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• W3070828650 abstract "•Ameboid microglia invade the corpus callosum and engulf OPCs during development•Majority of OPCs engulfed by ameboid microglia in the corpus callosum are viable•Fractalkine receptor-deficient microglia exhibit a reduction in engulfment of OPCs•Fractalkinereceptor-deficient mice have reduced myelin thickness in adulthood Oligodendrogenesis occurs during early postnatal development, coincident with neurogenesis and synaptogenesis, raising the possibility that microglia-dependent pruning mechanisms that modulate neurons regulate myelin sheath formation. Here we show a population of ameboid microglia migrating from the ventricular zone into the corpus callosum during early postnatal development, termed “the fountain of microglia,” phagocytosing viable oligodendrocyte progenitor cells (OPCs) before onset of myelination. Fractalkine receptor-deficient mice exhibit a reduction in microglial engulfment of viable OPCs, increased numbers of oligodendrocytes, and reduced myelin thickness but no change in axon number. These data provide evidence that microglia phagocytose OPCs as a homeostatic mechanism for proper myelination. A hallmark of hypomyelinating developmental disorders such as periventricular leukomalacia and of adult demyelinating diseases such as multiple sclerosis is increased numbers of oligodendrocytes but failure to myelinate, suggesting that microglial pruning of OPCs may be impaired in pathological states and hinder myelination. Oligodendrogenesis occurs during early postnatal development, coincident with neurogenesis and synaptogenesis, raising the possibility that microglia-dependent pruning mechanisms that modulate neurons regulate myelin sheath formation. Here we show a population of ameboid microglia migrating from the ventricular zone into the corpus callosum during early postnatal development, termed “the fountain of microglia,” phagocytosing viable oligodendrocyte progenitor cells (OPCs) before onset of myelination. Fractalkine receptor-deficient mice exhibit a reduction in microglial engulfment of viable OPCs, increased numbers of oligodendrocytes, and reduced myelin thickness but no change in axon number. These data provide evidence that microglia phagocytose OPCs as a homeostatic mechanism for proper myelination. A hallmark of hypomyelinating developmental disorders such as periventricular leukomalacia and of adult demyelinating diseases such as multiple sclerosis is increased numbers of oligodendrocytes but failure to myelinate, suggesting that microglial pruning of OPCs may be impaired in pathological states and hinder myelination. Historically, microglia have been surmised to principally play a role in the neuroinflammatory responses of disease and injury. Recent literature highlights their importance in performing surveillance and neuroprotective functions (Ueno et al., 2013Ueno M. Fujita Y. Tanaka T. Nakamura Y. Kikuta J. Ishii M. Yamashita T. Layer V cortical neurons require microglial support for survival during postnatal development.Nat. Neurosci. 2013; 16: 543-551Crossref PubMed Scopus (440) Google Scholar; Vinet et al., 2012Vinet J. Weering H.R. Heinrich A. Kälin R.E. Wegner A. Brouwer N. Heppner F.L. Rooijen N.v. Boddeke H.W. Biber K. Neuroprotective function for ramified microglia in hippocampal excitotoxicity.J. Neuroinflammation. 2012; 9: 27Crossref PubMed Scopus (184) Google Scholar; Davalos et al., 2005Davalos D. Grutzendler J. Yang G. Kim J.V. Zuo Y. Jung S. Littman D.R. Dustin M.L. Gan W.B. ATP mediates rapid microglial response to local brain injury in vivo.Nat. Neurosci. 2005; 8: 752-758Crossref PubMed Scopus (2492) Google Scholar; Nimmerjahn et al., 2005Nimmerjahn A. Kirchhoff F. Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (3569) Google Scholar). Moreover, three different innate immune receptors are expressed on microglia (triggering receptor expressed on myeloid cells 2, complement receptor 3, and fractalkine receptor) regulate synaptic pruning to modulate developmental brain connectivity (Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar’ Filipello et al., 2018Filipello F. Morini R. Corradini I. Zerbi V. Canzi A. Michalski B. Erreni M. Markicevic M. Starvaggi-Cucuzza C. Otero K. et al.The Microglial Innate Immune Receptor TREM2 Is Required for Synapse Elimination and Normal Brain Connectivity.Immunity. 2018; 48: 979-991.e8Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar; Paolicelli et al., 2011Paolicelli R.C. Bolasco G. Pagani F. Maggi L. Scianni M. Panzanelli P. Giustetto M. Ferreira T.A. Guiducci E. Dumas L. et al.Synaptic pruning by microglia is necessary for normal brain development.Science. 2011; 333: 1456-1458Crossref PubMed Scopus (2050) Google Scholar). Furthermore, microglial engulfment of neural precursor cells regulates the developing cerebral cortex (Cunningham et al., 2013Cunningham C.L. Martínez-Cerdeño V. Noctor S.C. Microglia regulate the number of neural precursor cells in the developing cerebral cortex.J. Neurosci. 2013; 33: 4216-4233Crossref PubMed Scopus (495) Google Scholar), and newborn neurons in the adult dentate gyrus are pruned during neurogenesis (Sierra et al., 2010Sierra A. Encinas J.M. Deudero J.J. Chancey J.H. Enikolopov G. Overstreet-Wadiche L.S. Tsirka S.E. Maletic-Savatic M. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis.Cell Stem Cell. 2010; 7: 483-495Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar) to modify synaptic transmission (Adlaf et al., 2017Adlaf E.W. Vaden R.J. Niver A.J. Manuel A.F. Onyilo V.C. Araujo M.T. Dieni C.V. Vo H.T. King G.D. Wadiche J.I. Overstreet-Wadiche L. Adult-born neurons modify excitatory synaptic transmission to existing neurons.eLife. 2017; 6: e19886Crossref PubMed Scopus (41) Google Scholar). Oligodendrogenesis (Kessaris et al., 2006Kessaris N. Fogarty M. Iannarelli P. Grist M. Wegner M. Richardson W.D. Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage.Nat. Neurosci. 2006; 9: 173-179Crossref PubMed Scopus (715) Google Scholar; Levison and Goldman, 1993Levison S.W. Goldman J.E. Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain.Neuron. 1993; 10: 201-212Abstract Full Text PDF PubMed Scopus (595) Google Scholar) occurs at an early postnatal time point, coincident with neurogenesis and synaptogenesis, suggesting that microglia-dependent homeostatic mechanisms that regulate neurons may also play an important role in myelin sheath formation, critical for transmission of action potentials (Smith and Koles, 1970Smith R.S. Koles Z.J. Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity.Am. J. Physiol. 1970; 219: 1256-1258Crossref PubMed Scopus (138) Google Scholar) and metabolic support to axons (Lee et al., 2012Lee Y. Morrison B.M. Li Y. Lengacher S. Farah M.H. Hoffman P.N. Liu Y. Tsingalia A. Jin L. Zhang P.W. et al.Oligodendroglia metabolically support axons and contribute to neurodegeneration.Nature. 2012; 487: 443-448Crossref PubMed Scopus (933) Google Scholar; Fünfschilling et al., 2012Fünfschilling U. Supplie L.M. Mahad D. Boretius S. Saab A.S. Edgar J. Brinkmann B.G. Kassmann C.M. Tzvetanova I.D. Möbius W. et al.Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.Nature. 2012; 485: 517-521Crossref PubMed Scopus (797) Google Scholar). Interestingly, during this same time period in development, microglia with an ameboid morphology migrate from the ventricular zone into the corpus callosum, originally documented as “the fountain of microglia” (Imamoto and Leblond, 1978Imamoto K. Leblond C.P. Radioautographic investigation of gliogenesis in the corpus callosum of young rats. II. Origin of microglial cells.J. Comp. Neurol. 