Drosophila FGFR/Htl signaling shapes embryonic glia to phagocytose apoptotic neurons.

Glial phagocytosis of apoptotic neurons is crucial for development and proper function of the central nervous system. Relying on transmembrane receptors located on their protrusions, phagocytic glia recognize and engulf apoptotic debris. Like vertebrate microglia, Drosophila phagocytic glial cells form an elaborate network in the developing brain to reach and remove apoptotic neurons. However, the mechanisms controlling creation of the branched morphology of these glial cells critical for their phagocytic ability remain unknown. Here, we demonstrate that during early embryogenesis, the Drosophila fibroblast growth factor receptor (FGFR) Heartless (Htl) and its ligand Pyramus are essential in glial cells for the formation of glial extensions, the presence of which strongly affects glial phagocytosis of apoptotic neurons during later stages of embryonic development. Reduction in Htl pathway activity results in shorter lengths and lower complexity of glial branches, thereby disrupting the glial network. Our work thus illuminates the important role Htl signaling plays in glial subcellular morphogenesis and in establishing glial phagocytic ability.

The phagocytic cyst cells in Drosophila testis eliminate germ cell progenitors via phagoptosis.

Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) "eat-me" signals on target cells initiate the process, the precise sequence of events is largely unknown. Here, we show that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, we demonstrate that cyst cell-derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors.

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila.

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non-professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.

Drosophila Skp1 Homologue SkpA Plays a Neuroprotective Role in Adult Brain.

Skp1, a component of the ubiquitin E3 ligases, was found to be decreased in the brains of sporadic Parkinson's disease (PD) patients, and its overexpression prevented death of murine neurons in culture. Here we expose the neuroprotective role of the Drosophila skp1 homolog, skpA, in the adult brain. Neuronal knockdown of skpA leads to accumulation of ubiquitinated protein aggregates and loss of dopaminergic neurons accompanied by motor dysfunction and reduced lifespan. Conversely, neuronal overexpression of skpA reduces aggregate load, improves age-related motor decline, and prolongs lifespan. Moreover, SkpA rescues neurodegeneration in a Drosophila model of PD. We also show that a Drosophila homolog of FBXO7, the F Box protein, Nutcracker (Ntc), works in the same pathway with SkpA. However, skpA overexpression rescues ntc knockdown phenotype, suggesting that SkpA interacts with additional F box proteins in the adult brain neurons. Collectively, our study discloses Skp1/SkpA as a potential therapeutic target in neurodegenerative diseases.

Glial phagocytosis in developing and mature Drosophila CNS: tight regulation for a healthy brain.

Glia are heterogeneous and multitasking cell type in the nervous system that supports neuronal function. One of the main glial tasks is removal of unneeded and potentially harmful material through phagocytosis. Glial phagocytosis is highly conserved throughout evolution, which makes genetic model organisms such as Drosophila of great value for investigating its molecular mechanisms. This mini review will focus on recent findings regarding the complex role of glial phagocytosis in Drosophila CNS during development as well as at the adult stage. We will summarize the current knowledge regarding partially redundant and overlapping signaling pathways underlying clearance of different targets in normal and diseased brain. In addition, we will highlight a recently emerged concept of glia-driven neurodegeneration that exposes the potentially harmful role of phagocytic glia. Studies on Drosophila glia provide new insights, which open new directions in glial cell biology with potential impact on healthy and diseased brain function.

Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain.

Glial phagocytosis is critical for the development and maintenance of the CNS in vertebrates and flies and relies on the function of phagocytic receptors to remove apoptotic cells and debris. Glial phagocytic ability declines with age, which correlates with neuronal dysfunction, suggesting that increased glial phagocytosis may prevent neurodegeneration. Contradicting this hypothesis, we provide experimental evidence showing that an elevated expression of the phagocytic receptors Six-Microns-Under (SIMU) and Draper (Drpr) in adult Drosophila glia leads to a loss of both dopaminergic and GABAergic neurons, accompanied by motor dysfunction and a shortened lifespan. Importantly, this reduction in neuronal number is not linked to neuronal apoptosis, but rather to phosphatidylserine-mediated phagoptosis of live neurons by hyper-phagocytic glia. Altogether, our study reveals that the level of glial phagocytic receptors must be tightly regulated for proper brain function and that neurodegeneration occurs not only by defective, but also excessive glial cell function.

