Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue.

Central nervous system myelin is a multilayered membrane sheath generated by oligodendrocytes for rapid impulse propagation. However, the underlying mechanisms of myelin wrapping have remained unclear. Using an integrative approach of live imaging, electron microscopy, and genetics, we show that new myelin membranes are incorporated adjacent to the axon at the innermost tongue. Simultaneously, newly formed layers extend laterally, ultimately leading to the formation of a set of closely apposed paranodal loops. An elaborated system of cytoplasmic channels within the growing myelin sheath enables membrane trafficking to the leading edge. Most of these channels close with ongoing development but can be reopened in adults by experimentally raising phosphatidylinositol-(3,4,5)-triphosphate levels, which reinitiates myelin growth. Our model can explain assembly of myelin as a multilayered structure, abnormal myelin outfoldings in neurological disease, and plasticity of myelin biogenesis observed in adult life.

A new gatekeeper to control oligodendrogenesis.

The diversity of oligodendrocyte precursor cells (OPCs) is not well understood and is actively discussed in the field. A new study in PLOS Biology describes a novel marker for an OPC subpopulation that controls oligodendrogenesis and myelination.

Insights into mechanisms of central nervous system myelination using zebrafish.

Myelin is the multi-layered membrane that surrounds most axons and is produced by oligodendrocytes in the central nervous system (CNS). In addition to its important role in enabling rapid nerve conduction, it has become clear in recent years that myelin plays additional vital roles in CNS function. Myelinating oligodendrocytes provide metabolic support to axons and active myelination is even involved in regulating forms of learning and memory formation. However, there are still large gaps in our understanding of how myelination by oligodendrocytes is regulated. The small tropical zebrafish has become an increasingly popular model organism to investigate many aspects of nervous system formation, function, and regeneration. This is mainly due to two approaches for which the zebrafish is an ideally suited vertebrate model--(1) in vivo live cell imaging using vital dyes and genetically encoded reporters, and (2) gene and target discovery using unbiased screens. This review summarizes how the use of zebrafish has helped understand mechanisms of oligodendrocyte behavior and myelination in vivo and discusses the potential use of zebrafish to shed light on important future questions relating to myelination in the context of CNS development, function and repair.

Regulation of oligodendrocyte precursor maintenance by chondroitin sulphate glycosaminoglycans.

Chondroitin sulfate proteoglycans (CSPGs) have been proven to inhibit morphological maturation of oligodendrocytes as well as their myelination capabilities. Yet, it remained unclear, whether CSPGs and/or their respective chondroitin sulfate glycosaminoglycan (CS-GAG) side chains also regulate the oligodendrocyte lineage progression. Here, we initially show that CS-GAGs detected by the monoclonal antibody 473HD are expressed by primary rat NG2-positive oligodendrocyte precursor cells (OPCs) and O4-positive immature oligodendrocytes. CS-GAGs become down-regulated with ongoing oligodendrocyte differentiation. Enzymatic removal of the CS-GAG chains by the bacterial enzyme Chondroitinase ABC (ChABC) promoted spontaneous differentiation of proliferating rat OPCs toward O4-positive immature oligodendrocytes. Upon forced differentiation, the enzymatic removal of the CS-GAGs accelerated oligodendrocyte differentiation toward both MBP-positive and membrane forming oligodendrocytes. These processes were attenuated on enriched CSPG fractions, mainly consisting of Phosphacan/RPTPß/ζ and to less extent of Brevican and NG2. To qualify CS-GAGs as universal regulators of oligodendrocyte biology, we finally tested the effect of CS-GAG removal on OPCs from different sources such as mouse cortical oligospheres, mouse spinal cord neurospheres, and most importantly human-induced pluripotent stem cell-derived radial glia-like neural precursor cells. For all culture systems used, we observed a similar inhibitory effect of CS-GAGs on oligodendrocyte differentiation. In conclusion, this study clearly suggests an important fundamental principle for complex CS-GAGs to regulate the oligodendrocyte lineage progression. Moreover, the use of ChABC in order to promote oligodendrocyte differentiation toward myelin gene expressing cells might be an applicable therapeutic option to enhance white matter repair.

Individual axons regulate the myelinating potential of single oligodendrocytes in vivo.