1978; 180: 139-163Crossref PubMed Scopus (164) Google Scholar; Ling, 1979Ling E.A. Transformation of monocytes into amoeboid microglia in the corpus callosum of postnatal rats, as shown by labelling monocytes by carbon particles.J. Anat. 1979; 128: 847-858PubMed Google Scholar). Using a genetically encoded fluorescent reporter, CX3CR1:GFP, for microglia, green ramified cells were found evenly dispersed throughout the brain on embryonic day 14 (E14) (Figure S1A), as described previously (Ginhoux et al., 2010Ginhoux F. Greter M. Leboeuf M. Nandi S. See P. Gokhan S. Mehler M.F. Conway S.J. Ng L.G. Stanley E.R. et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (2771) Google Scholar; Gomez Perdiguero et al., 2015Gomez Perdiguero E. Klapproth K. Schulz C. Busch K. Azzoni E. Crozet L. Garner H. Trouillet C. de Bruijn M.F. Geissmann F. Rodewald H.R. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1121) Google Scholar). On E20, a second population of CX3CR1:GFP cells with ameboid morphology emerged from the ventricular zone (Figure S1B) and increased in density on post-natal day 0 (P0) (Figure S2A). This population infiltrated the corpus callosum by P3 (Figure S2B), increasing in number on P5 (Figure S2C) and P7 (Figure S2D), forming the fountain of microglia (Imamoto and Leblond, 1978Imamoto K. Leblond C.P. Radioautographic investigation of gliogenesis in the corpus callosum of young rats. II. Origin of microglial cells.J. Comp. Neurol. 1978; 180: 139-163Crossref PubMed Scopus (164) Google Scholar; Ling, 1979Ling E.A. Transformation of monocytes into amoeboid microglia in the corpus callosum of postnatal rats, as shown by labelling monocytes by carbon particles.J. Anat. 1979; 128: 847-858PubMed Google Scholar). Ameboid microglia are predominantly located within the corpus callosum (Figures S1C and S1D). Cortical microglia (Figure S1E) are morphologically distinct from ameboid microglia (Figure S1F) found in the corpus callosum during this early postnatal time period. Colocalization with anti-Iba-1 confirmed that this population of ameboid CX3CR1:GFP cells in the corpus callosum is of the myeloid lineage (Figures S3A–S3F). To explore the possibility that the fountain of microglia is derived from peripheral circulating monocytes, CCR2:RFP reporter mice crossed with CX3CR1:GFP reporter mice were used. CCR2 (chemokine C-C motif receptor 2) is a member of the beta chemokine receptor family expressed exclusively by peripheral monocytes (Mizutani et al., 2012Mizutani M. Pino P.A. Saederup N. Charo I.F. Ransohoff R.M. Cardona A.E. The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.J. Immunol. 2012; 188: 29-36Crossref PubMed Scopus (246) Google Scholar; Saederup et al., 2010Saederup N. Cardona A.E. Croft K. Mizutani M. Cotleur A.C. Tsou C.L. Ransohoff R.M. Charo I.F. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.PLoS ONE. 2010; 5: e13693Crossref PubMed Scopus (371) Google Scholar). Confocal imaging of the corpus callosum on P4 and P7 showed green CX3CR1-expressing cells but no red CCR2-expressing cells (Figures S3G and S3H), as reported previously (Hagemeyer et al., 2017Hagemeyer N. Hanft K.M. Akriditou M.A. Unger N. Park E.S. Stanley E.R. Staszewski O. Dimou L. Prinz M. Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood.Acta Neuropathol. 2017; 134: 441-458Crossref PubMed Scopus (206) Google Scholar). The spleen served as a positive internal control. Ameboid microglia infiltrate the corpus callosum during a restricted time period that parallels oligodendrogenesis. To evaluate this potential interaction, NG2:Tom reporter mice were used to fate-map oligodendrocyte progenitor cells (OPCs) in the corpus callosum, and microglia were immunostained with Iba-1. Confocal 3D reconstruction was performed in the corpus callosum above the lateral ventricles (Figure 1A, white boxes). A high-powered view of a 0.3-μm plane from a z stack shows red OPCs within green microglia at P7 (Figures 1B–1D; Video S1). Representative