Modeling Parkinson's disease in adult Drosophila.

BACKGROUND: Protein aggregation in neurons is a prominent pathological mark of neurodegeneration. In Parkinson's disease (PD), inclusions of the α-Synuclein (α-Syn) protein form the Lewy bodies in dopaminergic (DA) neurons. Ectopic expression of human α-Syn inDrosophila neurons leads to the protein accumulation, degeneration of DA neurons and locomotor deterioration, and therefore constitutes the present fly PD model. Yet, this model does not enable to study the role of genes, which are essential for normal development, in neurodegeneration. THE NEW METHOD: Using the Gal80/Gal4/UAS system we optimized the current PD model, such that only the adult stage of the fly is affected by α-Syn expression in the brain. RESULTS: The symptoms of neurodegeneration typifying the classic model, including reduced locomotor ability, shortened lifespan and the loss of DA neurons, are significantly demonstrated in the novel adult fly PD model. COMPARISON WITH EXISTING METHOD: The neurodegeneration symptoms exhibited by the innovative model are very similar to those manifested in the recognized one. CONCLUSIONS: Specific expression of α-Syn in the adult fly brain enables the investigation of developmental genes involved in neurodegeneration, thereby deciphering gene functions and molecular mechanisms. It may further be used for addressing therapeutic targets and treatment platforms specifically during adult stages.

Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development.

The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.

Draper-mediated JNK signaling is required for glial phagocytosis of apoptotic neurons during Drosophila metamorphosis.

Development of the central nervous system involves elimination of superfluous neurons through apoptosis and subsequent phagocytosis. In Drosophila, this occurs mainly during three developmental stages: embryogenesis, metamorphosis and emerging adult. Two transmembrane glial phagocytic receptors, SIMU (homolog of the mammalian Stabilin-2) and Draper (homolog of the mammalian MEGF10 and Jedi), mediate glial phagocytosis of apoptotic neurons during embryogenesis. However, less is known about the removal of apoptotic neurons during later stages of development. Here we show that during metamorphosis, Draper plays a critical role in apoptotic cell clearance by glia, whereas SIMU, which is mostly expressed in pupal macrophages outside the brain, is not involved in glial phagocytosis. We found that Draper activates Drosophila c-Jun N-terminal kinase (dJNK) signaling predominantly in the ensheathing glia and astrocytes, where it is required for efficient removal of apoptotic neurons. Our data suggest that besides the dJNK pathway, Draper also triggers an additional signaling pathway capable of removing apoptotic neurons in the pupal brain. This study thus reveals that SIMU unexpectedly is not involved in glial phagocytosis of apoptotic neurons during metamorphosis and highlights the novel role of dJNK signaling in developmental apoptotic cell clearance downstream of Draper.

Drosophila GATA Factor Serpent Establishes Phagocytic Ability of Embryonic Macrophages.

During Drosophila embryogenesis, a large number of apoptotic cells are efficiently engulfed and degraded by professional phagocytes, macrophages. Phagocytic receptors Six-Microns-Under (SIMU), Draper (Drpr) and Croquemort (Crq) are specifically expressed in embryonic macrophages and required for their phagocytic function. However, how this function is established during development remains unclear. Here we demonstrate that the key regulator of Drosophila embryonic hemocyte differentiation, the transcription factor Serpent (Srp), plays a central role in establishing macrophage phagocytic competence. Srp, a homolog of the mammalian GATA factors, is required and sufficient for the specific expression of SIMU, Drpr and Crq receptors in embryonic macrophages. Moreover, we show that each of these receptors can significantly rescue phagocytosis defects of macrophages in srp mutants, including their distribution in the embryo and engulfment of apoptotic cells. This reveals that the proficiency of macrophages to remove apoptotic cells relies on the expression of SIMU, Crq and/or Drpr. However, Glial Cells Missing (GCM) acting downstream of Srp in the differentiation of hemocytes, is dispensable for their phagocytic function during embryogenesis. Taken together, our study discloses the molecular mechanism underlying the development of macrophages as skilled phagocytes of apoptotic cells.

Apoptotic Cell Clearance in Development.