The majority of axons in the central nervous system (CNS) are eventually myelinated by oligodendrocytes, but whether the timing and extent of myelination in vivo reflect intrinsic properties of oligodendrocytes, or are regulated by axons, remains undetermined. Here, we use zebrafish to study CNS myelination at single-cell resolution in vivo. We show that the large caliber Mauthner axon is the first to be myelinated (shortly before axons of smaller caliber) and that the presence of supernumerary large caliber Mauthner axons can profoundly affect myelination by single oligodendrocytes. Oligodendrocytes that typically myelinate just one Mauthner axon in wild type can myelinate multiple supernumerary Mauthner axons. Furthermore, oligodendrocytes that exclusively myelinate numerous smaller caliber axons in wild type can readily myelinate small caliber axons in addition to the much larger caliber supernumerary Mauthner axons. These data indicate that single oligodendrocytes can myelinate diverse axons and that their myelinating potential is actively regulated by individual axons.

Glial Cell Development and Function in the Zebrafish Central Nervous System.

Over the past decades the zebrafish has emerged as an excellent model organism with which to study the biology of all glial cell types in nervous system development, plasticity, and regeneration. In this review, which builds on the earlier work by Lyons and Talbot in 2015, we will summarize how the relative ease to manipulate the zebrafish genome and its suitability for intravital imaging have helped understand principles of glial cell biology with a focus on oligodendrocytes, microglia, and astrocytes. We will highlight recent findings on the diverse properties and functions of these glial cell types in the central nervous system and discuss open questions and future directions of the field.

Synaptic input and Ca2+ activity in zebrafish oligodendrocyte precursor cells contribute to myelin sheath formation.

In the nervous system, only one type of neuron-glial synapse is known to exist: that between neurons and oligodendrocyte precursor cells (OPCs), yet their composition, assembly, downstream signaling and in vivo functions remain largely unclear. Here, we address these questions using in vivo microscopy in zebrafish spinal cord and identify postsynaptic molecules PSD-95 and gephyrin in OPCs. The puncta containing these molecules in OPCs increase during early development and decrease upon OPC differentiation. These puncta are highly dynamic and frequently assemble at 'hotspots'. Gephyrin hotspots and synapse-associated Ca2+ activity in OPCs predict where a subset of myelin sheaths forms in differentiated oligodendrocytes. Further analyses reveal that spontaneous synaptic release is integral to OPC Ca2+ activity, while evoked synaptic release contributes only in early development. Finally, disruption of the synaptic genes dlg4a/dlg4b, gphnb and nlgn3b impairs OPC differentiation and myelination. Together, we propose that neuron-OPC synapses are dynamically assembled and can predetermine myelination patterns through Ca2+ signaling.

Myelination-independent functions of oligodendrocyte precursor cells in health and disease.

Oligodendrocyte precursor cells (OPCs) are a population of tissue-resident glial cells found throughout the CNS, constituting approximately 5% of all CNS cells and persisting from development to adulthood and aging. The canonical role of OPCs is to give rise to myelinating oligodendrocytes. However, additional functions of OPCs beyond this traditional role as precursors have been suggested for a long time. In this Perspective, we provide an overview of the multiple myelination-independent functions that have been described for OPCs in the context of neuron development, angiogenesis, inflammatory response, axon regeneration and their recently discovered roles in neural circuit remodeling.

Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells.

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.

Oligodendrocyte precursor cells sculpt the visual system by regulating axonal remodeling.

Many oligodendrocyte precursor cells (OPCs) do not differentiate to form myelin, suggesting additional roles of this cell population. The zebrafish optic tectum contains OPCs in regions devoid of myelin. Elimination of these OPCs impaired precise control of retinal ganglion cell axon arbor size during formation and maturation of retinotectal connectivity and degraded functional processing of visual stimuli. Therefore, OPCs fine-tune neural circuits independently of their canonical role to make myelin.

Clusters of neuronal neurofascin prefigure the position of a subset of nodes of Ranvier along individual central nervous system axons in vivo.

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It is assumed that node position is primarily driven by growing myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. Through longitudinal live imaging of node formation dynamics in the zebrafish central nervous system, we show that stable clusters of the cell adhesion molecule neurofascin a can accumulate at specific sites along axons prior to myelination. While some of these clusters are pushed into future node position by extending myelin sheaths, others are not and thus prefigure the position of where a mature node forms. Animals that lack full-length neurofascin a show increased internodal distances and less regular nodal spacing along single axons. Together, our data reveal the existence of an axonal mechanism to position nodes of Ranvier that does not depend on regulation by myelin sheath growth.

Innate Immune Pathways Promote Oligodendrocyte Progenitor Cell Recruitment to the Injury Site in Adult Zebrafish Brain.

The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration.

Regulatory mechanisms that mediate tenascin C-dependent inhibition of oligodendrocyte precursor differentiation.

