Oligodendrocytes are non-dividing cells, responsible for myelination in CNS. Mature oligodendrocytes form myelin sheath which provides critical insulation to facilitate axonal conduction by increasing the resistance and lowering the capacitance of the axonal membrane, resulting in faster conduction speed in myelinated axons than in unmyelinated axons of the same diameter ¹⁹. The number of oligodendrocytes in any region of the CNS broadly depends upon, the number of OPCs migrating toward that region, the proliferative potential of the resident OPCs prior to differentiation and number of cells lost. All these factors ultimately affect the smooth and effective myelination process²⁰.

Oligodendrocytes are mature glial cells present in CNS derived from their precursor cells, commonly called as Oligodendrocyte precursor cells (OPCs). During the formation of CNS, OPCs are derived from neural stem cells. Around embryonic day (E12.5) OPCs are derived from pMN domain located at ventral midline of caudal portion of neural tube ²¹. About 2 days later around E15, OPCs are also arises from dorsal spinal card. This is known as second wave of OPCs generation ^{22, 23} Third wave also developed after birth from cerebral cortex which give rise that most of the population of OPCs present in adult brain ^{24, 25}.

The development of Oligodendrocytes lineage cells from their precursors to mature form is a complex process during which the cells exhibit morphological and biochemical changes. These diversities help to identify OLs lineage cells in various stages beneficial to adjust different targets for the treatment of demyelinating diseases. The development and maturation of oligodendrocytes require a series of highly-coordinated events that organise the proliferation and differentiation of the OPCs as well as the spatio-temporal regulation of myelination. After differentiation from neural stem cells, first recognized stage of oligodendrocytes lineage cells is OPCs. In this stage cells exhibit various markers including, Nestin, PSA-NCAM, PDGFRα and NG2. Among them, platelet-derived growth factor receptor α (PDGFRα) transcript is unique for oligodendrocytes at very early stages of development ^{26, 27}. First signs of OPC maturation into oligodendrocytes and myelination can be observed around E17 and these processes continue after birth ^{28, 29}. As OPCs start process of maturation, they lose their proliferative and migratory capacity and their shape change to complex one ³⁰. Oligodendrocytes precursor cells when start differentiation, they express monoclonal antibody O4, and are termed pro-oligodendroblast ³¹. With further development OPCs are differentiated in immature oligodendrocytes. In this stage oligodendrocytes express galactocerebroside (GalC) and 2′,3′-cyclic nucleotide 3′-phosphodihydrolase (CNPase). From this pre-myelinating immature stage oligodendrocytes are differentiated in to mature myelin forming oligodendrocytes where they express Myelin Basic Protein (MBP), ProteoLipid Protein (PLP), Myelin-Associated Glycoprotein (MAG), and Myelin/Oligodendrocyte Glycoprotein, as well as other minor myelin proteins ³².

In the developmental myelination, OLs lineage cells are tightly regulated by various factors. These factors include growth factors, protein kinases, extracellular matrix, intracellular signaling cascade and soluble molecules. All these factors influence epigenetic modifications, transcriptional & translational regulation, and the actin cytoskeleton in oligodendrocytes ^{33, 34, 35, 36, 37}. Oligodendrocytes differentiation result from transcriptional changes in gene expression and is favoured by sonic hedgehog (Shh) (38, 39), thyroid hormone ^{38, 39, 40, 41}, IGF1^{42, 43} and retinoic acid ⁴⁴ and antagonized by mitogenic stimuli such as PDGF and FGF ^{40, 45, 46, 47}, Wnt ^{48, 49}, Notch ^{50, 51} and BMP4 ^{52, 53, 54, 55}. The progression from OPCs to mature oligodendrocytes occurs in stages and is characterized by complex cytoskeletal organization ²⁸ and progressive chromatin compaction ^{56, 57}. Various researchers have previously reported that changes of acetylation levels in nucleosomal histones, the basic unit of chromatin, are critical for oligodendrocyte differentiation in the developing brain and in repair after demyelination in adult mice ^{58, 59, 60, 61, 62}

Wingless and integration site (Wnt) signaling cascade are a group of  proteins, responsible for signals transmission from cell surface receptors to the nucleus where gene transcription carried out ⁶³. Wnt signaling is one of the fundamental mechanisms that direct cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis ⁶⁴. Therefore mutations in the Wnt signaling pathway are often related to congenital diseases, carcinomas and other diseases ⁶⁵.

