Developmental Cues and Molecular Drivers in Myelinogenesis: Revisiting Early Life to Re-Evaluate the Integrity of CNS Myelin

The mammalian central nervous system (CNS) coordinates its communication through saltatory conduction, facilitated by myelin-forming oligodendrocytes (OLs). Despite the fact that neurogenesis from stem cell niches has caught the majority of attention in recent years, oligodendrogenesis and, more specifically, the molecular underpinnings behind OL-dependent myelinogenesis, remain largely unknown. In this comprehensive review, we determine the developmental cues and molecular drivers which regulate normal myelination both at the prenatal and postnatal periods. We have indexed the individual stages of myelinogenesis sequentially; from the initiation of oligodendrocyte precursor cells, including migration and proliferation, to first contact with the axon that enlists positive and negative regulators for myelination, until the ultimate maintenance of the axon ensheathment and myelin growth. Here, we highlight multiple developmental pathways that are key to successful myelin formation and define the molecular pathways that can potentially be targets for pharmacological interventions in a variety of neurological disorders that exhibit demyelination.


Introduction
As the regulator of all cognitive, sensory, and motor activity, the nervous system is the most complex biological system in humans; the complexities of integrated neural networks are a hot area of intensive research that will require multidisciplinary investigations to address a variable array of neurological disorders that remain an unmet medical need. The main types of cells in the nervous system are neurons and glial cells with the latter performing vital supporting roles [1,2]. The glial/neuronal ratio differs uniformly across brain regions of mammalian species, underlining the pivotal role of interaction between glial cells and neurons for appropriate integration of the central nervous system (CNS) to coordinate neurophysiological and cognitive functions [3]. For propagation of action potentials to ensue in neurons, axonal myelination is crucial [4,5]. The cells responsible for myelination are the oligodendrocytes (OLs) in the CNS and Schwann cells in the peripheral nervous system (PNS). Rudolf Virchow initially designated the term "myelin" in 1854, named after the Greek word "marrow" (myelos), since it is especially plentiful in the brain's center, or marrow [6]. He posited that neurons produced myelin, but Pío del Río Hortega's The mammalian CNS emerges from an ectodermal, neuroepithelial lining of the neural tube in the developing embryo [9,18]. Multiple divisions give rise to radial glia (RG), a multipotent neural stem population that colonizes the newly-formed ventricular walls ( Figure 1) [18,19]. The VZ is the primary embryonic site for OPCs production through asymmetrical division of the RG cells. In mice, OPCs are firstly detected in the ventral VZ closer to the floor plate on embryonic day 12.5 (E12.5), and in humans at gestational week (GW) 6.5 (~E45) [20]. More specifically, the outer SVZ (oSVZ) is an enlarged cortical germinal zone only generated in humans [21]. In oSVZ, a distinct RG cell population termed as outer RG (oRG) is located peripherally and gives rise to a transit-amplifying population, which is an additional source of OPCs supplying the human cortex [19,21].

Molecular Signals Driving Myelinogenesis
As has been articulated from the experimental evidence, the inauguration of myelinogenesis necessitates the formation of OPCs from multipotent NSCs, which ultimately give rise to mature myelinating OLs through a multistep process ( Figure 1) [18,24]. A vital step herein lies in OPCs' ability to migrate toward miscellaneous sites and proliferate, based predominately on environmental stimuli. These cells become post-mitotic, exiting the cell cycle to express a substantial amount of myelin-associated proteins and differentiate into mature pre-myelinating OLs [23]. Following the proper recognition, targeting and ensheathing specific nerve fibers is the subsequent critical milestone where each pioneer process creates lamellar extensions that stretch and elaborate circumferentially around the target axon [24]. As a new membrane is generated at the leading edge of the forming myelin sheath's inner tongue, which starts to resemble a spiral cross-sectional shape, the sheath continues to spread along the axonal length. The secured stability and maintenance of a newly-formed myelin sheath is the concluding event. Specific developmental cues and molecular drivers regulate all the aforementioned cellular activities and are enlisted in full capacity in Tables A1-A3.