Here, we present mechanisms for the inhibition of oligodendendrocyte precursor cell (OPC) differentiation, a biological function of neural extracellular matrix (ECM). The differentiation of oligodendrocytes is orchestrated by a complex set of stimuli. In the present study, we investigated the signaling pathway elicited by the ECM glycoprotein tenascin C (Tnc). Tnc substrates inhibit myelin basic protein (MBP) expression of cultured rat oligodendrocytes, and, conversely, we found that the emergence of MBP expression is accelerated in forebrains of Tnc-deficient mice. Mechanistically, Tnc interfered with phosphorylation of Akt, which in turn reduced MBP expression. At the cell surface, Tnc associates with lipid rafts in oligodendrocyte membranes, together with the cell adhesion molecule contactin (Cntn1) and the Src family kinase (SFK) Fyn. Depletion of Cntn1 in OPCs by small interfering RNAs (siRNAs) abolished the Tnc-dependent inhibition of oligodendrocyte differentiation, while Tnc exposure impeded the activation of the tyrosine kinase Fyn by Cntn1. Concomitant with oligodendrocyte differentiation, Tnc antagonized the expression of the signaling adaptor and RNA-binding molecule Sam68. siRNA-mediated knockdown or overexpression of Sam68 delayed or accelerated oligodendrocyte differentiation, respectively. Inhibition of oligodendrocyte differentiation with the SFK inhibitor PP2 could be rescued by Sam68 overexpression, which may indicate a regulatory role for Sam68 downstream of Fyn. Our study therefore uncovers the first signaling pathways that underlie Tnc-induced, ECM-dependent maintenance of the immature state of OPCs.

Proteome Profile of Myelin in the Zebrafish Brain.

The velocity of nerve conduction along vertebrate axons depends on their ensheathment with myelin. Myelin membranes comprise specialized proteins well characterized in mice. Much less is known about the protein composition of myelin in non-mammalian species. Here, we assess the proteome of myelin biochemically purified from the brains of adult zebrafish (Danio rerio), considering its increasing popularity as model organism for myelin biology. Combining gel-based and gel-free proteomic approaches, we identified > 1,000 proteins in purified zebrafish myelin, including all known constituents. By mass spectrometric quantification, the predominant Ig-CAM myelin protein zero (MPZ/P0), myelin basic protein (MBP), and the short-chain dehydrogenase 36K constitute 12%, 8%, and 6% of the total myelin protein, respectively. Comparison with previously established mRNA-abundance profiles shows that expression of many myelin-related transcripts coincides with the maturation of zebrafish oligodendrocytes. Zebrafish myelin comprises several proteins that are not present in mice, including 36K, CLDNK, and ZWI. However, a surprisingly large number of ortholog proteins is present in myelin of both species, indicating partial evolutionary preservation of its constituents. Yet, the relative abundance of CNS myelin proteins can differ markedly as exemplified by the complement inhibitor CD59 that constitutes 5% of the total zebrafish myelin protein but is a low-abundant myelin component in mice. Using novel transgenic reporter constructs and cryo-immuno electron microscopy, we confirm the incorporation of CD59 into myelin sheaths. These data provide the first proteome resource of zebrafish CNS myelin and demonstrate both similarities and heterogeneity of myelin composition between teleost fish and rodents.

Completion of neuronal remodeling prompts myelination along developing motor axon branches.

Neuronal remodeling and myelination are two fundamental processes during neurodevelopment. How they influence each other remains largely unknown, even though their coordinated execution is critical for circuit function and often disrupted in neuropsychiatric disorders. It is unclear whether myelination stabilizes axon branches during remodeling or whether ongoing remodeling delays myelination. By modulating synaptic transmission, cytoskeletal dynamics, and axonal transport in mouse motor axons, we show that local axon remodeling delays myelination onset and node formation. Conversely, glial differentiation does not determine the outcome of axon remodeling. Delayed myelination is not due to a limited supply of structural components of the axon-glial unit but rather is triggered by increased transport of signaling factors that initiate myelination, such as neuregulin. Further, transport of promyelinating signals is regulated via local cytoskeletal maturation related to activity-dependent competition. Our study reveals an axon branch-specific fine-tuning mechanism that locally coordinates axon remodeling and myelination.

Systemic loss of Sarm1 protects Schwann cells from chemotoxicity by delaying axon degeneration.

Protecting the nervous system from chronic effects of physical and chemical stress is a pressing clinical challenge. The obligate pro-degenerative protein Sarm1 is essential for Wallerian axon degeneration. Thus, blocking Sarm1 function is emerging as a promising neuroprotective strategy with therapeutic relevance. Yet, the conditions that will most benefit from inhibiting Sarm1 remain undefined. Here we combine genome engineering, pharmacology and high-resolution intravital videmicroscopy in zebrafish to show that genetic elimination of Sarm1 increases Schwann-cell resistance to toxicity by diverse chemotherapeutic agents after axonal injury. Synthetic degradation of Sarm1-deficient axons reversed this effect, suggesting that glioprotection is a non-autonomous effect of delayed axon degeneration. Moreover, loss of Sarm1 does not affect macrophage recruitment to nerve-wound microenvironment, injury resolution, or neural-circuit repair. These findings anticipate that interventions aimed at inhibiting Sarm1 can counter heightened glial vulnerability to chemical stressors and may be an effective strategy to reduce chronic consequences of neurotrauma.