Since last two decades, Wnt/β-catenin signaling has emerged an important pathway involved in many cellular processes. Wnt signaling pathway is of two types, canonical and non-canonical. Canonical Wnt signaling pathway plays essential roles in cell proliferation, migration, invasion and metastasis⁶⁶ while non-canonical Wnt pathway activates T cell related transcription to modulate cytoskeleton rearrangement, cell adhesion, migration and tissue separation⁶⁷. Non-canonical Pathway again has been divided into two types, the planar cell polarity pathway and Wnt/Ca²⁺ pathway. In the planar cell polarity pathway, frizzled activates JNK and directs asymmetric cytoskeletal organization and coordinated polarization of cells within the plane of epithelial sheets. The Wnt/Ca²⁺ pathway leads to release of intracellular Ca^2+, possibly via G-proteins. Elevated Ca²⁺ can activate the phosphatase calcineurin, which leads to dephosphorylation of the transcription factor NF-AT in the nucleus, where it causes the transcription of many genes. Role of canonical pathway in myelination and remyelination has been well investigated. Therefore in this review we mainly focused on canonical Wnt signaling pathway.

In mammals, canonical Wnt signaling pathway consist of four components, Wnt ligands/proteins family (extracellular), frizzled receptors (located at surface membrane) along with low density lipoprotein receptor related protein-5 & 6 (LRP-5/6), β-catenin (cytoplasmic) and LEF/TCF transcription factors (intranuclear) ⁶⁸. Wnt pathway is activated by binding of Wnt ligand to surface receptors, seven-transmembrane domain Frizzled receptors (Fzd) and single-transmembrane domain co-receptor Lrp5/6⁶⁹. This binding causes the degradation of “β-catenin destruction complex”, which consists of adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3 β (Gsk3- β), and casein kinase 1 (CK1). β-catenin destruction complex degradation results in accumulation of β-catenin in the cytoplasm and starts entering into the nucleus where it binds to TCF4 and activates downstream gene e.g CyclinD, c-myc, CD44⁷⁰. Finally this results in cell proliferation, differentiation, metastasis, chemo-resistance and other biological effects. When Wnt is not activated, cytoplasmic β-catenin is degraded by β-catenin destruction complex. This degradation results from phosphorylation of β-catenin at amino terminal region by CK1 and GSK3-β. After phosphorylation β-catenin is recognized by β-Trcp, an E3 ubiquitin ligase subunit, followed by β-catenin ubiquitination and proteasomal degradation ⁷¹. In the absence of β-catenin, Wnt target genes are repressed by the DNA-bound T cell factor/lymphoid enhancer factor (TCF/ LEF) family of proteins.

Wnt/β-catenin signaling regulates proliferation, fate specification and differentiation in different developmental stages and various adult tissues homeostasis. Therefore Wnt target genes show diversity ⁹⁹ and cell- and context-specificity ⁶⁴. Wnt signaling components including Fz, LRP6, Axin2, TCF/LEF, Dkk1, and Rspo, are often regulated positively or negatively by TCF/β-catenin ^{64, 84, 100, 101}. Wnt induction of Axin2, Dkk1, Naked and suppression of Fz and LRP6 constitute negative feedback loops that suppressed Wnt signaling ⁶⁴. Contrary, Wnt induction of Rspo and TCF/LEF genes constitute positive feedback and reinforce Wnt signaling ^{102, 103}. These various Wnt pathway self-regulatory loops are mostly utilized in a cell-specific manner, providing further complexity in the control of amplitude and duration of Wnt responses.

Wnt/β-catenin signaling pathway plays a critical role in development and homeostasis. Therefore, there is no surprise that its mutations would be associated with many hereditary disorders, demyelinating diseases, carcinomas and other diseases (Table 1). These mutations involve various Wnt ligands, agonists and antagonists, and affect on Wnt regulation of human development leading to various disease processes. For example, RSPO1 mutations result in XX sex reversal ¹⁰⁴, a condition having similar features to patients with WNT4 mutations ¹⁰⁵. FZ4 or LRP5 mutations are associated with familial exudative vitreo-retinopathy (FEVR) ¹⁰⁶, manifested by defective retinal vascularization ⁸³. LRP5 loss-of-function mutations has been identified in patients with osteoporosis pseudo-glioma syndrome (OPPG), a recessive disorder characterized by low bone mass and abnormal eye vasculature ¹⁰⁷, while LRP5 missense (‘gain-of-function’) mutations has been observed in patients with autosomal dominant high bone mass (HBM) diseases ^{108, 109}.

