Stages Of Oligodendrocyte Differentiation Research Articles

OLIG2 regulates lncRNAs and its own expression during oligodendrocyte lineage formation

BackgroundOligodendrocytes, responsible for axon ensheathment, are critical for central nervous system (CNS) development, function, and diseases. OLIG2 is an important transcription factor (TF) that acts during oligodendrocyte development and performs distinct functions at different stages. Previous studies have shown that lncRNAs (long non-coding RNAs; > 200 bp) have important functions during oligodendrocyte development, but their roles have not been systematically characterized and their regulation is not yet clear.ResultsWe performed an integrated study of genome-wide OLIG2 binding and the epigenetic modification status of both coding and non-coding genes during three stages of oligodendrocyte differentiation in vivo: neural stem cells (NSCs), oligodendrocyte progenitor cells (OPCs), and newly formed oligodendrocytes (NFOs). We found that 613 lncRNAs have OLIG2 binding sites and are expressed in at least one cell type, which can potentially be activated or repressed by OLIG2. Forty-eight of them have increased expression in oligodendrocyte lineage cells. Predicting lncRNA functions by using a “guilt-by-association” approach revealed that the functions of these 48 lncRNAs were enriched in “oligodendrocyte development and differentiation.” Additionally, bivalent genes are known to play essential roles during embryonic stem cell differentiation. We identified bivalent genes in NSCs, OPCs, and NFOs and found that some bivalent genes bound by OLIG2 are dynamically regulated during oligodendrocyte development. Importantly, we unveiled a previously unknown mechanism that, in addition to transcriptional regulation via DNA binding, OLIG2 could self-regulate through the 3′ UTR of its own mRNA.ConclusionsOur studies have revealed the missing links in the mechanisms regulating oligodendrocyte development at the transcriptional level and after transcription. The results of our research have improved the understanding of fundamental cell fate decisions during oligodendrocyte lineage formation, which can enable insights into demyelination diseases and regenerative medicine.

Could the inhibitor of DNA binding 2 and 4 play a role in white matter injury?

White matter injury (WMI) prevents the normal development of myelination, leading to central nervous system myelination disorders and the production of chronic sequelae associated with WMI, such as chronic dyskinesia, cognitive impairment and cerebral palsy. This results in a large emotional and socioeconomic burden. Decreased myelination in preterm infant WMI is associated with the delayed development or destruction of oligodendrocyte (OL) lineage cells, particularly oligodendrocyte precursor cells (OPCs). The development of cells from the OL lineage involves the migration, proliferation and different stages of OL differentiation, finally leading to myelination. A series of complex intrinsic, extrinsic and epigenetic factors regulate the OPC cell cycle withdrawal, OL lineage progression and myelination. We focus on the inhibitor of DNA binding 2 (ID2), because it is widely involved in the different stages of OL differentiation and genesis. ID2 is a key transcription factor for the normal development of OL lineage cells, and the pathogenesis of WMI is closely linked with OL developmental disorders. ID4, another family member of the IDs protein, also plays a similar role in OL differentiation and genesis. ID2 and ID4 belong to the helix-loop-helix family; they lack the DNA-binding sequences and inhibit oligodendrogenesis and OPC differentiation. In this review, we mainly discuss the roles of ID2 in OL development, especially during OPC differentiation, and summarize the ID2-mediated intracellular and extracellular signaling pathways that regulate these processes. We also discuss ID4 in relation to bone morphogenetic protein signaling and oligodendrogenesis. It is likely that these developmental mechanisms are also involved in the myelin repair or remyelination in human neurological diseases.

Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes.

Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin.

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75NTR antagonists

LINGO-1 is a potent negative regulator of oligodendrocyte differentiation and hence may play a pivotal restrictive role during remyelination in demyelinating diseases such as multiple sclerosis. However, little is known as to which stages of oligodendrocyte differentiation are inhibited by LINGO-1, which domains of the protein are involved and whether accessory proteins are required. Here, we show that LINGO-1 expression in the human oligodendroglial cell line MO3.13 inhibited process extension and this was reversed by an anti-LINGO-1 antibody or the antagonist LINGO-1-Fc. LINGO-1 expression was also found to inhibit myelin basic protein transcription in the rat oligodendroglial cell line CG4. Both of these inhibitory actions of LINGO-1 were abrogated by deletion of the entire ectodomain or cytoplasmic domains but, surprisingly, were unaffected by deletion of the leucine-rich repeats (LRRs). As in neurons, LINGO-1 physically associated with endogenous p75NTR in MO3.13 cells and, correspondingly, its inhibition of process extension was reversed by antagonists of p75NTR. Thus, LINGO-1 inhibits multiple aspects of oligodendrocyte differentiation independently of the LRRs via a process that requires p75NTR signalling.