confocal 3D reconstruction at P4 demonstrates ameboid microglia with bushy processes in contact with OPCs (Figure 1E). At P7, ameboid microglia with few processes are engulfing OPCs (Figure 1F). At P9, engulfment is decreased (Figure 1G), and by P11, many microglia begin to assume a ramified morphology (Figure 1H). At P15, microglia are predominantly ramified (Figure 1I), which persists at P30 (Figure S1G), consistent with morphology found in adult brains. To assess OPC engulfment and contact by microglia, Imaris software (Bitplane) was used to analyze 3D confocal reconstructions, as described previously (Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar), in three areas of the corpus callosum (Figure 1A). Engulfment, defined as 15% or more of NG2:Tom OPC volume internalized within microglia, peaked at P7 (Figure 1J). Contacting was defined as less than 15% of NG2:Tom OPC volume internalized within microglia (Figure S1H). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjZjNkNTVjMDdjNjFkYzIxMTQ2YTkzZmJhMmU2ZmQwNSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ3MzA5OTAxfQ.oSGskysU7O7ylMgHkA6I3HdgJmHJ_ZyuFICY4E__bUsLAf1ccUOGfNLQFq2q8WBMn9HFCugzwlJz0JNWEwdyBS4ND-kjQ-emm9G_xR_ZEWZhsozAMR56V67XAXYcZgNpjdJQHF_YrLCn7HtNv-1tFKNhJxGndYOXj6qxlboUa4ZNLc2VJ_4vlf_ckerIKc8KqE2VMtN9QvZ_bRcVvjuplXVKRAplm9L9GyJ1l7-jhCcJHLEbuwW993QOqZQzI2iAmEAWPWt5xJGDzikw1qcrnOu_tDzKih-kldv2m6dyWz17mc0egU4GvBYQEcpxR7FL2C7PldGzgPN1n24jFmHFBg Download .mp4 (0.96 MB) Help with .mp4 files Video S1. Microglia Engulf OPCs in the Corpus Callosum on P7, Related to Figure 1Representative 3D reconstruction of a confocal z stack of engulfed NG2:Tom OPCs (red) inside Iba-1 labeled microglia (green). Image stacks were taken using 40x magnification and 0.3μm steps on a Nikon C2 confocal microscope. To further investigate whether microglia contained encapsulated cells consistent with data from 3D confocal imaging (Figure 1), ultrastructural assessment of the corpus callosum at P7 was performed. Microglia were identified as cells with a dark, irregularly shaped nucleus (Figures 2A–2D, asterisk) and densely populated with coarse chromatin and cytoplasm (Figures 2A–2D, pseudo-colored green) with a prominent Golgi apparatus (Mori and Leblond, 1969Mori S. Leblond C.P. Identification of microglia in light and electron microscopy.J. Comp. Neurol. 1969; 135: 57-80Crossref PubMed Scopus (287) Google Scholar; Sturrock, 1981Sturrock R.R. Microglia in the prenatal mouse neostriatum and spinal cord.J. Anat. 1981; 133: 499-512PubMed Google Scholar; Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar). Microglia were observed beginning to phagocytose a cell (Figure 2A, arrowhead). Microglia were identified with one or two fully encapsulated cells (Figures 2A–2D; arrows) consistent with confocal imaging (Figure 1). Fully engulfed cells were at varying degrees of degradation, signifying phagocytosis (Figures 2A–2D, arrows). Microglia in contact with each other (Figure 2D) were also detected, similar to confocal imaging (Figure 1F). A marker of lysosomes specific to the myeloid lineage, CD68, was used to confirm phagocytosis (Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar). Confocal 3D reconstruction shows CX3CR1:GFP microglia (Figure 2E) engulfing NG2:Tom cells (Figure 2F; merge in Figure 2G). CD68 staining (Figure 2H) shows localization of engulfed OPCs within the lysosome of microglia (Figure 2I; Video S2) and colocalization with bisbenzimide, a marker of cell nuclei (Figures 2J and 2K). The percent volume of CX3CR1:GFP microglia containing CD68+ lysosome was 56.07% ± 8.20%, and that of NG2:Tom OPCs was 29.48% ± 1.39% (Figure 2L). The percentage of engulfed NG2:Tom OPCs that colocalized with CD68 was 90.77% ± 2.32%, and the percentage of engulfed NG2:Tom OPCs that did not colocalize with CD68 was 9.23% ± 2.32% (Figure 2M). Thus, more than half the volume of ameboid microglia is occupied by the CD68+ lysosome, and more than 90% of OPCs being engulfed are inside the lysosomal compartment. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwNDYxZTEyZjU5YmE3ZjRjNTc1YWVkY2NhYTlkMjlkOSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ3MzA5OTAxfQ.lS7OJM7ddOtkdLPgKV2QOKIcWcDpdNkoJfnM6ANibt3dSEf-vnE7ok4wXUkbyfgTTM8sCxd34yq73TTAT24eo3gQgx9oJzA29ER1-QWJP2umlmepm2Za8UGup5VyK2OpQtdxkS4zEvw9_KmhO2joS_wrtEjMUVK612dyrmt7vsshgehYwSQIAkTUAWhWDY7Wk0_6LW-KVTvsgchFdpj7DMUAPiSy_7wsGLD37s7W2yEews8JM4RrbHC2P_u21AopuXxewVKF_j23S4B8tDZuniSYniybdz9zDsG4eSzn0w6dsvcuSbc6oAJsDr5O5J4ScnlslauBGx96o1KPAFNfrQ Download .mp4 (1.12 MB) Help with .mp4 files Video S2. Engulfed OPCs Are Located within the Lysosome of Microglia, Related to Figure 2Representative 3D reconstruction shows NG2:Tom OPCs (red) inside of the CD68+ lysosome (pink) of amoeboid microglia (green) in the P7 corpus callosum. Apoptosis of cells or synapses signals microglial phagocytosis, regulating neural development (Sierra et al., 2010Sierra A. Encinas J.M. Deudero J.J. Chancey J.H. Enikolopov G. Overstreet-Wadiche L.S. Tsirka S.E. Maletic-Savatic M. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis.Cell Stem Cell. 2010; 7: 483-495Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar; Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar). A representative confocal 3D reconstruction of CX3CR1:GFP microglia (Figure 2N) engulfing an NG2:dsRed OPC (Figure 2O; merge in Figure 2P) in the corpus callosum shows that the majority of OPCs are negative for caspase-3 (Figure 2Q; merge in Figure 2R), with bisbenzimide showing in blue (Figure 2S; merge in Figure 2T). The percentage of NG2:dsRed OPCs engulfed that were immunopositive for caspase-3 was 10.16% ± 2.33% (Figures 2U and S4A), whereas the percentage of NG2:dsRed OPCs engulfed that were negative for caspase-3 was 89.84% ± 2.33% (Figure 2U). Similar results were obtained for microglia contacting OPCs (Figure 2V; caspase-3-positive (+), 14.96% ± 2.99%; caspase-3-negative (−), 85.04% ± 2.99%). Therefore, the majority of OPCs engulfed or contacted by microglia were not apoptotic. NG2:dsRed-negative cells engulfed by microglia were immunopositive for caspase-3 in the cortex on P3 (Figure S4B), serving as a positive internal control because neuronal apoptosis is documented during development (Ahern et al., 2013Ahern T.H. Krug S. Carr A.V. Murray E.K. Fitzpatrick E. Bengston L. McCutcheon J. De Vries G.J. Forger N.G. Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: effects of age and sex.J. Comp. Neurol. 2013; 521: 2551-2569Crossref PubMed Scopus (40) Google Scholar). Additionally, caspase-3 staining was detected in the subventricular zone on P3 in a few NG2:dsRed OPCs engulfed by microglia (Figure S4C) as well as a rare NG2:dsRed OPC not in close proximity to microglia (Figure S4D). Phosphatidylserine (PS) is expressed on cells in early stages of apoptosis or cellular stress, known as an “eat me” signal in neuronal pruning (Brown and Neher, 2014Brown G.C. Neher J.J. Microglial phagocytosis of live neurons.Nat. Rev. Neurosci. 2014; 15: 209-216Crossref PubMed Scopus (455) Google Scholar). Confocal 3D reconstruction of engulfed OPCs (Figures 2W–2X; merge in Figure 2Y) shows that the majority of OPCs are negative for anti-PS (Figure 2Z; merge in Figure 2Aa), with bisbenzimide showing in blue (Figure 2Bb; merge in Figure 2Cc). The percentage of engulfed NG2:dsRed OPCs immunopositive for anti-PS was 11.65% ± 4.77% (Figures 2Dd and S4E), whereas the percentage of NG2:dsRed OPCs engulfed that were negative for anti-PS was 88% ± 4.77% (Figure 2Dd). Similar results were obtained for microglia contacting OPCs (Figure 2Ee; anti-PS+, 15.88% ± 3.24; PS–, 84.12% ± 3.24%). In the subventricular zone at P7, a rare NG2:dsRed cell is immunopositive for anti-PS but not engulfed by microglia (Figure