TCF7L2 has strong relationship with diabetes mellitus type II ¹¹⁰. Although the diabetes-associated TCF7L2 gene polymorphism does not alter protein coding, but its association with the disease has been confirmed in numerous populations ¹¹¹. Some studies have suggested that the predisposing TCF7L2 variant causes a decreased insulin secretion from pancreatic β-cells, but other pathogenic mechanisms involving additional tissues/organs or endocrine functions remain to be elusive.

Demyelinating diseases also caused by dysregulation of Wnt signaling pathway. Wnt controls multiple aspects of OL development including the specification of OPCs, OPCs differentiation, myelination, and remyelination. Here Wnt shows paradoxal role. It’s promoting and inhibitory effects are under great debate along with the timing and intensity of Wnt activation. Wnt signaling dysregulation with cancer has been well documented, particularly with colorectal cancer ¹¹². Constitutively activated β-catenin signaling, prevent its degradation, leads to excessive stem cell renewal/proliferation that predisposes cells to tumorigenesis ¹¹³.

Nkx2.2 regulates the expression of myelin structure genes and oligodendrocyte differentiation. It is plausible that the Nkx2.2 homeodomain transcription factor may directly bind to the promoters of MBP and PLP genes and subsequently regulate their expression ¹¹⁴. Common binding sites for Nkx2.2 are found in PLP and MBP promoters, thus overexpression of Nkx2.2 transcription factor can induce gene expression from the PLP promoter in transient transfection assays ¹¹⁴. In Nkx2.2 knockout mice, the number of myelin basic protein (MBP)-positive and proteolipid protein (PLP)-positive oligodendrocytes is drastically reduced and delayed in both the spinal cord and the brain. PLP and MBP expression is not inhibited completely, which indicates that Nkx2.2 may have a partially redundant function with other transcription factors such as Olig1/Olig2 or Sox10 which are co-expressed in oligodendrocyte progenitors. It is possible that Nkx2.2 may enhance or modulate the activities of these transcription factors. Wnt signaling pathway also plays its role in expression of Nkx2-2. Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated positively by Shh signaling and negatively by TCF7l2 repressor activity.

OPCs Differentiation results from the integration of extracellular signals and the chromatin state of a cell. OPCs are characterized by euchromatin and amenable to “accept” environmental signals (i.e Wnt, Shh, Bmp4, Notch) to modulate gene expression and differentiation. Impaired OPCs generation after Shh loss-of function ¹¹⁵ and ectopic oligodendrogenesis after Shh gain-of function has been reported ^{38, 116}. BMP4 increases the number of astrocytes at the expenses of oligodendrocytes ⁵², inhibits differentiation at later stage ⁵³ and decreases myelin genes expression ^{54, 117, 118, 119}. BMP4 signals activate Smad1/5/8 proteins (R-Smads), associate with the common mediator-Smad 4 (co-Smad) ^{120, 121} and activate gene expression by interacting with transcription co-activators ^{122, 123}. In OPCs competitive mechanism between Shh and BMP4 has been identified ¹¹⁸. It involved the functional sequestration of Shh target molecules (i.e. Olig2) by the inhibitors of Id2 and Id4 (differentiation protein), which are induced by BMP4 ^{124, 125, 126, 127}. In mouse cerebellar cultures, in contrast, BMPs interfere with the Shh-induced proliferation by decreasing the levels of Gli1 and Smo ¹²⁸, while in the chick spinal cord, BMP has been shown to repress Shh target genes Olig2 and Nkx2.2 ¹²⁹. In the adult brain the inhibitory effect of BMP4 on oligodendrogliogenesis has been attributed to the effect of Smad4 on the expression of Olig2. In a series of elegant studies on mice lacking Smad4 in neural stem cells, higher numbers of Olig2+ cells and oligodendrocytes were detected, a finding that was replicated by infusion of noggin (BMP inhibitor) ¹³⁰. High levels of BMP4 have been reported in human brains from Multiple Sclerosis patients ¹³¹, in animal models of spinal cord injury, ¹³², in mice with induced experimental allergic encephalomyelitis ¹³³ and in the cuprizone-induced demyelination model and they correlated with a dose-dependent increase of the astrocyte number at the expenses of oligodendrocytes ¹³⁴. The inhibition of BMP4 signaling by infusion of noggin reversed the effect and reduced the astrocyte numbers ¹³⁴ while promoting oligodendrocyte regeneration and remyelination ¹³⁵.

