Motor Exit Point (MEP) Glia: Novel Myelinating Glia That Bridge CNS and PNS Myelin

Oligodendrocytes (OLs) and Schwann cells (SCs) have traditionally been thought of as the exclusive myelinating glial cells of the central and peripheral nervous systems (CNS and PNS), respectively, for a little over a century. However, recent studies demonstrate the existence of a novel, centrally-derived peripheral glial population called motor exit point (MEP) glia, which myelinate spinal motor root axons in the periphery. Until recently, the boundaries that exist between the CNS and PNS, and the cells permitted to cross them, were mostly described based on fixed histological collections and static lineage tracing. Recent work in zebrafish using in vivo, time-lapse imaging has shed light on glial cell interactions at the MEP transition zone and reveals a more complex picture of myelination both centrally and peripherally

The Neuromodulator Adenosine Regulates Oligodendrocyte Migration at Motor Exit Point Transition Zones

During development, oligodendrocyte progenitor cells (OPCs) migrate extensively throughout the spinal cord. However, their migration is restricted at transition zones (TZs). At these specialized locations, unique glial cells in both zebrafish and mice play a role in preventing peripheral OPC migration, but the mechanisms of this regulation are not understood. To elucidate the mechanisms that mediate OPC segregation at motor exit point (MEP) TZs, we performed an unbiased small-molecule screen. Using chemical screening and in vivo imaging, we discovered that inhibition of A2a adenosine receptors (ARs) causes ectopic OPC migration out of the spinal cord. We provide in vivo evidence that neuromodulation, partially mediated by adenosine, influences OPC migration specifically at the MEP TZ. This work opens exciting possibilities for understanding how OPCs reach their final destinations during development and identifies mechanisms that could promote their migration in disease

The Neuromodulator Adenosine Regulates Oligodendrocyte Migration at Motor Exit Point Transition Zones

During development, oligodendrocyte progenitor cells (OPCs) migrate extensively throughout the spinal cord. However, their migration is restricted at transition zones (TZs). At these specialized locations, unique glial cells in both zebrafish and mice play a role in preventing peripheral OPC migration, but the mechanisms of this regulation are not understood. To elucidate the mechanisms that mediate OPC segregation at motor exit point (MEP) TZs, we performed an unbiased small-molecule screen. Using chemical screening and in vivo imaging, we discovered that inhibition of A2a adenosine receptors (ARs) causes ectopic OPC migration out of the spinal cord. We provide in vivo evidence that neuromodulation, partially mediated by adenosine, influences OPC migration specifically at the MEP TZ. This work opens exciting possibilities for understanding how OPCs reach their final destinations during development and identifies mechanisms that could promote their migration in disease

Functional analysis of two genes, ndrg4 and elmo1, in the peripheral nervous system development of zebrafish