S4F), serving as a positive internal control. Also, a few cells negative for NG2:dsRed but positive for anti-PS were found within microglia in the subventricular zone on P7 (Figure S4G). Additionally, the cell volumes of OPCs as well as their sphericity (Figures S1I and S1J) are not different based on their interaction with microglia (engulfed or contacted) or no interactions with microglia (not contacted or engulfed). Therefore, the majority of OPCs engulfed or contacted by microglia did not appear to be undergoing cellular stress. To capture the engulfment process as well as further explore the cellular integrity of OPCs being engulfed or contacted by microglia, live 3D confocal time-lapse imaging was performed on acute ex vivo slices of the P7 corpus callosum from CX3CR1:GFP crossed with NG2:dsRed mice. Video S3 shows two different interactions of microglia with OPCs. In the first interaction, microglia contact an OPC on several occasions, but no engulfment occurs (Video S3, arrow). In the second interaction, microglia contact an OPC one or more times before engulfment (Video S3, arrowhead). 3D confocal reconstructed images from time-lapse microscopy of the corpus callosum in a P7 mouse brain show microglia engulfing an OPC (Figure 3A). When microglia begin to engulf an OPC, complete encapsulation occurs within 20 min (Video S4). Next, live 3D confocal time-lapse imaging of the P7 corpus callosum was performed in ex vivo slices from CX3CR1:GFP;NG2:dsRed mice in the presence of PSVue, a fluorescent dye that binds to negatively charged phospholipids such as PS, which is expressed on cells in early stages of apoptosis or cellular stress, known as an “eat me” signal in neuronal pruning (Brown and Neher, 2014Brown G.C. Neher J.J. Microglial phagocytosis of live neurons.Nat. Rev. Neurosci. 2014; 15: 209-216Crossref PubMed Scopus (455) Google Scholar). Quantification of 3D reconstructed images every 3 min over a 12-h period from four videos showed only 3 of 32 NG2:dsRed OPCs became PSVue+ before engulfment, providing evidence that the majority of OPCs were viable prior to engulfment. Only one cell became PSVue+ after engulfment; however, this happened very quickly, suggesting that it may have been in poor health prior to engulfment. The majority of cells, 28 of 32, never expressed PSVue at any point during the engulfment process. Representative 3D confocal images show that microglial engulfed OPCs (Figure 3B and 3C, arrows; merge in Figure 3D) do not express PSVue (Figure 3E; merge with dsRed only in Figure 3F; merge of all in Figure 3G; Video S5). PSVue cell debris that was not expressing dsRed was found in microglia (Figures 3B–3G, asterisks) as well as adjacent to NG2:dsRed OPCs, indicating that the dye penetrated the tissue (Video S5). To further explore the viability of OPCs during contact and engulfment by microglia, we performed 3D confocal reconstruction of ex vivo brain slices incubated with oxazole blue, a cell-impermeant nucleic acid stain for dead cells, and DilC1(5), a carbocyanine dye with a far-red fluorescence signal produced in the presence of mitochondrial membrane potential for viable cells. Representative 3D reconstructed images show CX3CR1:GFP microglia engulfing NG2:dsRed OPCs (Figures 3H and 3I; merge in Figure 3J) in the P7 corpus callosum, which are negative for oxazole blue (Figure 3K; merge in Figure 3L). To show viable cells, DilC1(5) (Figure 3M; merge in Figure 3N), was used simultaneously, showing that engulfed OPCs are viable (Figure 3O). Arrows indicate engulfed NG2:dsRed OPCs, which are negative for oxazole blue but positive for DilC1(5). The percentage of engulfed DilC(1)5+ NG2:dsRed OPCs was 86.37% ± 4.94%, and the percentage of engulfed oxazole blue+ NG2:dsRed OPCs was 13.63% ± 4.94% (Figure 3P), providing evidence that the majority of engulfed OPCs are viable. Consistent with these data, the