The Wnt pathway is a key signaling mechanism that controls multiple aspects of OL development including the specification of OPCs, OPCs differentiation, myelination, and remyelination. Wnt signaling via the canonical pathway is transiently activated in OPCs at the time of initial differentiation and then down-regulated when oligodendrocytes get mature ^{48, 136}. Initial specification of the oligodendrocyte lineage requires coordination of various upstream and downstream transcription factors ^{23, 137} including Tcf7l2, for the generation of mature, post-mitotic oligodendrocytes ⁵⁸. In contrast to other signaling pathways, Wnt shows paradoxal role in OPCs proliferation and differentiation. Effect of Wnt depends upon stage of maturation at the time of activation and intensity of activation.

The Wnt signaling pathway was initially described as an OL development inhibitor in embryonic spinal cord cultures ¹³⁸. Later on multiple in vivo studies confirmed that Wnt plays an inhibitory role in OPC differentiation during early postnatal development ^{48, 58, 139}. Azim et al in their study observed the negative impact of Wnt3a ligand on the OPCs differentiation. By the addition of Wnt3a agonist, Sox10⁺ OPCs and OLs were increased while significant decrease was observed in PLP⁺ OLs ¹⁴⁰. Lee et al reported that Wnt signaling, when antagonised by Apcdd1, which binds to LRP6 receptor and blocked its function, promotes OPCs differentiation ¹⁴¹. Daam2 is required for canonical Wnt signaling during patterning in the dorsal spinal cord, functioning through the clustering and formation of Wnt receptor signalosomes ¹⁴². Suppression of Wnt signalling pathway in Daam 2 knockout mice resulted in increased number of MBP⁺ and PLP⁺ OLs, further supporting that Wnt plays an inhibitory role in differentiation process ¹⁴³. OPC differentiation was also delayed following Wnt pathway activation by expressing a dominant-active β-catenin specifically in cells of the OL lineage ^{48, 139}. Loss of APC or Axin2, stabilizes the β-catenin and exerts an inhibitory effect on myelination ^{144, 145, 146, 147}. Similarly pharmacological agent (XAV939)¹⁴⁸ that stabilized by the Axin2 protein, resulting in increased MBP expression and number of PLP⁺OLs in comparison of control group ⁹. Together, these studies demonstrate that both genetic and pharmacological activation of Wnt/β-catenin impair OPC differentiation, thereby preventing cells from maturing and producing myelin sheaths.

TCF7l2, important component of Wnt, also affects the differentiation of OPCs. Various studies suggest that β-catenin/TCF7l2 complex is inhibitory for OPC differentiation and subsequent myelination. Fancy et al described that Olig2-Cre/DA-Cat mice expressed TCF7L2, an important nuclear binding partner of β-catenin ⁴⁸. Two other independent studies demonstrated that deletion of Tcf7l2 inhibited OPC differentiation in the spinal cord, suggesting that TCF7L2 is actually necessary for OPC differentiation (58, 136). From these studies it can be assumed that β-catenin may mediate its negative effect on OPC differentiation at least in part by recruiting TCF7L2 to regulate the transcription of important Wnt pathway target genes. Fancy et al demonstrated that Tcf7l2 is normally expressed in OLs during postnatal development but not in adulthood, potentially rendering constitutively active β-catenin ineffectual as the mice aged, and providing a potential mechanism for the delay but not complete block in OPC differentiation in the DA-cat mice ⁴⁸.