Formation of OPCs
OPCs being generated from the ventral VZ are under the influence of the morphogen molecule Sonic hedgehog (SHH) secreted from the notochord, while the dorsal counterparts are SHH-independent [25,26]. SHH signalling drives NSCs into a neuronal or OLs lineage fate superseding the effect of bone morphogenetic proteins (BMPs) which favour astroglial generation ( Figure 1) [27,28]. Early secretion of SHH promotes motor neuron lineage formation, while interaction in later time periods promotes OLs differentiation [29]. Interestingly, the concentration of SHH can be controlled by sulfatase 1 expression in the ventral neuroepithelium prior to OPCs specification [30], whereas fibroblast growth factor (FGF) signalling is of paramount importance for further OLs differentiation, especially in the spinal cord [31,32].

Proliferation
Specific driver molecules that participate in migration, such as PDGFA and PDGFRα, contribute additionally to the OPC proliferation [47,71]. Interestingly, in the spinal cord, the mitogenic effect of PDGF was enhanced by chemokine CXCL1 and CXCR2 [44,72], while CXCL12 had a proliferative effect on OPCs, mediated by its receptor CXCR4 [73]. More growth factors, such as FGF2, brain-derived neurotrophic factor (BDNF), and epidermal growth factor (EGF) are shown to play a vital role in OPCs proliferation [74][75][76].

Differentiation
OLIG1 and OLIG2 are heavily involved in the post-proliferating step of myelinogenesis, defining the initiation of OPCs differentiation ( Figure 1) [51,53,104], while BMPs seem to inhibit this process by downregulating myelin protein expression [105]. The effect can be reversed by using a physiological antagonist of BMP4, such as noggin, which may restore differentiation [91,106,107]. OLIG2 appears to interact with a variety of factors, such as ASCL1, BRG1, transcription factor 4 (TCF4), and SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) to ensure proper OPCs differentiation [108][109][110][111][112]. G proteincoupled receptor 17 (GPR17) can act as a downregulator of OLIG1 that negatively controls the maturation and coordinates the generation of myelinating OLs from pre-myelinating OLs through ID proteins [113]. Although overexpression of ID2 and ID4 both regulate myelin gene expression by inhibiting OLs differentiation [89,114], they are not the major in vivo repressors of differentiation [115]. Moreover, decreased levels of OLIG1 and myelin regulatory factor (MYRF) were observed under early growth response 1 (EGR1) and SOX11 overexpression, delineating the inhibitory action of the latest in OPCs differentiation [116,117]. Intriguingly, MYRF is a unique regulator participating in the late stages of OLs maturation and myelination, while the action of the other OLs' lineage transcription factors is restricted on OPCs specification or initial differentiation of OLs [118].
SOX family proteins are also participating in the OLs differentiation. In particular, SOX2 and SOX3, through negative regulation of miR145, promote OLs maturation [119], while SOX5 and SOX6 increase PDGFRα expression, maintaining OLs in their immature state [44]. For the terminal differentiation of OLs, SOX8, SOX9, and SOX10 are required [82,[120][121][122]. The state of myelinogenesis-associated gene expression is uniformly affected by NKX2-2 and NKX2-6 [40,123,124]. Ji et al. suggested a mechanism regarding NKX2-2-mediated inhibition of OLs differentiation via regulation of sirtuin 2 (SIRT2), which generally is a positive cue for OLs maturation [125]. Similarly, sirtuin 1 (SIRT1) participates in the differentiation of OPCs during development [126] through cytoskeleton-related OLs proteins. The Kruppel-like factor 6 (KLF6) is another transcription factor promoting OPCs differentiation through glycoprotein 130 (GP130)-signal transducer and transcription activator 3 (STAT3) signalling [127]. Growth factor-wise, BDNF is a regulator of OLs differentiation operating via binding to tyrosine receptor kinase B (TrkB) and enhancing the MAPK pathway to upregulate gene expression during OLs maturation [75,77,128]. Evidently, NT-3 is important for the transition of immature OLs to myelin-forming cells by recruiting c-Fos protein-activating protein kinase C (PKC) and tyrosine kinase activities [129,130]. Insulin-like growth factor 1 (IGF-1) is another main factor in assisting the development of OPCs to mature OLs [131]. In accordance with that, GRB2 associated binding protein 1 (GAB1) absence decreased OLs differentiation, acting as a novel target of PDGF [132]. Incidentally, Canoll et al. suggested that NRG1 is a negative regulator of OPCs differentiation [101], while Brinkmann et al. later demonstrated that NRG1 is required for OPCs differentiation [103].
As far as metabolism is concerned, quaking homolog, KH domain RNA binding (QKI)-5 forms a complex with sterol regulatory element-binding transcription factor 2 (SREBF2) that regulates the transcription of genes responsible for cholesterol biosynthesis in OLs during differentiation [133]. Lack of transactive response DNA-binding protein 43 (TDP-43) results in lower SREBF2 and low-density lipoprotein receptor (LDLR) expression and cholesterol levels in vitro and in vivo, indicating the potential role of TDP-43 in cholesterol homeostasis in OLs, which is linked with the proper completion of OLs development [134]. In the same manner, ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) participates in OLs maturation via a supplement of OLs with choline [135]. Most importantly, triiodothyronine (T3) is a key molecule for blocking OPCs proliferation and promoting their differentiation into mature OLs [136,137]. Thyroid hormone receptor alpha (TRα) is found both in OPCs and mature OLs, whilst thyroid hormone receptor isoform beta 1 (TRβ1) is located only in mature OLs [138]. The OPCs differentiation is mediated by the TRα, while TRβ1 is responsible for promoting myelinogenesis in later stages [77]. Overexpression of HES5 decreases the levels of TRβ1 receptors, while ASCL1 increases them, demonstrating their role in regulating OLs differentiation timing [139]. The neurogenic locus notch homolog protein 1 (NOTCH1) is another receptor that also regulates the differentiation timing [140]. Interestingly, JAG1 is a receptor's ligand responsible for inhibiting OLs differentiation, while contactin 1 (CNTN1) is another ligand with the opposite function [79,141].

