full color figures throughout. Neglected in research for years, it is now evident that the brain only functions in a concerted action of all the cells, namely glia and neurons. Seventy one chapters comprehensively discuss virtually every aspect of normal glial cell anatomy, physiology, biochemistry and function, and consider the central roles of these cells in neurological diseases including stroke, Alzheimer disease, multiple sclerosis, Parkinson's disease, neuropathy, and psychiatric conditions. With more than 20 new chapters it addresses the massive growth of knowledge about the basic biology of glia and the sophisticated manner in which they partner with neurons in the course of normal brain function.
Glial cells constitute about 10 % of the Drosophila nervous system. The development of genetic and molecular tools has helped greatly in defining different types of glia. Furthermore, considerable progress has been made in unraveling the mechanisms that control the development and differentiation of Drosophila glia. By contrast, the role of glia in adult Drosophila behavior is not well understood. We here summarize recent work describing the role of glia in normal behavior and in Drosophila models for neurological and behavioral disorders.
Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes.
The genetic tools available in Drosophila have facilitated our understanding of how apoptosis is regulated and executed in the context of the developing organism. All embryonic apoptosis is initiated by the activity of three genes, rpr, grim and hid. Each of these genes is independently regulated, allowing developmental apoptosis to be finely controlled. These initiators in turn activate the core apoptotic machinery, including the caspases. Drosophila counterparts to other conserved components of the apoptotic machinery have been recently identified, and we discuss how these may be integrated into the process of normal developmentally regulated cell death. We also outline the role that phagocytosis plays in the final stages of apoptosis and consider the molecular mechanisms guiding the elimination of apoptotic corpses.
Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.
This resource is the long-awaited new revision of the most highly regarded reference volume on glial cells, and has been completely revised, greatly enlarged, and enhanced with full color figures throughout. Neglected in research for years, it is now evident that the brain only functions in a concerted action of all the cells, namely glia and neurons. Seventy one chapters comprehensively discuss virtually every aspect of normal glial cell anatomy, physiology, biochemistry and function, and consider the central roles of these cells in neurological diseases including stroke, Alzheimer disease, multiple sclerosis, Parkinson's disease, neuropathy, and psychiatric conditions. With more than 20 new chapters it addresses the massive growth of knowledge about the basic biology of glia and the sophisticated manner in which they partner with neurons in the course of normal brain function.
To facilitate the investigation of glial development inDrosophila, we present a detailed description of theDrosophila glial cells in the ventral nerve cord. A GAL4 enhancer-trap screen for glial-specific expression was performed. Using UAS-lacZ and UAS-kinesin-lacZ as reporter constructs, we describe the distribution and morphology of the identified glial cells in the fully differentiated ventral nerve cord of first-instar larvae just after hatching. The three-dimensional structure of the glial network was reconstructed using a computer. Using the strains with consistent GAL4 expression during late embryogenesis, we traced back the development of the identified cells to provide a glial map at embryonic stage 16. We identify typically 60 (54–64) glial cells per abdominal neuromere both in embryos and early larvae. They are divided into six subtypes under three categories: surface-associated glia (16–18 subperineurial glial cells and 6–8 channel glial cells), cortex-associated glia (6–8 cell body glial cells), and neuropile-associated glia (8–10 nerve root glial cells, 14–16 interface glial cells, and 3–4 midline glial cells). The proposed glial classification system is discussed in comparison with previous insect glial classifications.
Mammalian macrophage scavenger receptors exhibit unusually broad binding specificity and are implicated in atherosclerosis and host defense. Scavenger receptor-like endocytosis was observed in Drosophila melanogaster embryos and in primary embryonic cell cultures. This receptor activity was expressed primarily by macrophages. The Drosophila Schneider L2, but not the Kc, cell line also exhibited a scavenger receptor-mediated endocytic pathway similar to its mammalian counterpart. L2 receptors mediated high-affinity internalization and subsequent temperature- and chloroquine-sensitive degradation of 125I-labeled acetylated low density lipoprotein and displayed characteristic ligand specificity. These findings suggest that scavenger receptors mediate important, well-conserved functions and raise the possibility that they may be pattern recognition receptors that arose early in the evolution of host defense mechanisms. They also establish additional systems for the investigation of endocytosis in Drosophila and scavenger receptor function in disease, host defense, and development.
Mononuclear phagocytes which express the HIV entry receptor CD4 have been implicated as possible sites of virus replication in brain, but there is still considerable uncertainty as to which cells in the CNS express CD4 Ag. Although it is not susceptible to HIV infection the rat provides a model to define expression of the CD4 Ag on MO in brain. We report that the CD4 epitopes W3/25 and OX35 are found only on monocytes, MO, microglia, and occasional lymphocytes and not on neurons, other glia, or endothelium. CD4 Ag levels are modulated during microglial differentiation, after reactivation after local inflammation, and within the intact blood brain barrier. MO and microglia also express other potential plasma membrane binding and entry sites for HIV viz Fc and complement receptors that are regulated independently of CD4.