percentage of engulfed DilC(1)5− NG2:dsRed OPCs was 13.56% ± 4.94% (Figure 3Q), and the percentage of engulfed oxazole blue− NG2:dsRed OPCs was 85.86% ± 4.94% (Figure 3R). Likewise, the majority of contacted NG2:dsRed OPCs were DilC1(5)+ but not oxazole blue+ (Figure 3S; 76.29% ± 6.75% and 23.71% ± 6.75%, respectively), supporting that engulfed and contacted OPCs by microglia are predominantly viable. Quantification of the percentage of contacted DilC1(5)− NG2:dsRed OPCs (Figure 3T; 23.71% ± 6.75%) and oxazole blue− NG2:dsRed OPCs (Figure 3U; 76.29% ± 6.75%) also corroborate that the majority of contacted OPCs are viable. Of note, no OPCs were positive for both markers, indicating specificity of the respective dyes. As a control for ex vivo brain sections, the percentage of engulfed OPCs quantified from 3D confocal images was compared with perfusion-fixed brain sections from CX3CR1:GFP;NG2:dsRed mice. There was no difference in engulfment from ex vivo brain sections compared with perfusion-fixed sections, which demonstrates that 39.18% ± 6.20% of the total NG2:dsRed OPCs are being engulfed by microglia in the corpus callosum (Figures S4H and S4I). Additionally, analysis of dye penetration in all OPCs and microglia is shown in Figures S4J and S4K. These data, in conjunction with live imaging data for PSVue (Figures 3B–3G) and lack of immunopositive staining for caspase-3 (Figures 2N–2V) and anti-PS (Figures 2W–2Ee) provide evidence that OPCs in the corpus callosum are viable before engulfment by microglia. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiNTUyM2I3Njc5ODAwODFlYWI5OGJhMjY2ODdhNmJjMCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ3MzA5OTAxfQ.QP2qurS5JZprZaKpJZNlE3HEPBOqvMKmjWbz5oEnVsuBMyoR1FOYCn1BOE2vwBr9sn16xgKjGATs44t0UcD2qyG6X9umMxJHRxrLldIO3ktVPxijL-xuEy_2NESv-kbI_Dfodtb6sueSBM-bQ35Zz4gbTKB_n6vDDMSYmKcDZxXBCmeO8hia6O1l7P3IBAVbO1h_Rlw9VlLvOZKaWwkln73lEHQeHSjiW36bbVPwjxhigBMrFD5lG2SuN0MgojrQKNvuSxHNyKK13k9wESW_0GTxAIFfBFMSVhLpPC1RyFyVZvpR9LFHMC3knZLDVqAl_a4vjPpXX063DOkYQd3-kw Download .mp4 (7.49 MB) Help with .mp4 files Video S3. 3D Confocal Time-Lapse Microscopy of the Corpus Callosum in Ex Vivo Brain Slices from a CX3CR1:GFP;NG2:dsRed Mouse on P7, Related to Figure 3Representative video shows amoeboid microglia (green) actively surveying their environment, contacting and engulfing OPCs (red). Arrowhead shows one example of microglia engulfing an OPC. Arrow shows another example of a microglial cell contacting one OPC but not engulfing it, then moving to another OPC and engulfing it instead. Image stacks were collected every 4 minutes using a Leica TCS-SP confocal microscope at 40x magnification from n = 2 mice in two fields of view over 12 hours. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhMjA4NjEyYTI3NzgwNTkwMzQyOTE4MjE3MWJiZDE4NiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ3MzA5OTAxfQ.aSj8rpDtPVG9FWpjQnoACalcB2MNM1ISxnkAEQtg5q1nPe6ez7JBSeJkVyKH9X7JKDj6qGE_MLtGLywpBmLfISG2KTnUWS-mTCJ0hp47X57gPRc-FAt48ixU4RRNOcsWfCeBB5CzM_ofuTI95LWvC6M9GrmaqLnzmahXY8b5UihfrvqEg-Kz2xnRuGaQ-Lvg5AMMyyyA2LdFrGDkwtq8srDmkK4aZzir20Ac0SQ7pM5D62dABbx7CewVkoGEhdA4qj_WVXggv7ZtxroYU_pSKNPnuqE42H2sP8EEZkNrYPdUEa941AgI8VVAq3MfcZwi7kRysnq1-lwK2DhwAGHJtQ Download .mp4 (0.04 MB) Help with .mp4 files Video S4. Cropped View of 3D Confocal Time-Lapse Microscopy, Showing a Microglial Cell Actively Engulfing an OPC, Related to Figure 3Representative video from live imaging experiment shown in Figure 3A of microglial cell (green) engulfing an OPC (red) in a duration of 20 minutes. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwZDk5NTk2MDcxNTA4ZWU5MzFlNmM0YWE1OTQwZjYwNyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ3MzA5OTAxfQ.PVXd9FWAyhnNJxialUNGJvrC4tzEUxYoUHbYx6ypuOZOdWI52G3-bB8rjSaJJNEcAh_CHGyituWDv7zG7obemwrEc6R92m6xaPLjyoCs5Hsgf7l72MFh1Hl9D4YEq6X-_VXNDNf1C7IPh-Eh2aGszYguCQMhl8dGbihKhyO1id-Z239" @default.