MicroRNA Expression Profiling of Oligodendrocyte Differentiation from Human Embryonic Stem Cells

BackgroundCells of the oligodendrocyte (OL) lineage play a vital role in the production and maintenance of myelin, a multilamellar membrane which allows for saltatory conduction along axons. These cells may provide immense therapeutic potential for lost sensory and motor function in demyelinating conditions, such as spinal cord injury, multiple sclerosis, and transverse myelitis. However, the molecular mechanisms controlling OL differentiation are largely unknown. MicroRNAs (miRNAs) are considered the “micromanagers” of gene expression with suggestive roles in cellular differentiation and maintenance. Although unique patterns of miRNA expression in various cell lineages have been characterized, this is the first report documenting their expression during oligodendrocyte maturation from human embryonic stem (hES) cells. Here, we performed a global miRNA analysis to reveal and identify characteristic patterns in the multiple stages leading to OL maturation from hES cells including those targeting factors involved in myelin production.Methodology/Principal FindingsWe isolated cells from 8 stages of OL differentiation. Total RNA was subjected to miRNA profiling and validations preformed using real-time qRT-PCR. A comparison of miRNAs from our cultured OLs and OL progenitors showed significant similarities with published results from equivalent cells found in the rat and mouse central nervous system. Principal component analysis revealed four main clusters of miRNA expression corresponding to early, mid, and late progenitors, and mature OLs. These results were supported by correlation analyses between adjacent stages. Interestingly, the highest differentially-expressed miRNAs demonstrated a similar pattern of expression throughout all stages of differentiation, suggesting that they potentially regulate a common target or set of targets in this process. The predicted targets of these miRNAs include those with known or suspected roles in oligodendrocyte development and myelination including C11Orf9, CLDN11, MYTL1, MBOP, MPZL2, and DDR1.Conclusions/SignificanceWe demonstrate miRNA profiles during distinct stages in oligodendroglial differentiation that may provide key markers of OL maturation. Our results reveal pronounced trends in miRNA expression and their potential mRNA target interactions that could provide valuable insight into the molecular mechanisms of differentiation.

Olig2 Overexpression Induces the In Vitro Differentiation of Neural Stem Cells into Mature Oligodendrocytes

Differentiation induction of neural stem cells (NSCs) into oligodendrocytes during embryogenesis is the result of a complex interaction between local induction factors and intracellular transcription factors. At the early stage of differentiation, in particular, the helix-loop-helix transcription factors Olig1 and Olig2 have been shown to be essential for oligodendrocyte lineage determination. In view of the possible application of NSCs as a source for remyelinating cell transplants in demyelinating diseases (e.g., multiple sclerosis), in vitro procedures need to be developed to drive the oligodendrocyte differentiation process. Mere culture in medium supplemented with major embryonic oligodendrogenic induction factors, such as Sonic hedgehog, results in oligodendrocyte differentiation of only about 10% of NSCs. We previously showed that induction of Olig1 expression by gene transfection could indeed initiate the first stage of oligodendrocyte differentiation in NSCs, but appeared to be unable to generate fully mature, functional oligodendrocytes. In this study, we transfected NSCs isolated from the embryonic mouse brain with the Olig2 gene and found that the introduced overexpression of Olig2 could induce the development of fully mature oligodendrocytes expressing the transcription factor Nkx2.2 and all major myelin-specific proteins. Moreover, Olig2-transfected NSCs, in contrast to nontransfected NSCs, developed into actively remyelinating oligodendrocytes after transplantation into the corpus callo-sum of long-term cuprizonefed mice, an animal model for demyelination. Our results show that transfection of genes encoding for oligodendrogenic transcription factors can be an efficient way to induce the differentiation of NSCs into functional oligodendrocytes.

Proteolipid Promoter Activity Distinguishes Two Populations of NG2-Positive Cells throughout Neonatal Cortical Development

Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ. EGFP(+)/NG2(+) cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.

Expression of molecular chaperones and vesicle transport proteins in differentiating oligodendrocytes.

The major stages of oligodendrocyte differentiation are defined by the presence or absence of certain myelin-specific proteins. Events leading to the successful processing of these proteins, such as the folding, assembly, and trafficking of these proteins through the biosynthetic pathway, are largely undefined. In the present study, we have examined both cultured primary oligodendrocytes and immortalized oligodendrocyte cell lines for the presence of molecular chaperones and/or vesicle transport proteins. We find that a select set of these proteins are expressed relatively early in oligodendrocyte differentiation, whereas a characteristically different set of proteins are expressed at later stages of oligodendrocyte differentiation. In other systems, these proteins participate in the folding and assembly of protein complexes, in the prevention of protein aggregation, as well as the trafficking of proteins via vesicles to specific subcellular destinations including the plasma membrane. Some of the chaperones and/or vesicle transport proteins investigated in this study may play a pivotal role in the certain aspect of myelin biogenesis.