We have identified the neurons that pioneer the major CNS axon tracts in the Drosophila embryo and determined their trajectory and fasciculation choices using serial section electron microscopy. Although Drosophila pioneer neurons make choices similar to those of their grasshopper homologs, there are interesting differences that reflect the much smaller nervous system size and the much faster rate of development characteristic of Drosophila. For example, where 2 longitudinal tracts are pioneered independently in grasshopper, only one is formed in Drosophila. This change is due to a change in fasciculation affinity of the pCC growth cone. Additionally, the intersegmental (IS) nerve is pioneered by a different neuron in Drosophila (aCC) than in the grasshopper (U1) because the smaller Drosophila CNS places the IS nerve within filopodial reach of the aCC soma, while in the grasshopper it is not. Drosophila growth cones explore a much more confined neuropil volume than do grasshopper growth cones but can also sample a larger fraction of the CNS as well. For this reason, some cell-cell recognition events critical to pathfinding in the grasshopper embryo may not be as essential in Drosophila. Nevertheless, many specific cellular affinities have been retained through the evolutionary divergence of these 2 species.
A monoclonal antibody (Mab5B12) demonstrating specificity for glial cells within the central and peripheral nervous systems of Drosophila has been used in combination with neural-specific antibodies to study the early organization of the Drosophila embryo. The embryonic central nervous system of Drosophila contains cells within the ventral midline that are recognized by monoclonal antibody 5B12. These cells are not recognized by either a polyclonal antiserum to horse radish peroxidase, which recognizes several antigens on the surface of Drosophila neurons, or Mab22C10, which recognizes an antigen specific to the peripheral nervous system. Mab5B12-positive cells lie dorsal both to the developing anterior and posterior commissures in each thoracic and abdominal segment and to the supraoesophageal commissure. They ensheath these commissures in later stage embryos. Other Mab5B12-positive cells lie dorsolateral to the CNS and send processes laterally to the lateral sensilla during axonogenesis in the PNS. These cells surround the axons of the intersegmental and segmental nerves. Other cells that line the advancing ectoderm during dorsal closure and surround the anal pads also express the Mab5B12 antigen. Neuronal cell cultures derived from Drosophila gastrulae contain cells expressing the Mab5B12 antigen. These cells can be found separate or in close association with neuronal clusters and their axons.
Three classes of glial cells are present early in embryogenesis and appear to play a major role in axon pathway formation in the Drosophila CNS. Six longitudinal glial (LG) cells are present over the longitudinal connective on each side of each segment. Six midline glia (MG) cells surround the anterior and posterior commissures of each segment. Finally, the intersegmental nerve root is covered by a glial cell: the segment boundary cell (SBC). All 3 classes of glial cells are present in their final position before axon outgrowth and their pattern prefigures the first axon pathways. The pioneer growth cones that establish the first axon pathways in the longitudinal connective and intersegmental nerve extend along the elongate surface of the LG and SBC glial cells; the pioneer growth cones for the anterior and posterior commissures extend toward and make close contact with the end feet of the MG glial cells. Later, all 3 classes of glial cells enwrap the axon tracts in much the same way as vertebrate oligodendrocytes. The results suggest that these early glial cells provide guidance cues for the first growth cones in the Drosophila CNS. More than simply providing a permissive substrate, the differential extension of specific early growth cones towards either the MG cells or along the LG cells suggests an active role for these glia in growth cone guidance.
The embryonic lethal abnormal visual system (elav) locus in Drosophila melanogaster, a vital gene mapping within the 1B5-1B9 region of the X-chromosome has been cloned and analysed. Previous developmental analyses have shown that in addition to the embryonic requirement there is a post-embryonic requirement for elav function in the cells of the visual system. A DNA segment containing elav+ function was defined through germ line transformation experiments. This region encodes three embryonic poly(A)+ RNAs and two adult transcripts which are preferentially expressed in the head. In situ hybridization experiments clearly demonstrate that the embryonic expression of elav is restricted to the nervous system.
The events which occur in the death of visceromotor neurons of the cervical region of the chick embryo's spinal cord have been analyzed by electron microscopy. These normal degenerative events are compared with those in the lumbosacral cord where nerve cell death was induced by removal of peripheral organs. The initial set of degenerative changes include a decrease in nuclear size, the clumping of chromatin beneath the nuclear envelope, an increase in electron opacity of the cells, the disappearance of Golgi bodies, and the disaggregation of polysomes. These events are followed by the loss of the nuclear envelope and most of the endoplasmic reticulum, the appearance of bundles of filaments, and the formation of many ribosome crystals. Ribosome crystals are seen only in the dying cells. Their abundance may indicate a drastic reduction in RNA synthesis as one of the initial events which lead to the death of these neurons. The neurons are finally subdivided and engulfed by cells of the normal glial population, and further breakdown of the cell fragments occurs in large phagocytic vesicles of the gliocytes.