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• W3070828650 date "2020-08-01" @default.
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• W3070828650 title "Fractalkine-Dependent Microglial Pruning of Viable Oligodendrocyte Progenitor Cells Regulates Myelination" @default.
• W3070828650 cites W1546401219 @default.
• W3070828650 cites W1589868346 @default.
• W3070828650 cites W1963696719 @default.
• W3070828650 cites W1975265782 @default.
• W3070828650 cites W1978924462 @default.
• W3070828650 cites W1980286364 @default.
• W3070828650 cites W1980471744 @default.
• W3070828650 cites W1980789245 @default.
• W3070828650 cites W1985286185 @default.
• W3070828650 cites W1996055705 @default.
• W3070828650 cites W2001888569 @default.
• W3070828650 cites W2005303635 @default.
• W3070828650 cites W2015412022 @default.
• W3070828650 cites W2018338123 @default.
• W3070828650 cites W2027467386 @default.
• W3070828650 cites W2029190354 @default.
• W3070828650 cites W2033617297 @default.
• W3070828650 cites W2038118492 @default.
• W3070828650 cites W2038811384 @default.
• W3070828650 cites W2042698977 @default.
• W3070828650 cites W2054275187 @default.
• W3070828650 cites W2058358434 @default.
• W3070828650 cites W2058915525 @default.
• W3070828650 cites W2069929255 @default.
• W3070828650 cites W2071042477 @default.
• W3070828650 cites W2071099949 @default.
• W3070828650 cites W2085029391 @default.
• W3070828650 cites W2091159687 @default.
• W3070828650 cites W2094660330 @default.
• W3070828650 cites W2095467094 @default.
• W3070828650 cites W2096423240 @default.
• W3070828650 cites W2097849969 @default.
• W3070828650 cites W2103279601 @default.
• W3070828650 cites W2104003768 @default.
• W3070828650 cites W2111447973 @default.
• W3070828650 cites W2117719082 @default.
• W3070828650 cites W2117723028 @default.
• W3070828650 cites W2120911948 @default.
• W3070828650 cites W2122828560 @default.
• W3070828650 cites W2142242761 @default.
• W3070828650 cites W2148898943 @default.
• W3070828650 cites W2150724604 @default.
• W3070828650 cites W2160779535 @default.
• W3070828650 cites W2165730834 @default.
• W3070828650 cites W2309640507 @default.
• W3070828650 cites W2337852945 @default.
• W3070828650 cites W2411898393 @default.
• W3070828650 cites W2462084918 @default.
• W3070828650 cites W2514150090 @default.
• W3070828650 cites W2581847505 @default.
• W3070828650 cites W2653495475 @default.
• W3070828650 cites W2728530348 @default.
• W3070828650 cites W2741386819 @default.
• W3070828650 cites W2753593979 @default.
• W3070828650 cites W2756092850 @default.
• W3070828650 cites W2796365598 @default.
• W3070828650 cites W2799868609 @default.
• W3070828650 cites W2801885674 @default.
• W3070828650 cites W2900856701 @default.
• W3070828650 cites W2902676103 @default.
• W3070828650 cites W2911063894 @default.
• W3070828650 cites W2938531047 @default.
• W3070828650 cites W2949996247 @default.
• W3070828650 doi "https://doi.org/10.1016/j.celrep.2020.108047" @default.
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