Ensheathment
Multiple positive cues are important for the inauguration of ensheathment ( Figure 2). Amongst the prime ones with a positive effect on axon-glial junction maintenance is NCAD, which regulates the interaction between OLs processes and axons [165]. The L1 cell adhesion molecule (L1-CAM) and laminin expressed in axons bind to contactin and integrin located in OLs [166]. Upon the formation of the first loops/wraps, neurofascin 155 (NF155), located in paranodal loops, forms a well-defined complex with contactin-associated protein (CASPR) and CNTN1, transmembrane proteins which are expressed in axons [167][168][169]. The activation of this complex has a pivotal role in myelin targeting, sheath growth, organisation of paranodal loops and, therefore, supporting the axoglial junction [170,171]. However, CASPR does not participate in myelin targeting [170]. In juxtaparanodes, the axoglial junction is strengthened when transient axonal glycoprotein-1 (TAG-1), a crucial molecule for maintaining enrichment of Kv1.1/Kv1.2 channels [172], interacts with CASPR2. Regarding internodal axoglial adhesion, glial cell adhesion molecule (CADM) 4 binds to axonal CADM2 and CADM3, facilitating myelin targeting, axon wrapping, and myelin sheath growth [173]. Similarly, CADM1b strongly binds to axonal CADM2, positively regulating ensheathment and strengthening the junction [174]. In the same region of the myelin sheath, MAG binds to ganglioside in axons, especially ganglioside GD1a and GT1b, and enforces the junction's stability [175,176].
Based on several studies, ephrins (A, B) and cognate receptors (A, B) have dual roles that rely on location and expression. While ephrin receptor (Eph) A4 in OLs is activated by axonal ephrin-A1 ligand, which inhibits the stability of axoglial junctions needed for ensheathment, EphA4, expressed in the axon surface, interacts with ephrin-B, promoting myelin sheet formation [177,178]. In addition, EphB1 of axons is activated through ephrin-B in OLs, which in turn stimulates myelinogenesis [178]. The axonal ephrinB2 via binding with EphB OLs receptor influences integrin activation, reducing myelin sheet formation [178]. The list of negative cues includes LINGO-1, which is located in both axons and OLs, and self-interacts in trans to control the number of targeted axons inhibiting myelinogenesis [143,179]. The NCAM is a cell adhesion molecule negatively regulating myelinogenesis. The downregulation of this protein is essential for promoting myelin formation during development, as myelinogenesis occurs only on NCAM negative axons [180].
A somatodendritic protein, junctional adhesion molecule 2 (JAM2) inhibits oligodendroglial interaction, suppressing myelinogenesis [181]. Apart from the somatodendritic molecules, GAL-4 is expressed only to unmyelinated segments of neurons in hippocampal and cortical regions; this protein is demonstrated as the first identified inhibitor of myelinogenesis in axons [182]. Of particular interest is the possible role of OLIG1 in axonal recognition during myelinogenesis (Table A2) [183]. B in OLs, which in turn stimulates myelinogenesis [178]. The axonal ephrinB2 via binding with EphB OLs receptor influences integrin activation, reducing myelin sheet formation [178]. The list of negative cues includes LINGO-1, which is located in both axons and OLs, and self-interacts in trans to control the number of targeted axons inhibiting myelinogenesis [143,179]. The NCAM is a cell adhesion molecule negatively regulating myelinogenesis. The downregulation of this protein is essential for promoting myelin formation during development, as myelinogenesis occurs only on NCAM negative axons [180]. A somatodendritic protein, junctional adhesion molecule 2 (JAM2) inhibits oligodendroglial interaction, suppressing myelinogenesis [181]. Αpart from the somatodendritic molecules, GAL-4 is expressed only to unmyelinated segments of neurons in hippocampal and cortical regions; this protein is demonstrated as the first identified inhibitor of myelinogenesis in axons [182]. Of particular interest is the possible role of OLIG1 in axonal recognition during myelinogenesis (Table A2) [183].