In the developing mouse retina degenerating neurons can be observed initially in the ganglion cell layer followed by a phase of cell death in the inner nuclear layer. Using an immunohistochemical method to localize the mouse macrophage specific antigen F4/80, we show that macrophages migrate from the vascular supply overlying the developing retina and phagocytose the degenerating neurons. The macrophages subsequently differentiate to become the microglia of the retina and form a regularly spaced distribution across the retina in the inner and outer plexiform layers. These experiments provide strong evidence for the mesodermal origin of central nervous system microglia.
We report the identification of RK2, a glial-specific homeodomain protein. RK2 is localized to the nucleus of virtually all embryonic and imaginal glial cells, with the exception of midline glia. Embryos mutant for the gene encoding RK2 are embryonic lethal but normal for early gliogenesis (birth, initial divisions and migration of glia) and axonogenesis (neuronal pathfinding and fasciculation). However, later in development, there are significantly fewer longitudinal glia that are spatially disorganized; in addition, there is a slight disorganization of axon fascicles and a defective nerve cord condensation. This suggests that RK2 is not required for early glial determination, but rather for aspects of glial differentiation or function that are required for embryonic viability.
We have examined cell death within lineages in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages died by apoptosis after separation of the commissural axon tracts. Glial apoptosis is blocked in embryos deficient for reaper, where greater than wild-type numbers of midline glia (MG) are present after stage 12. Quantitative studies revealed that MG death followed a consistent temporal pattern during embryogenesis. Apoptotic MG were expelled from the central nervous system and were subsequently engulfed by phagocytic haemocytes. MGA and MGM survival was apparently dependent upon proper axonal contact. In embryos mutant for the commissureless gene, a decrease in axon-glia contact correlated with a decrease in MGA and MGM survival and accelerated the time course of MG death. In embryos mutant for the slit gene, MGA and MGM maintained contact with longitudinally and contralaterally projecting axons and MG survival was comparable to that in wild-type embryos. The initial number of MG within individual ventral nerve cord segments was increased by ectopic expression of the rhomboid gene, without changing axon number. Extra MGA and MGM were eliminated from the ventral nerve cord by apoptosis to restore wild-type numbers of midline glia. Ectopic rhomboid expression also shifted MGA and MGM cell death to an earlier stage of embryogenesis. One possible explanation is that axon-glia contact or communication promotes survival of the MG and that MG death may result from a competition for available axon contact.
In the developing mouse retina degenerating neurons can be observed initially in the ganglion cell layer followed by a phase of cell death in the inner nuclear layer. Using an immunohistochemical method to localize the mouse macrophage specific antigen F4/80, we show that macrophages migrate from the vascular supply overlying the developing retina and phagocytose the degenerating neurons. The macrophages subsequently differentiate to become the microglia of the retina and form a regularly spaced distribution across the retina in the inner and outer plexiform layers. These experiments provide strong evidence for the mesodermal origin of central nervous system microglia.
A Summary of Drosophila Embryogenesis.- Stages of Drosophila Embryogenesis.- Mesoderm Development.- Musculature.- Circulatory System and Fat Body.- Macrophages.- The Gut and its Annexes.- Epidermis.- Peripheral Nervous System.- The Peripheral Nervous System.- Central Nervous System.- Tracheal Tree.- The Gonads.- The Pattern of Embryonic Cell Divisions.- Morphogenetic Movements.- Cephalogenesis.- Some Aspects of Segmentation.- A Fate Map of the Blastoderm.
The glial cells of the prothoracic ganglion of the hawk moth Manduca sexta were studied in histological sections of several postembryonic stages and classified according to cell morphology, size, staining properties, and topographical relationships. In general, each glial cell type was found to be confined to one of the major ganglionic domains and each of these domains (i.e., perineurium, cell body rind, glial cover of the neuropil, and neuropil) was found to comprise specific cell types. Some types of glia were recognized in both larval and later stages, but other types were found exclusively from late pupal stages. It is proposed that the higher morphological diversity expressed by the glia of the pharate adult is attained by differentiation of new cell types during metamorphosis. Before the differentiation of new cell types, extensive cell death and cell proliferation seem to occur within some glial subpopulations.
The onset and sequence of cell death in the lateral motoneurons of the lumbar spinal cord of the chick embryo was studied between 4 and 18 days of incubation by light and electron microscopy. The naturally occurring degeneration of a few motoneurons begins already on day 4 of incubation, but the cell loss becomes much more frequent from day 5.5 to day 9, during which 40% of the neuron population degenerates. In the light microscope, the degenerating neurons are seen to undergo shrinkage and condensation. Ultrastructurally, two types of degeneration can be recognized. In the first, termed Type I, the polyribosomes are dissociated and appear free in the cytoplasm. Ribosomes also become detached from rough endoplasmic reticulum, although small pieces of cisternae of rough endoplasmic reticulum are still recognizable. Most of the mitochondria are vacuolized. These changes are accompanied by the appearance of a pyknotic nucleus which contains condensed chromatin masses. The most characteristic feature in what we term Type II degeneration is the striking dilatation of the endoplasmic reticulum, the nuclear envelope and the Golgi apparatus. Ribosomes still form distinctly rosette-like polyribosomes. A few mitochondria show signs of degeneration. The nuclear profile in most cells of this type is rounded whereas the chromatin is becoming condensed. In the late stages of Type II degeneration, the dilated membrane systems break down into numerous vesicles some of which still have ribosomes attached. Only late in the sequence of Type II degeneration do polyribosomes then dissociate to free monoribosomes. The degeneration process in both types leads finally to cell death and complete cellular breakdown: the entire process being due to an autolytic mechanism. The nuclear envelope breaks down and the nuclear content becomes mixed with lytic cytoplasm. The dying cell finally either condenses into one big globule or several smaller fragmented globules. All the globular debris are highly osmiophilic and compact. Only at this stage of breakdown is the cell debris phagocytozed by radial ependymal processes and mononuclear leukocytes. The entire degenerative process in these immature neurons is strikingly rapid.