Programmed cell death and its specific form apoptosis play an important role during development of multicellular organisms. They are crucial for morphogenesis and organ sculpting as well as for adjusting cell number in different systems. Removal of apoptotic cells is the last critical step of apoptosis. Apoptotic cells are properly and efficiently recognized and eliminated through phagocytosis, which is performed by professional and nonprofessional phagocytes. Phagocytosis of apoptotic cells or apoptotic cell clearance is a dynamic multistep process, involving interactions between phagocytic receptors and ligands on apoptotic cells, which are highly conserved in evolution. However, this process is extremely redundant in mammals, containing multiple factors playing similar roles in the process. Using model organisms such as Caenorhabditis elegans, Drosophila melanogaster, zebrafish, and mouse permits addressing fundamental questions in developmental cell clearance by a comprehensive approach including powerful genetics and cell biological tools enriched by live imaging. Recent studies in model organisms have enhanced significantly our understanding of the molecular and cellular basis of apoptotic cell clearance during development. Here, we review the current knowledge and illuminate the great potential of the research performed in genetic models, which opens new directions in developmental biology.

The role of Drosophila TNF Eiger in developmental and damage-induced neuronal apoptosis.

Eiger, the sole Drosophila TNF-alpha homolog, causes ectopic apoptosis through JNK pathway activation. Yet, its role in developmental apoptosis remains unclear. eiger mutant flies are viable and fertile but display compromised elimination of oncogenic cells and extracellular bacteria. Here we show that Eiger, specifically expressed in embryonic neurons and glia, is not involved in developmental neuronal apoptosis or in apoptotic cell clearance. Instead, we provide evidence that Eiger is required for damage-induced apoptosis in the embryonic CNS through regulation of the pro-apoptotic gene hid independently of the JNK pathway. Our study thus reveals a new requirement for Eiger in eliminating damaged cells during development.

Drosophila model for studying phagocytosis following neuronal cell death.

During central nervous system (CNS ) development, a large number of neurons die by apoptosis and are efficiently removed through phagocytosis. Since apoptosis and apoptotic cell clearance are highly conserved in evolution, relatively simple and easily accessible Drosophila embryonic CNS provides a good model to study molecular and cellular mechanisms of these processes. Here, we describe how to assess neuronal apoptosis and glial phagocytosis of apoptotic neurons using immunohistochemistry of whole fixed embryos and live imaging of developing embryonic CNS. Combination of these different strategies allows a comprehensive analysis of neuronal cell death in vivo.

Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis.

The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.

Live imaging of apoptotic cell clearance during Drosophila embryogenesis.

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes--macrophages and dendritic cells to 'non-professional'--tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Caspase activity is required for engulfment of apoptotic cells.

Clearance of apoptotic cells by phagocytic neighbors is crucial for normal development of multicellular organisms. However, how phagocytes discriminate between healthy and dying cells remains poorly understood. We focus on glial phagocytosis of apoptotic neurons during development of the Drosophila central nervous system. We identified phosphatidylserine (PS) as a ligand on apoptotic cells for the phagocytic receptor Six Microns Under (SIMU) and report that PS alone is not sufficient for engulfment. Our data reveal that, additionally to PS exposure, caspase activity is required for clearance of apoptotic cells by phagocytes. Here we demonstrate that SIMU recognizes and binds PS on apoptotic cells through its N-terminal EMILIN (EMI), Nimrod 1 (NIM1), and NIM2 repeats, whereas the C-terminal NIM3 and NIM4 repeats control SIMU affinity to PS. Based on the structure-function analysis of SIMU, we discovered a novel mechanism of internal inhibition responsible for differential affinities of SIMU to its ligand which might prevent elimination of living cells exposing PS on their surfaces.

Keeping the CNS clear: glial phagocytic functions in Drosophila.

Elimination of unwanted and potentially harmful matter is crucial for nervous system development and function. Glia are the main cleaners of the CNS that perform their function through engulfment and degradation of dying neurons and degenerating neuronal branches, developing excessive axons and synapses. Recent studies in Drosophila melanogaster have enhanced significantly our understanding of the phagocytic functions of glia and demonstrated that Drosophila provides an excellent model for investigating the molecular and cellular basis of glial phagocytosis. The current knowledge and great potential of this model, which is reviewed here, can open new directions in mammalian glial biology.

Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.

The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. Here we report the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. Our study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as "semiprofessional" phagocytes in the nervous system.