Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.     

Emergence of three myelin proteins in oligodendrocytes cultured without neurons

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.     

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.     

The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.     

Myelin under construction -- teamwork required

Myelinating glial cells synthesize specialized myelin proteins and deposit them in the growing myelin sheath that enwraps axons multiple times. How do axons and myelinating glial cells coordinate this spectacular cell–cell interaction? In this issue, Trajkovic et al. (p. 937) show that neuronal signaling regulates cell surface expression of the myelin proteolipid protein in cultured oligodendrocytes in unexpected ways that may also contribute to myelination in situ.Myelination is a stunning example of how multiple cells cooperate to build a complex structure. Understanding how myelinating glia and neurons work together to achieve this feat is thus a challenging and important problem. Trajkovic et al. (p. 937) investigate the regulation of the trafficking of a major myelin protein, proteolipid protein (PLP), to the plasma membrane (PM) of cultured oligodendrocytes (OLs). When initially expressed in cultured OLs, PLP resides in a compartment with characteristics of a late endosome/lysosome (LE/L). Co-culture with neurons leads to an increase of PLP on the PM and a disappearance from the LE/L. This increased surface expression of PLP is due to at least two distinct mechanisms: a decrease in PLP endocytosis from the PM and an increase in exocytosis from the LE/L. The relative contributions of these two mechanisms (and possibly additional ones?) remain open questions for the future.The cells that produce myelin are highly specialized glial cells, Schwann cells in the peripheral nervous system (PNS) and OLs in the central nervous system (CNS). Myelin consists of many wrappings of glial cell membrane around the axon with little or no cytoplasm left between adjacent wraps. This compact myelin region insulates the axon from the extracellular medium and allows saltatory conduction along axons. Each successive myelin wrap creates at its lateral margins a membrane loop containing some cytoplasm. These so-called paranodal loops make up part of the noncompact myelin. Each paranodal loop forms a specialized cell junction with the axon, the axoglial apparatus. The paranodal loops, in turn, flank Nodes of Ranvier, gaps in the myelin where voltage-gated sodium channels cluster and regenerate the action potential (for review see Sherman and Brophy, 2005).Myelination is a supreme example of differential protein distribution. During myelination, glia elaborate distinct domains (such as soma and compact and noncompact myelin) with distinct lipids and protein components. At the same time, axonal membrane proteins also accumulate in distinct regions, such that the Node of Ranvier contains different proteins than the paranodal region (underlying the paranodal loops) or the juxtaparanode (flanking the paranode). Much work on who signaled whom, when, and why, revealed that neurons and myelinating glia communicate with each other bidirectionally in multiple ways to orchestrate myelination (Sherman and Brophy 2005). For instance, glial cells signal to neurons to influence axonal diameter, neurofilament spacing, and phosphorylation (Hsieh et al., 1994). Additionally, nodal, paranodal, and juxtaparanodal domains on axons form as a result of interactions with glial cells. Mutations in genes encoding paranodal proteins lead to aberrant paranodal loops and mislocalization of paranodal and juxtaparanodal components in the axon (for review see Poliak and Peles, 2003; Salzer, 2003). Somewhat surprisingly, nodal proteins still cluster in these mice, leading to the suggestion that nodal assembly might be intrinsic to axons or (in the CNS) driven by diffusible glial-derived factors (Kaplan et al., 1997). New work argues that glial cell processes which contact the node itself could direct nodal assembly. In the PNS, the node is contacted directly by microvilli of the myelinating Schwann cell. Mice lacking Schwann cell dystroglycan or laminin have aberrant microvilli and poorly clustered voltage-gated sodium channels (Saito et al., 2003; Occhi et al., 2005). Gliomedin, identified by the Peles lab, is expressed in Schwann cell microvilli and required for clustering of nodal axonal components (Eshed et al., 2005). In the CNS, Colman''s group localized the outgrowth-inhibitory molecule Omgp to distinct glial cells that can encircle nodes (Huang et al., 2005). Omgp knock-out mice show wider and disorganized nodes as well as aberrant sprouting of branches from nodes. These findings highlight the importance of node-encircling glial cells for organizing the axon.Do neurons in turn give instructions to glial cells? Oligodendrocyte precursor cells (OPCs) in the CNS migrate into developing white matter where they differentiate into postmitotic OLs and produce the myelin sheath. The differentiation of OPCs in terms of changes in gene expression and in morphology has been studied extensively in vitro and in vivo (for reviews see Pfeiffer et al., 1993; Barres