In the case of induced cell death by removal of the limb bud on day 2.5, we found that both types of degeneration described above also occur in the peripherally deprived lateral motor column (LMC). The only obvious difference is that limb bud removal increases the speed and the number of cells undergoing degeneration. By day 10, about 90% of the neuron population in the deprived LMC have completely disappeared.
The rapid development of the study of insect neurobiology, which is currently occurring principally because individual neurons can be re-identifled and because their activities can be recorded in situ and related to behavior, is generating a demand for more knowledge concerning insect glial cells and their functional relationships with neurons. This study examines the ultrastructure of glial cells in locust metathoracic ganglia in relation to general locale within the ganglion and also to specific identified neurons and neuron types. Seven major types of glial cell form are recognized, with subdivisions, requiring a new scheme for classification. Glial invaginations into neurons are of four different kinds: regular, chunky, filigree, and ridge (found only at axon hillocks). They also range from only intrusive to fully reciprocal. In addition, some neurons make projections of various lengths into surrounding glia and between neighboring neuron somata, and some glia make long, branched projections into other glial cells. The differences show that insect glial cells develop highly specific functional specializations; they may not be interchangeable. The complexity and intimacy of relationships of glia with neurons suggest that some glial cells may have roles other than that of nursemaids, possibly in modulation of behaviordetermining neural activity, and in learning and other adaptive acts.
The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. I have examined the differentiation of the LG in six mutations in which the longitudinal tracts were absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero had no longitudinal tracts, and glial differentiation remained arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia failed to form an oriented scaffold, but cytological differentiation of the LG was unperturbed. The longitudinal tracts in embryos mutant for slit fused at the VNC midline and scaffold formation was normal, except that it was medially displaced. Longitudinaltracts had intersegmental interruptions in embryos mutant for hindsight and midline. In hindsight, there were intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracted after initial extension. LG morphogenesis during axonogenesis was abnormal in midline. Commitment to glial identity and glial differentiation also occurred before scaffold formation. In all mutants examined, the early distribution of the glycoprotein neuroglian was perturbed. This was indicative of early alterations in VNC pattern present before LG scaffold formation began. Therefore, some changes in scaffold formation may have reflected changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation preceded longitudinal axon tract formation. © 1993 John Wiley & Sons, Inc.
This paper deals with morphological aspects of early neurogenesis inDrosophila, in particular with the segregation of neuroblasts from the neurogenic region of the ectoderm and the pattern formed by those wells within both the germ band and the procephalic lobe. The neurogenic ectoderm was found to contain neural precursors intermingled with epidermal precursors, extending from the midline up to the primordia of the tracheal tree along the germ band and laterodorsally in the procephalic lobe. Germ band neuroblasts segregate from the neurogenic ectoderm during a period of several hours according to characteristic spatial and temporal patterns. During the first half of the segregation process the pattern of germ band neuroblasts was found to be the same in different animals in both spatial arrangement and number of cells; this permitted the identification of individual neuroblasts from different embryos. Later in development several difficulties were encountered which precluded an exact description of the neuroblast pattern. The constitution of the neurogenic region is discussed in relation to the phenotype of mutants affecting neurogenesis.
Monoclonal antibodies and enhancer trap insertions that mark subsets of neurons have been valuable tools in the study of Drosophila neuronal development. Similarly, glial specific cell markers could prove to be valuable in investigating the development and function of glia. Here we characterize the architecture and development of distinct sets of Drosophila embryonic glia, using a reporter gene driven by fushi tarazu homeodomain binding sites. Reporter expresssion in glia is dependent on the orientation and spacing of the homeodomain binding sites, revealing potential differences in glial determination. These studies suggest that the use of transcription factor binding sites to drive reporter gene expression may prove to be a generally useful means of generating additional cell type-specific markers.
The pattern of neuroblast divisions was studied in thoracic and abdominal neuromeres of wild-type Drosophila melanogaster embryos stained with a monoclonal antibody directed against a chromatin-associated antigen. Since fixed material was used, our conclusions are based upon the statistical evaluation of a large number of accurately staged embryos, covering the stages between the formation of the cephalic furrow up to shortened germ band. Our observations point to a rather stereotypic pattern of proliferation, consisting of several parasynchronous cycles of division. The data suggest that all SI neuroblasts divide at least eight times, all SII neuroblasts six or seven times and all SIII neuroblasts at least five times. This conclusion is based on the mapping of mitotic neuroblasts and is supported by the progressive reduction of the neuroblast volume and by the results of cell countings performed on embryos of increasing age. No conclusive evidence was obtained concerning the fate of the neuroblasts after their last mitosis, i.e. it cannot be decided whether the neuroblasts degenerate or become incorporated as inconspicuous cells in the larval ventral cord. The duration of the cycles of division of the neuroblasts was found to be 40–50 min each, while in the case of ganglion mother cells about 100 min are required to complete one cell cycle.