Functionally distinct subgroups of oligodendrocyte precursor cells integrate neural activity and execute myelin formation.

Recent reports have revealed that oligodendrocyte precursor cells (OPCs) are heterogeneous. It remains unclear whether such heterogeneity reflects different subtypes of cells with distinct functions or instead reflects transiently acquired states of cells with the same function. By integrating lineage formation of individual OPC clones, single-cell transcriptomics, calcium imaging and neural activity manipulation, we show that OPCs in the zebrafish spinal cord can be divided into two functionally distinct groups. One subgroup forms elaborate networks of processes and exhibits a high degree of calcium signaling, but infrequently differentiates despite contact with permissive axons. Instead, these OPCs divide in an activity- and calcium-dependent manner to produce another subgroup, with higher process motility and less calcium signaling and that readily differentiates. Our data show that OPC subgroups are functionally diverse in their response to neurons and that activity regulates the proliferation of a subset of OPCs that is distinct from the cells that generate differentiated oligodendrocytes.

Tenascin C and tenascin R similarly prevent the formation of myelin membranes in a RhoA-dependent manner, but antagonistically regulate the expression of myelin basic protein via a separate pathway.

Membrane formation and the initiation of myelin gene expression are hallmarks of the differentiation of oligodendrocytes from their precursors. Here, we compared the roles of the two related extracellular matrix (ECM) glycoproteins Tenascin C (Tnc) and Tenascin R (Tnr) in oligodendrocyte differentiation. Oligodendrocyte precursors from Tnr-deficient mice exhibited reduced differentiation, as revealed by retarded expression of myelin basic protein (MBP) in culture. This could be rescued with purified Tnr. In contrast, when we cultured oligodendrocytes on a Tnc-containing, astrocyte-derived ECM, they barely expressed MBP. This inhibition could be overcome when we used ECM from astrocytes deficient for Tnc, suggesting that Tnc inhibits differentiation. In contrast to their antagonistic effect on differentiation, both Tnc and Tnr similarly inhibited morphologic maturation. When oligodendrocytes were cultured on the purified glycoproteins, process elaboration and membrane expansion were reduced. Both Tnc and Tnr interfered with the activation of the small GTPase RhoA. Conversely, RhoA and Rac1 activation induced by cytotoxic necrotizing factor 1 (CNF1) increased the formation of myelin membranes, whereas Y27632-mediated inhibition of the Rho-cascade prevented it without, however, affecting the fraction of MBP-expressing cells. Because Tnc and Tnr play antagonistic roles for differentiation and comparably inhibit morphologic maturation, we conclude that independent molecular pathways regulate these processes.

Novel conserved oligodendrocyte surface epitope identified by monoclonal antibody 4860.

Oligodendrocytes are the myelinating cells of the central nervous system. They differentiate from oligodendrocyte precursor cells through several intermediate states that can be followed by characteristic morphological changes and the expression of marker molecules. However, most oligodendrocyte lineage markers demarcate either the precursor or the differentiated oligodendrocyte in restricted subcellular compartments. Here, we describe a novel marker of the oligodendrocyte lineage recognised by the monoclonal antibody clone 4860. It selectively labels the surfaces of differentiated oligodendrocytes in culture and clearly differs from other oligodendrocyte markers. Importantly, the 4860 epitope highlights developing white matter tracts in rodent and avian brains and thus represents a useful and conserved feature. The 4860 epitope is not associated with protein backbones as revealed by the related 487/L5 antibody. Furthermore, the epitope disappears upon lipid extraction from cryosections or inhibition of sphingolipid synthesis in cultured oligodendrocytes. Thus, we conclude that mAb 4860 represents a novel lipid-based oligodendrocyte marker.

Visualization and Time-Lapse Microscopy of Myelinating Glia In Vivo in Zebrafish.

In vivo time-lapse microscopy provides important information about the kinetics of cellular events and their control by interactions with neighboring cells. Here, we describe the generation and use of transgenic zebrafish to visualize dynamics of myelinating glia using cell type-specific expression and microscopy of genetically encoded fluorescent proteins. With this method, we are able to simultaneously separate and trace up to three different colors over time.