and Raff, 1999). Because OPCs differentiate normally in axon-free culture and express myelin components, a role for neurons was not immediately apparent. In vivo, on the other hand, few OLs develop after transection of the optic nerve and subsequently, axons were shown to be required for survival and differentiation of OLs (Barres and Raff, 1999). OPCs and newly born OLs require astrocyte-derived factors such as PDGF, but OLs become dependent on axonal signals later. Axonal signaling to OLs occurs on at least two levels (Barres and Raff, 1999; Coman et al., 2005). Electrical activity (mediated by extrasynaptic release of adenosine [Stevens et al., 2002]) is required for proliferation of OPCs. Additionally, contact-mediated neuronal signals play important roles in OPC and Schwann cell differentiation and myelination (Corfas et al., 2004). Salzer and colleagues recently showed that the levels of neuregulin 1 type III expressed on axons determine the ensheathment fate of axons in the PNS (Taveggia et al., 2005).Compact myelin has a very specific composition of 70% lipids by dry weight (mostly composed of galactoceramide and cerebroside) with 80% of the protein mass comprised of only two proteins, myelin basic protein MBP and proteolipid protein PLP/DM20 (for review see Kramer et al., 2001). Studies have therefore focused on how OLs synthesize MBP and PLP and incorporate them into the growing myelin sheath. MBP is synthesized on free ribosomes, but its mRNA is localized to the myelin sheath (Colman et al., 1982). PLP on the other hand is a membrane-spanning protein that traverses the ER and Golgi. The role for axonal signaling for production of the myelin sheath is not well understood. For instance, OPCs in culture undergo differentiation and start to synthesize myelin components in the absence of neurons (Pfeiffer et al., 1993). Early reports from cultured rat OLs concluded that PLP was synthesized and incorporated into the PM without neurons (Hudson et al., 1989). Interestingly though, PM expression of PLP could not be detected for many days after intracellular pools of PLP were clearly detectable. The delayed PM expression of PLP raised the possibility that axonal signaling could speed up PM expression.The paper by Simons and colleagues in this issue demonstrates neuronal control of PLP trafficking (Trajkovic et al., 2006). Primary OLs, as well as two OL cell lines, contain PLP in a LE/L (as well as on the PM). This LE/L pool of PLP persists if neurons are absent from the culture. When OLs are cocultured with neurons, PLP is found with LE/L initially, but later disappears from there and increased amounts can be detected on the PM. When brain sections were costained against lysosomal markers and PLP, high colocalization of PLP with LE/L was detected in P7 mice while in P60 brains PLP did not colocalize with LE/L. Therefore, PLP localizes (at least partially) with LE/L in vivo and disappears from there upon myelination. This finding assuages much of the fear that PLP-containing LE/L are just a culture phenomenon or due to inappropriate expression levels (Kramer et al., 2001; Simons et al., 2002). The authors tested three explanations to account for their observations: increased proteolysis of PLP, decreased endocytosis, and/or increased exocytosis from LE/L. Proteolysis of PLP was found to be unaffected by neuronal coculture. Endocytosis (via a clathrin-independent, cholesterol-dependent, actin-dependent, and RhoA-dependent pathway), on the other hand, was decreased. Using PLP-GFP and lysotracker to mark LE/L in live OLs, the authors also found that the LE/L became much more mobile in the presence of neurons. To determine whether the moving LE/L in cocultured OLs can fuse with the PM and potentially deliver PLP sequestered in LE/L, the authors used total internal reflection fluorescence microscopy (TIRFM) on lysotracker-labeled OLs. Without neurons present, the LE/L was not found within 100 nm of the PM and was therefore invisible to TIRFM. When neurons were present, many LE/L were found near the PM and events suggestive of fusion could be observed at a rate of 1–2 events/min. Lastly, the authors determined that diffusible neuronal factors were sufficient to induce increased PLP surface expression. Addition of a membrane-permeable cAMP analogue to OLs in the absence of neurons led to increased PLP on the surface as well as high mobility of lysotracker pools containing PLP-GFP.These results suggest that diffusible neuronal factors (currently unknown) could activate cAMP signaling in OLs and regulate endocytosis and exocytosis of PLP. Exocytosis from LE/L is a regulated pathway in other cells as well (Blott and Griffiths, 2002). In OLs, at least some of the PLP could be stored in LE/L until neuronal promyelinating signals are received. Because many proteins arrive in the LE/L from the TGN, it would be interesting to investigate the potential neuronal regulation of PLP sorting events in the Golgi. Although we still await a complete quantitative account of what proportion of PLP is transported where and when, this paper presents an exciting advance in our understanding of the neuronal control of OL membrane traffic.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