Injection of physiologically inert particles (fluorescent microspheres) has a profound effect on neural repair of central nervous connectives of the cockroach Periplaneta americana following selective glial disruption. The injected particles, which do not gain direct access to the central nervous tissues, are taken up by a relatively small proportion (< 10%)="" of="" the="" haemocytes.="" this="" interference="" with="" haemocyte="" function="" virtually="" abolishes="" the="" appearance="" of="" the="" granule-containing="" cells="" (which="" are="" prominently="" involved="" in="" normal="" glial="" repair)="" and="" produces="" abnormal="" reorganization="" of="" the="" superficial="" glial="" elements.="" these="" results="" are="" interpreted="" as="" evidence="" that="" the="" granule-containing="" cells="" are="" derived="" from="" haemocytes="" which="" are="" critically="" involved="" in="" glial="">
Ultrastructural aspects of the natural degeneration of a group of six motor neurons in the fourth abdominal ganglion of Manduca sexta are described. These motor neurons innervate intersegmental muscles that degenerate and disappear immediately after adult eclosion. The first detectable changes in the cell bodies appear 12 h after eclosion and include disruption of the endoplasmic reticulum and an increase in the size and number of lamellar bodies. At 32 h the nuclear membranes rupture, and the membranous and granular cytoorganelles segregate in different parts of the cell. At that stage the surrounding glial cells participate in the digestion of material from the degenerating neurons. From 72 h onward the remaining neuronal structures become disrupted, and are finally transformed into a single, large lamellar body (residual body) within the glial profile. The degeneration pattern differs significantly from that of embryonic vertebrate neurons.
Autoradiographs of tritiated thymidine uptake and subsequent light- and electron-microscopical examination revealed an onset of perineurial glial cell proliferation 3 days after injury to the CNS. The number of cells labelled increased rapidly until 7 days post-lesioning. At 2 weeks, the labelled cells equalled the number of nuclei present in the perineurium. No label was seen in the subperineurial cells, possibly because of the inability of the label to penetrate into a region where localised division is taking place.Prior to the onset of thymidine uptake, the damaged nerve cord was invaded by an exogenous reactive cell. The number of these cells increased rapidly in the first 48 h, then decreased as a negative exponential, very few remaining after 7 days. We suggest that this cell type must either return to the haemocoel or transform into a functional glial cell class.The repair of the insect central nervous system can be divided into three phases which show striking similarities to vertebrate repair sequences. These include: initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, the longer term flux of cell numbers, their distribution and the time scale of events. This suggests that the insect CNS might provide a system for examining common cellular mechanisms and events.
The appearance and distribution of microglia in the developing cerebellum has been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. This distribution has in turn been correlated with that of pyknotic figures in the same Nissl-counterstained sections, in order to gain an understanding of the role of microglia in the developing cerebellum. Round and ameboid microglia may be recognised in the fetal cerebellum as early as E11. Numbers of microglia increase steadily from that time, with initial concentrations in the region of the dorsal and ventricular surfaces. By P1, concentrations of both pyknotic figures and ameboid microglia begin to appear in the region of the future cerebellar medulla. Ameboid microglia are recognisable in the cerebellar medulla until P10, with particular concentrations where folia branch and in the rostral cerebellar peduncles. After this time only resting microglia are found in the cerebellum. Concentrations of microglia largely match the positions of pyknotic figures throughout development, except at P10 and P14, when cell death is found in the external granular layer without an accompanying concentration of microglia. Electron microscopic examination of the phagosomes of ameboid microglia at P5 and P6 indicates that these cells are mainly concerned with the phagocytosis of entire cells rather than axons. Cell death in the cerebellar medulla may serve to clear pathways for developing cortical afferents and efferents, or to increase the mechanical plasticity of the medulla during cortical folding.
It has been suggested that microglia, a type of glial cells in the central nervous system, play various important roles in normal and pathologic brains. In this article, we discussed the association or roles of microglia in injury and in brain diseases such as Alzheimer's disease, AIDS dementia complex, multiple sclerosis and ischemia. Further-more, microglia-derived cytotoxic products and other secretory factors were summarized. In addition to the pathological aspects, secretory factors that showed neurotrophic effects were described with special reference to their physiological significance in the neuronal growth, neuronal function and regeneration processes. Accumulated evidence suggests that microglia are associated with not only brain pathology but also normal physiology in the brain.
One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.