PDGF-α Receptor and Myelin Basic Protein mRNAs Are Not Coexpressed by Oligodendrocytesin Vivo:A Doublein SituHybridization Study in the Anterior Medullary Velum of the Neonatal Rat

Platelet-derived growth factor (PDGF) is a growth-regulatory dimer with A and B subunits. PDGF-AA, acting via PDGF receptors of the α-unit subtype (PDGF-αR), is implicated in the differentiation of oligodendrocyte precursors and in the survival of newly formed oligodendrocytes, which gradually lose expression of PDGF-αR. However, it is unclear whether terminally differentiated oligodendrocytes express PDGF-αRin vivo.To address this question, and to help clarify the role of PDGF-AA in late oligodendrocyte differentiation, we have used doublein situhybridization with digoxigenin- and fluorescein-labeled riboprobes to relate PDGF-αR mRNA and myelin basic protein (MBP) mRNA expression in the isolated intact anterior medullary velum (AMV) of rats ages Postnatal Day (P) 10–12 and P30–32. In parallel experiments, AMV were immunolabeled with the oligodendrocyte-specific monoclonal antibody Rip to provide information on oligodendrocyte development and the extent of myelination. At P10, the AMV contained tracts in which axons ranged from unmyelinated to fully myelinated, whereas myelination was complete in P30–32 AMV. The first oligodendrocytes to express MBP mRNA or Rip were promyelinating oligodendrocytes, which had a “star-burst” morphology and had not yet begun to form myelin sheaths. As myelination proceeded, MBP mRNA became dispersed throughout oligodendrocyte units, comprising cell somata, processes, and internodal myelin sheaths. By P30–32, MBP mRNA had been redistributed to the myelin sheaths only, reflecting a change in the site of protein synthesis in mature myelinated axon tracts. At no stage of oligodendrocyte differentiation did we observe cellular coexpression of mRNA for PDGFαR and MBP. Our results indicated that oligodendrocytes lost the expression of PDGFαR prior to gaining that of myelin gene products, and preclude an action of PDGF-AA on Rip+/MBP+star-burst promyelinating oligodendrocytes. The spatial and temporal expression of PDGF-αR mRNA in the AMV was inversely related to the pattern of maturation of both myelin and oligodendrocytes, and is consistent with PDGF-αR being expressed by pro-oligodendrocytes. A notable finding was the high level of expression of PDGF-αR mRNA in the AMV of juvenile rats, localized to cell bodies within the myelinated axon tracts, strongly suggesting that oligodendrocyte precursors persisted in the mature velum.

Oligodendrocyte progenitors in the embryonic spinal cord express DM‐20

Oligodendrocyte progenitors, originating in the ventral ventricular zone of the embryonic rodent spinal cord, migrate and differentiate into the oligodendrocytes myelinating the future white matter. Transcripts for the dm-20 isoform of the proteolipid protein (plp) gene are detectable initially in cells of the ventral ventricular region of the embryonic central canal and subsequently throughout the white matter. The dm-20+ cells are present several days before oligodendrocytes or myelin sheaths are detectable. The purpose of the present study was to determine if DM-20 protein is present and whether DM-20+ cells can be linked to the oligodendrocyte lineage in the mouse spinal cord. Expression of plp and dm-20 transcripts and product was monitored using reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization and immunostaining of cryosections and associated cultures. Cell identification was performed using antigenic markers characterizing different stages of oligodendrocyte differentiation. We show a temporal and spatial progression of cells expressing dm-20 transcripts and product from the ventral ventricular zone at embryonic day 13 (E13.0), via the lateral borders of the floor plate to the ventral pia and white matter. The cells, initially devoid of myelin basic protein (MBP) and PLP, co-express these myelin proteins at approximately E16.5/17.0. Some DM-20+ cells co-label with definitive markers of the early oligodendrocyte lineage, are capable of mitosis and subsequently differentiate into oligodendrocytes. Other DM-20+ cells may represent earlier precursor cells. The expression of DM-20 in oligodendrocyte progenitors is consistent with a postulated role in glial cell development.

Distinctive ganglioside patterns revealed by anti-ganglioside antibody binding to differentiating CG-4 oligodendrocytes.

Oligodendrocytes are central nervous system glial cells responsible for myelination of neuronal axons. During brain development oligodendrocyte progenitor cells progress through a series of morphologically and immunohistochemically distinct differentiation steps leading to mature myelin-producing oligodendrocytes. Much of this same differentiation sequence is expressed in vitro by primary oligodendrocyte progenitor cells, and by the clonal progenitor cell line CG-4. We report the use of highly specific monoclonal antibodies against GM1, GD1a, GD1b, GT1b, and GQ1b to determine major brain ganglioside expression and morphological distribution during CG-4 differentiation in vitro. Prominent anti-GD1b antibody stained defined a highly arborized intermediate stage of oligodendrocyte differentiation. In contrast, anti-GT1b antibody bound to discrete patches on the cell bodies of early progenitor cells and more mature oligodendrocytes, and to sites of progenitor arborization. The other anti-ganglioside antibodies tested did not bind above background levels. Cells with anti-GD1b antibody binding and morphology similar to those in differentiating CG-4 cells were detected in rat brain primary cell cultures enriched in oligodendrocyte precursors. The remarkably distinctive ganglioside immunoreactivity on differentiating oligodendrocytes suggests the possibility of a functional role for their surface expression.