Although the inhibitory effects of Wnt signaling pathway on OPC differentiation are well accepted, however some studies conflict this inhibitory effect. Those researchers have described Wnt as a positive regulator of OPCs development. Tawk et al showed that addition of Wnt1 or Wnt3a in OPCs culture increased the expression of PLP⁺OLs by 3.5-fold and 2-fold, respectively ¹⁴⁹. Fancy et al described that microarray profiling has shown up regulation of Wnt ligand and receptors in lesions of MS and MS animal models ⁴⁸. Conditional knockout of β-catenin resulted in significantly decreased PLP⁺ and MPB⁺ OLs ¹⁵⁰. Knocking down of β-catenin and TCF molecules (all four types) decreased PLP promoter gene activity by 70%, reciprocally over expression of β-catenin increased PLP promoter gene activity in OLs lineage cells ^{89, 149}. Knockout of TCF4 caused a myelin deficient phenotype ¹³⁶. Some other studies showed that in TCF4 knockdown mouses expression of PLP & MBP was undetectable in comparison to control, indicating that TCF4 is essential for oligodendrocytes lineage differentiation and maturation ^{58, 151}. Microarray analysis of tissues from patients with multiple sclerosis revealed high expression of TCF4 in active lesion compared normal appearing white matter and silent chronic lesion ¹⁵².

From these controversial results described by various studies, it has been proposed that TCF7L2/β-catenin level must be tightly controlled for proper myelination ¹⁵¹. Therefore the extent to which an experimental model increases the level of intranuclear β-catenin correlates with the effect on OL maturation ¹⁵¹.

Oligodendrocyte precursor cells (OPCs) are generated from progenitor zones in the forebrain beginning at various times during embryonic development ²⁴. The mechanisms regulating the spatial and temporal production of OPCs have not been clearly elucidated. Sonic Hedgehog regulates the production of OPCs from ventral progenitor zones ²⁵ while In the telencephalon Wnt signaling plays a prominent dorsalizing role ^{153, 154}. Wnt signaling has been observed significantly decreased in the cortical progenitor domain at embryonic period when OPCs are generated ^{48, 58, 138}. Langseth et al described that Wnt signaling negatively regulates the specification of OPCs from neural progenitors and that inhibition of Wnt signaling increased the production of OPCs in the cortex ¹⁵⁵. Kessaris et al described that Wnt is very high in cortical progenitors at E17.5 (before cortical OPC production) and decreased by P5 when OPCs production is prominent ²⁴. Wnt signalling via the canonical pathway is transiently activated in OPCs concurrently with the initiation of terminal differentiation. Both β-catenin activity and the expression of Tcf7l2 are subsequently down-regulated in mature oligodendrocytes ^{48, 136}. Shimizu et al, described that addition of Wnt3a supernatant to CG4 cells (an OL progenitor strain) and to the dissociated primary cultured cells resulted in inhibition of differentiation step from OL progenitor to O4-positive stage ¹³⁸. Deletion of the Wnt effector Tcf7l2 blocks oligodendrocyte differentiation ^{58, 136} . These results have potential relevance for remyelination in human disease given that Wnt signaling components are present in MS lesions, suggesting that dysregulated Wnt/β-catenin signaling could contribute to the lack of remyelination, often seen in this disease ⁴⁸.

These findings indicate that Wnt negatively regulate the OPCs production ¹⁵⁰ at early developmental stage but at later stage promotes the differentiation of already established OPCs ^{155, 156} and finally down regulated when oligodendrocytes becomes mature ¹⁵⁷.

Along with activation of Wnt at various developmental stages, intensity of Wnt activation also plays an important role in oligodendrocytes development. During myelin formation, TCF7L2 must be associated with moderate level of β-catenin and either high or low levels of TCF7L2/β-catenin are detrimental for myelination. Either increased or decreased levels of TCF4/β-catenin expression have inhibitory effects on myelination process ¹⁵¹. Dai et al observed the decreased number of Olig1⁺ and Olig2⁺ cells in β-catenin activated mice (Cat^G/+) in comparison to control, and expression of Sox10 and PDGFRα was also impaired. Along with the number, distribution of these cells also affected, at E13.5 cells were only detected in the ventral ventricular zone of spinal cord, in contrary to their wide distribution in control tissues. Fancy et al also demonstrated that DA β-catenin signalling is sufficient to impede the remyelination process in mice. Similar results were obtained in mice lacking a copy of the β-catenin antagonist, APC. Together, these findings suggest that constitutive expression of β-catenin in OPCs correlates with a significant impairment of the remyelination process ⁴⁸. On other hand β-catenin inactivation (Cat^L/L) resulted in increased production of precocious OPCs (E12.5) ¹⁵⁰.