Transient arrest of the cerebral circulation leads to neuronal cell death in selectively vulnerable regions of the central nervous system. It has recently been shown at the light microscopical level that neuronal necrosis is accompanied by a rapid microglial reaction in ischemia (Gehrmann et al. (1992) J. Cereb. Blood Flow Metab. 12:257-269). In the present study we have examined the postischemic microglial reaction in the dorsal rat hippocampus at the ultrastructural level using immuno-electron microscopy. Global ischemia was produced by 30 min of four-vessel occlusion and the microglial reaction then studied after 8, 24 and 72 h. In sham-operated controls microglial cells were not phagocytic; they were randomly distributed throughout the neuropil and occasionally made contacts with other structures such as dendrites in CA1. Ultrastructural signs of activation were observed from 1 day postlesion onward. Reactive microglial cells were consistently seen to phagocytose degenerating neurons particularly in the CA1 stratum pyramidale and in the CA4 sector. They were sometimes interposed between two morphologically distinct types of CA1 neurons, i.e., "dark" (degenerating) and "pale" (surviving) types of neurons. Phagocytic microglial cells also became positive for major histocompatibility complex (MHC) class II antigens at these locations from 1 day after ischemia onward. Furthermore, activated microglial cells were frequent along degenerating dendrites in the stratum radiatum of CA1. After survival times of up to 72 h microglial cells, but not astrocytes, were occasionally observed to undergo mitosis. In addition to their random distribution across the neuropil, microglial cells were frequently observed in a perivascular position under normal conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Dead cells are observed in many developing animal tissues, but the causes of these normal cell deaths are mostly unknown. We show that about 50% of oligodendrocytes normally die in the developing rat optic nerve, apparently as a result of a competition for limiting amounts of survival signals. Both platelet-derived growth factor and insulin-like growth factors are survival factors for newly formed oligodendrocytes and their precursors in culture. Increasing platelet-derived growth factor in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes in 4 days. These results suggest that a requirement for survival signals is more general than previously thought and that some normal cell deaths in nonneural tissues may also reflect competition for survival factors.
In recent years much progress has been made toward a better understanding of the nature and function of microglial cells. This review summarizes new developments and attempts to provide a perspective for future avenues to take in microglial research. Microglia are considered to play an active role in a variety of neurological diseases. Their function in forming a network of immune competent cells within the CNS is discussed.
Phagocyte recognition of cells that have undergone apoptosis (programmed cell death) is an event of broad biological significance. Characterized by endogenous endonuclease activation, which results in chromatin fragmentation and nuclear condensation, apoptosis leads to swift ingestion of intact but 'senescent' or 'unwanted' cells by phagocytes in processes as diverse as the physiological involution of organs, the remodelling of embryonic tissues, and metamorphosis. The cell-surface mechanisms by which macrophages recognize apoptotic cells as 'senescent-self' have remained obscure. Here we report that macrophage recognition of apoptotic cells (both neutrophils and lymphocytes) is mediated by the vitronectin receptor, a heterodimer belonging to the beta 3 or cytoadhesin family of the integrins. Previously, the functions of the vitronectin receptor were believed to be limited to cell anchorage, but our findings indicate that the receptor has a novel and direct role in self-senescent-self intercellular recognition leading to macrophage phagocytosis of cells undergoing apoptosis.
The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system. Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages. A product of this gene, the ELAV protein, has homology to known RNA binding proteins. The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli. In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia. These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen. We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance.
Enhancer trap lines have been used to generate a collection of molecular lineage markers specific for different subsets of glia in the Drosophila embryo. Using these markers, we have been able to describe the diversity and pattern of glia along the major axon pathways in the embryonic central and peripheral nervous system. Just as these and other studies show the great diversity of embryonic glia, so too the enhancer trap lines described here point to a remarkable degree of molecular heterogeneity, and probably a concomitant functional specificity, of the embryonic glia.
fasiclin II (fas II), a member of the immunoglobulin superfamily, was previously characterized and cloned in grasshopper. To analyze the function of this molecule, we cloned the Drosophila fas II homolog and generated mutants in the gene. In both grasshopper and Drosophila, fasciclin II is expressed on the MP1 fascicle and a subset of other axon pathways. In fas II mutant Drosophila embryos, the CNS displays no gross phenotype, but the MP1 fascicle fails to develop. The MP1, dMP2, and vMP2 growth cones fail to recognize one another or other axons that normally join the MP1 pathway. During their normal period of axon out-growth, these growth cones stall and do not join any other neighboring pathway. Thus, fasciclin II functions as a neuronal recognition molecule for the MP1 axon pathway. These studies serve as molecular confirmation for the existence of functional labels on specific axon pathways in the developing nervous system.