Les cellules gliales qui forment les segments de myéline du système nerveux périphérique (SNP) sont appelées cellules de Schwann. Elles assurent aux nerfs un soutien fonctionnel et permettent une conduction rapide et efficace de l'influx nerveux. Cette fonction requiert une communication réciproque entre les neurones et les cellules gliales qui les entourent. Une perturbation de cette interaction entraine le plus souvent une situation pathologique comme les neuropathies périphériques dont la plus connue est la maladie de Charcot-Marie-Tooth. Cependant, les mécanismes qui conduisent à ces pathologies sont encore peu connus et leur compréhension demande au préalable l'élucidation des mécanismes physiologiques qui contrôlent le développement du SNP. Ce travail a consisté en l'analyse de nouvelles fonctions des gènes ndrg4 et elmo1, dans le développement du système nerveux périphérique, chez le poisson zèbre. Nous avons montré que ndrg4 (n-myc downstream regulated gene) est un facteur neuronal qui régule le développement de la myéline périphérique en contrôlant le regroupement des canaux sodiques aux nœuds de Ranvier et l'expression de la mbp. Ndrg4 semble moduler l'échange vésiculaire entre les axones et les cellules de Schwann, en contrôlant l'expression de certaines protéines de relargage vésiculaire comme SNAP25, membre du complexe SNARE.Ce travail décrit par ailleurs une nouvelle fonction de elmo1 (engulfment and cell motility 1) dans le développement du SNP du poisson zèbre, où il favorise la survie des neurones dans lesquels il est exprimé. Nous avons montré qu'elmo1 protège les neurones de l'apoptose et que cette fonction est contrôlée par la voie nétrine1/unc5b en amont de Rac1. De ce fait, elmo1 est requis pour le développement du ganglion de la ligne latérale postérieure et des axones qui en émergent pour donner un système nerveux fonctionnel. Ainsi, ces travaux contribuent à une meilleure connaissance du développement du SNP et élucident pour la première fois une nouvelle voie de signalisation requise pour la survie des neurones dans le SNP.The glial cells that form myelin segments in the peripheral nervous system (PNS) are called Schwann cells (SCs). They provide functional support for nerves and allow a fast and efficient conduction of the action potentials. This requires a bilateral communication between axons and the associated glial cells. Disruption of this interaction often leads to peripheral neuropathies e.g. Charcot-Marie-Tooth disease. However, the mechanisms underlying these diseases remain poorly known and their understanding requires, at first, the elucidation of the physiological mechanisms responsible for PNS development. This work consists of a functional analysis of two genes, ndrg4 and elmo1, in the PNS development, using the zebrafish model. We showed that the neuronal factor ndrg4 (n-myc downstream regulated gene 4) regulates nodes of Ranvier organization and mbp expression along the Posterior Lateral Line nerve (PLLn). This is achieved, most likely, by the ability of ndrg4 to modulate vesicular exchange between axons and SCs. Indeed, the expression of some key proteins involved in vesicle docking and release such as SNAP25, a member of the SNARE complex, are significantly dependent on ndrg4.Moreover, this work describes a novel role for neuronal elmo1 (engulfment and cell motility 1) in PNS development by promoting neuronal survival within the PLL ganglion. We showed that elmo1 has protective role against apoptosis and that its function is controlled by the netrin1/unc5b signalling upstream of Rac1 and independently of macrophages role in apoptotic clearance. Therefore, elmo1 is required for the proper development of the PLL ganglion and the axonal development of the PLLn. Thus, this study further contributes to our understanding of PNS development and unravels a novel molecular pathway required for neuronal survival in the PNS

Neuronal Ndrg4 Is Essential for Nodes of Ranvier Organization in Zebrafish

International audienceAxon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery

ndrg4 is required for sodium channel and neurofascin clustering in the peripheral nervous system.

<p>(A-C) Lateral view of <i>mbp</i> RNA expression in control (A), ndrg4 mutant (B) and morphant (C) embryos at 4 dpf. Arrows indicate <i>mbp</i>-expressing cells along the PLLn. Scale bars = 200μm. (D-L) Acetylated tubulin (ac tub; red) and sodium channels (NaCh; green) immunohistochemistry of a (D-F) control, ndrg4 mutant (G-I) and ndrg4 morphant (J-L) PLLn at 4 dpf. Scale bars = 5μm. (M) High magnification of three nodes of Ranvier (arrowheads) from a control nerve. Scale bar = 100nm. (N) A significant decrease in the number of sodium channels clusters is observed in ndrg4 mutants and morphants in comparison to controls (p<0.001). Acetylated tubulin (ac tub; red) and FIGQY (green) immunohistochemistry of a (D’-F’) control, ndrg4 mutant (G’-I’) and ndrg4 morphant (J’-L’) PLLn at 4dpf. Scale bars = 5 μm. (O,P) A significant decrease in the number of FIGQY labeled clusters is observed in ndrg4 mutants and morphants in comparison to controls (p<0.001).</p

ndrg4 is not required for axonal outgrowth or early Schwann cell development.