Niemann-Pick type C (NPC) disease is a lysosomal storage disease in which endocytosed cholesterol becomes sequestered in late endosomes/lysosomes (LEs/Ls) because of mutations in either the NPC1 or NPC2 gene. Mutations in either of these genes can lead to impaired functions of the NPC1 or NPC2 proteins and progressive neurodegeneration as well as liver and lung disease. NPC1 is a polytopic protein of the LE/L limiting membrane, whereas NPC2 is a soluble protein in the LE/L lumen. These two proteins act in tandem and promote the export of cholesterol from LEs/Ls. Consequently, a defect in either NPC1 or NPC2 causes cholesterol accumulation in LEs/Ls. In this review, we summarize the molecular mechanisms leading to NPC disease, particularly in the CNS. Recent exciting data on the mechanism by which the cholesterol-sequestering agent cyclodextrin can bypass the functions of NPC1 and NPC2 in the LEs/Ls, and mobilize cholesterol from LEs/Ls, will be highlighted. Moreover, the possible use of cyclodextrin as a valuable therapeutic agent for treatment of NPC patients will be considered.     

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.     

Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles

Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.     

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta- galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

Embryonic Expression of the Soma-Restricted Products of the Myelin Proteolipid Gene in Motor Neurons and Muscle

In addition to classic proteolipid protein (PLP) and DM20, the mouse myelin proteolipid gene produces the sr-PLP and sr-DM20 proteins. The sr-isoforms are localized to the cell bodies of both oligodendrocytes and neurons. However, they are expressed to a greater extent in neurons than they are in glia. In this study, we examined expression of the sr-proteolipids in the mouse embryo using immunohistochemistry with an sr-PLP/DM20 specific antibody. Widespread expression of the sr-proteins was found in many nonmyelinating cell types. In particular, strong immunoreactivity was detected in motor neurons of both the autonomic and somatic nervous systems as well as in striated muscle. This pattern of expression persisted throughout the embryonic period studied. Thus, the sr-proteolipids are expressed prior to the onset of myelination and in a much broader array of cell types than their classic counterparts. These results support the conclusion that the sr-isoforms of the PLP gene have a biological role independent of myelination.     

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N-terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X-100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10-day-old rats.     

Peculiar and typical oligodendrocytes are involved in an uneven myelination pattern during the ontogeny of the lizard visual pathway

We studied the myelination of the visual pathway during the ontogeny of the lizard Gallotia galloti using immunohistochemical methods to stain the myelin basic protein (MBP) and proteolipid protein (PLP/DM20), and electron microscopy. The staining pattern for the PLP/DM20 and MBP overlapped during the lizard ontogeny and was first observed at E39 in cell bodies and fibers located in the temporal optic nerve, optic chiasm, middle optic tract, and in the stratum album centrale of the optic tectum (OT). The expression of these proteins extended to the nerve fiber layer (NFL) of the temporal retina and to the outer strata of the OT at E40. From hatching onwards, the labeling became stronger and extended to the entire visual pathway. Our ultrastructural data in postnatal and adult animals revealed the presence of both myelinated and unmyelinated retinal ganglion cell axons in all visual areas, with a tendency for the larger axons to show the thicker myelin sheaths. Moreover, two kinds of oligodendrocytes were described: peculiar oligodendrocytes displaying loose myelin sheaths were only observed in the NFL, whereas typical medium electron-dense oligodendrocytes displaying compact myelin sheaths were observed in the rest of the visual areas. The weakest expression of the PLP/DM20 in the NFL of the retina appears to be linked to the loose appearance of its myelin sheaths. We conclude that typical and peculiar oligodendrocytes are involved in an uneven myelination process, which follows a temporo-nasal and rostro-caudal gradient in the retina and ON, and a ventro-dorsal gradient in the OT.     

Expression of myelin genes in the developing chick retina

In submammalian animals including chicks, the retina contains oligodendrocytes (OLs), and axons in the optic fiber layer are wrapped with compact myelin within the retina; however, the expression of myelin genes in the chick retina has not been demonstrated yet. In the present study, we examined the expression of three myelin genes (proteolipid protein, PLP; myelin basic protein, MBP; cyclic nucleotide phosphodiesterase, CNP) and PLP in the developing chick retina, in comparison to the localization of Mueller cells. In situ hybridization demonstrated that all three myelin genes began to be expressed at E14 in the chick embryo retina. They are mostly restricted to the ganglion cell layer and the optic fiber layer, with a few exceptions in the inner nuclear layer where Mueller cells reside; however, PLP mRNA+ cells do not express glutamine synthetase, or vice versa. The present results elucidate that myelin genes are expressed only by OLs that are mostly localized in the innermost layer of the developing chick retina.