Cultures of astroglia from C3H/HeJ mice, which are resistant to bacterial cell wall polysaccharide (LPS), initiated from embryos of Theiler stage 14 (9 days of gestation) up to Theiler stage 25 (17 days of gestation) as well as newborn animals, when subjected to nutritional deprivation, i.e. non-feeding of cultures, form large numbers of macrophage-like cells. These cells express Mac-1, Mac-3, F4/80 and Fc antigens. The cells are negative for GFAP, positive for vimentin, express Ia antigen and take up DiL-Ac-LDL. They are positive to non-specific esterase, secrete lysozyme and are phagocytic. Their morphology and ultrastructure closely resemble those of macrophages. Cultures initiated from neuroepithelium of Theiler stage 13 (8.5 days of gestation), before vascularization, when subjected to nutritional deprivation, also produce macrophage-like cells. Using spleen colony assay and methyl cellulose cultures, we were unable to detect the presence of hemopoietic (macrophage) precursor cells in astroglia cultures. This supports the hypothesis that the macrophage-like cells are of neuroectodermal origin and probably correspond to resident microglia of the CNS. Using nutritionally deprived astroglia cultures, a procedure was developed for isolation of macrophage-like cells and production of highly enriched macrophage-like (microglia) cultures.
There is a growing recognition, stemming from work with both vertebrates and invertebrates, that the capacity for neuronal regeneration is critically dependent on the local microenvironment. That environment is largely created by the non-neuronal elements of the nervous system, the neuroglia. Therefore an understanding of how glial cells respond to injury is crucial to understanding neuronal regeneration. Here we examine the process of repair in a relatively simple nervous system, that of the insect, in which it is possible to define precisely the cellular events of the repair process. This repair is rapid and well organised; it involves the recruitment of blood cells, the division of endogenous glial elements and, possibly, migration from pre-existing glial pools in adjacent ganglia. There are clear parallels between the events of repair and those of normal glial development. It seems likely that the ability of the insect central nervous system to repair resides in the retention of developmental capacities throughout its life and that damage results in the activation of this potential.
Regressive phenomena are common during the development of the nervous tissue. Among them, naturally occurring cell death has been observed in several regions of the nervous system. Cell death in the somatosensory cortex and medial cortical regions (hind limb, frontal cortex 1, frontal cortex 2, retrosplenial agranular, retrosplenial granular [Zilles K. et al. (1980) Anat. Embryol. 159, 335-360]) as well as in the cortical subplate (future subcortical white matter) in the rat mainly occurs during the first 10 days of postnatal life with peak values of 3.1 dead cells per 1000 live neurons at the end of the first week. Cell death progresses from birth to day 7 with a predominance of dead cells in the subplate and in layers II-III. Later, dead cells are more dispersed in the cerebral cortex, but a significant amount is still present in the subcortical white matter. This pattern correlates with the arrival and settlement of cortical afferents at the different cortical levels, as described in other studies, and points to the likelihood that transitory cellular populations are important clues in the modelling of the cerebral cortex during normal development. Transitory populations of macrophages (amoeboid or nascent microglial cells) that appear in great numbers during the same period and in the same regions are involved in the removal of dead cells.
The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an alpha-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.
Why might macrophages be of interest to neurobiologists? Recent evidence shows that macrophages play a role in tissue homeostasis as well as in defence and repair of tissues. We will review here the possible functions of resident and recruited macrophages in the developing and adult nervous system and examine what contribution these cells might make to repair mechanisms in the central and peripheral nervous systems. There is increasing evidence that macrophages form an important component of the non-neuronal cell population in the nervous system and the tools are becoming available that allow us to study these cells in situ.
The Bicaudal-D (Bic-D) gene is essential for the differentiation of the oocyte in Drosophila. Dominant gain-of-function mutations result in the formation of double abdomen embryos. The Bic-D gene was cloned and identified using restriction fragment length polymorphisms, Northern analysis, and transformation rescue. Bic-D RNA accumulates in the oocyte during the earliest stages of oogenesis and is localized anteriorly in later stages. The predicted protein contains several extended amphipathic helices, and its similarity to myosin heavy chain tails, paramyosin, and kinesin suggests a similar type of coiled-coil protein interaction.
The present review summarizes recently acquired data in vivo, which support a role of CNS microglia as a source of defense cells in the CNS capable of carrying out certain immune functions autonomously. We have kept the following discussion restricted to microglial cells and have not included work on the immunological functions of astrocytes, which has been recently reviewed elsewhere (Fontana et al.: Immunological Reviews 137:3521-3527, 1987). Resting microglia are scattered uniformly throughout the CNS forming a network of potential immunoeffector cells, which can be activated by stimuli ranging from peripheral nerve injury over viral infections to direct mechanical brain trauma. The term "activated microglia" is used here to describe proliferating cells that demonstrate changes in their immunophenotype but have not undergone transformation into brain macrophages. Such a transformation can be stimulated by neuronal death but not by sublethal neuronal injury. Microglia may function as antigen-presenting cells and may thus represent the effector cell responsible for the recruitment of lymphocytes to the brain resulting in an inflammatory reaction. The recent developments in the understanding of microglial cell function may lead to a redefinition of the often cited "immune privilege" of the brain.
In normal human white matter the predominant cell type expressing MHC Class II is the microglia. This population of cells reacts with the pan macrophage marker, EBM/11, and constitutes about 13% of the glial cell population. The intensity of staining was enhanced and the absolute number of Class II+ microglia increased in normal appearing white matter from multiple sclerosis (MS) brain. As T cell activation in MS may occur in the brain the upregulation of microglia bearing MHC Class II may reflect their function as antigen presenting cells in the development of inflammatory lesions.
Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.
To identify candidates for neuronal recognition molecules in Drosophila, we used monoclonal antibodies to search for surface glycoproteins expressed on subsets of axon bundles (or fascicles) during development. Here we report on the characterization and cloning of fasciclin III, which is expressed on a subset of neurons and axon pathways in the Drosophila embryo. Fasciclin III is also expressed at other times and places including transient segmentally repeated patches in the neuroepithelium and segmentally repeated stripes in the body epidermis. Antisera generated against each of four highly related forms of the protein were used for cDNA expression cloning to identify a single gene, which was confirmed to encode fasciclin III by tissue in situ hybridization and genetic deficiency analysis.
Macrophages and microglia in the developing and adult mouse brain have been identified by immunohistochemical localization of the macrophage-specific antigen F4/80 and monoclonal antibodies to the FcIgG1/2b (2.4G2) and type-three complement (Mac-1) receptors. In the adult mouse there are two classes of F4/80-positive cells; those associated with the choroid plexus, ventricles and leptomeninges and the microglia. The cells bearing Fc and complement receptors are indistinguishable, by their morphology and distribution, from those revealed by F4/80. During development macrophages invade the brain and can be followed through a series of transitional forms as they differentiate to become microglia. Macrophage invasion occurs when naturally dying cells are observed in large numbers and this is consistent with the idea that dying neurons and axons provide a stimulus for macrophage infiltration. Our results provide strong support for the hypothesis that the microglia are derived from monocytes and show that microglia possess receptors which would allow them to play a part in the immune defence of the nervous system.
In this article the author will first describe degenerating neurons and show how the morphological expression of cel death is closely related to a neuron's state of maturity. The timing of natural cell death also will be considered in order to define where natural neuron death fits into other aspects of neural development and neuron differentiation. Experimental studies will show the dependence a neuron has on the cell populations with which it ultimately connects. Finally, the possible functions of cell death will be considered in an attempt to provide a more comprehensive explanation for this phenomenon.
Hemocytes of Drosophila melanogaster and Drosophila yakuba larvae have been defined in terms of their ultrastructure and functions in "coagulation", wound healing, encapsulation, phenol-oxidase activity, and phagocytosis. The position of these cells among the classical hemocyte types of insects is determined. We distinguish two plasmatocyte types (macrophage-plasmatocytes and lamellocytes) which do not seem to belong to the same lineage, and oenocytoids which are the crystal cells of the literature.
The compound eye and the two most distal optic neuropils (lamina ganglionaris and medulla externa) of the Drosophila mutant w rdgB
KS222were examined with transmission electron microscopes at conventional (60 kV) and high (0.8–1 MV) voltages. Eye tissue was sampled in the newly emerged and at 3, 7, and 21 days following eclosion. This mutant is known to show a light-induced degeneration of the peripheral retinular cells (R 1–6); the spectral sensitivity is altered and the threshold is increased reflecting the function of the central cells (R7, 8) which do not degenerate.
A totally normal appearing visual system (peripheral retina and optic neuropiles) was found in newly emerged adults. After 3 days the somata of some of the peripheral retinal cells are affected and all of their axons show degeneration. At one week the R 1–6 pathology is well advanced in both somal and axonal regions. In affected cells the cytoplasm is more or less uniformly electron dense and contains liposomes, lysosome-like bodies, myeloid figures and vacuoles suggesting autophagy. Such cytoplasm (noted at 3 and 7 days post-eclosion) exhibits an electron dense reticulum and degenerate mitochondria. Microvilli become more electron dense. Retinular axon terminals are electron opaque and lack synaptic vesicles with few if any presynaptic structures. Mitochondrial remains are barely recognizable. Transsynaptic degeneration was not found. After 3 weeks, the structure of R 1–6 in the peripheral retina (somata and rhabdomeres) is greatly reduced or lost while R7 and R8 and higher order neurons are not affected. The debris from cell bodies and axon terminals of R 1–6 seems diminished, so that some phagocytosis probably takes place along with gliosis in the lamina.
Nerve injury that severs axons also disrupts ensheathing glial cells. Specifically, crushing or cutting the leech nerve cord separates the glial cell's nucleated portion from an anucleate recording, by intracellular injection of Lucifer Yellow dye and horseradish peroxidase (HRP) as tracers, and by electron microscopy. The nucleated portion of the glial cell did not divide, degenerate, or grow appreciably. The severed glial stump remained isolated from the nucleated portion but maintained its resting potential and normal morphology for months. Stumps typically began to deteriorate after 3 months. Small macrophage-like cells, or 'microglia' increased in number after injury and ensheathed axons, thus partially replacing the atrophying glial stump. Some axons in the nerve cord degenerated; the remainder appeared morphologically and physiologically normal. Thus, both nucleated and anucleate glial segments persisted throughout the one to two months required for axons to regenerate functional connections. Glial cells in the leech are therefore available to guide physically the growing axons or to contribute in other ways to nerve regeneration.