<p>Acetylated tubulin expression in control (A), ndrg4 mutant (C) and morphant (B) embryos at 4 dpf showing the PLLn nerve. Scale bar = 45μm. (d-F) Whole mount <i>in situ</i> hybridization of a (d) control embryo, ndrg4 mutant (F) and ndrg4 morphant (E) showing <i>sox10</i> expression in PLLn SCs (arrows) at 3 dpf. Scale bar = 200μm. Lateral view of a control foxd3::GFP embryo (G), a ndrg4 morphant (H) at 3 dpf showing SCs (arrows) along the PLLn. Transmission electron micrographs showing cross-section through (I) control and ndrg4 mutant (J). Control PLLn shows an average of 10.7 myelinated axons (blue asterisks). (J) An average of 5.36 myelinated axons (blue asterisks) is observed in the ndrg4 mutant’s PLLn. (S: Schwann cell). Scale bars = 0.5μm. (K,L) Quantification of the total number of axons and the number myelinated axons in controls, ndrg4 mutants and ndrg4 morphants. NS: Non Significant.</p

Chimeric embryos show evidence of ndrg4 requirement in neurons for sodium channel clustering.

<p>(A) ndrg4MO mCherry labeled PLL neurons shown by arrows. (b,e) ndrg4MO mCherry labeled axons of the PLLn in two different chimeric embryos; (c,f) sodium channels along the PLLn of ndrg4MO mCherry labeled (arrow) and of control (arrowheads) axons; (d,g) merge of the two labelings. Sodium channel clustering is absent in ndrg4MO axons (mCherry labeled) while control ones in the same PLLn show normal clustering. (H) Control mCherry labeled neurons indicated by arrows. The dashed line indicates the margin of the PLLg. (I) Control mCherry labeled axons (arrows). (J) Sodium channel clusters along the PLLn in control labeled (arrows) and other non-labeled (arrowheads) axons. (K) Merge of the two labelings. For (a, H) scale bars = 5μm. For (b-G; I-K) scale bars = 10μm. (L-N) WT mCherry labeled axons in ndrg4 morphant embryos are shown in (L, arrow) and the corresponding sodium channels in (M, arrows). Note the clustering of the nodes in the WT labeled axons while the other ndrg4 deficient axons show no sign of sodium channel clustering. (N) Merge of the two labelings. Scale bar = 7 μm. (O-Q) WT mcherry labeled axons in ndrg4-/- are shown in (O) and the corresponding sodium channels in (P, arrows). (Q) Merge of the two labelings showing clustered sodium channels along the WT axons (arrows). Scale bar = 5 μm.</p

Characterization of the ndrg4 mutant.

<p>(A) Schematic representation of the <i>ndrg4</i> genomic locus. The extended region on the <i>exon 2</i> represents the sequence targeted by the CRISPR/Cas9 system. Red: sgRNA binding site. Blue: PAM sequence. <i>ndrg4</i>^<i>+</i> corresponds to the wild-type allele; <i>ndrg4</i> *³¹ and <i>ndrg4</i> *³⁴ are the loss-of-function alleles used in this study. (B) Schematic of ndrg4 protein product. In <i>ndrg4</i> *³¹ and <i>ndrg4</i> *³⁴ mutant fish, the deletions result in a frameshift generating a premature STOP codon at the level of the amino acids 31 and 34 (of 352) in the ndr family domain. Lateral views of a control (C) and a ndrg4 mutant (D) embryos at 72 hpf. The arrows point to the heart, note the pronounced heart edema (white asterisk) observed in the ndrg4 mutant. Lateral view of <i>ndrg4</i> mRNA expression in a control (E) and a ndrg4-/- embryo (F) at 48hpf. Note the absence of <i>ndrg4</i> expression in the mutant. Scale bar = 200 μm.</p