Myelin Sheath Growth and Preservation
The long-term membrane expansion and maintenance of the newly-formed myelin sheath is the final step in completing myelinogenesis and is utterly controlled by the major myelin proteins (Table A3). The most abundant myelin proteins are PLP (>50%) and MBP (~15%), having a significant role in the stabilization of the myelin structure [24,52,184].

Myelin Sheath Growth and Preservation
The long-term membrane expansion and maintenance of the newly-formed myelin sheath is the final step in completing myelinogenesis and is utterly controlled by the major myelin proteins (Table A3). The most abundant myelin proteins are PLP (>50%) and MBP (~15%), having a significant role in the stabilization of the myelin structure [24,52,184]. The disruption of PLP gene expression presents impaired membrane compaction [185]. MAG, on the other side, is the third most abundant protein in CNS myelin (~5%), and does not seem to contribute to maintenance as much as it does to the previously described initial interaction between OLs and axons [147,186]. Interestingly, myelin oligodendrocyte glycoprotein (MOG) [187,188], CNPase [52,189], myelin-associated oligodendrocyte basic protein (MOBP) [190], and OMgp [86,191,192], all minor CNS myelin proteins (<1%), need more investigation on how they influence the formation and maintenance of myelin sheaths in compact myelin.
Proper cholesterol biosynthesis is prioritized in myelinogenesis, with QKI regulating this cholesterol production via SREBF2. Specifically, QKI-5 acts synergistically with peroxisome proliferator-activated receptor beta (PPARβ)-retinoid X receptor alpha (RXRα) activating transcription of the response in fatty acid metabolism genes. This operation of QKI-5 is significant for maintaining myelin homeostasis [133]. The ceramide galactosyl transferase (CGT) is a key enzyme for catalyzing GALC synthesis, while ceramide sulfotransferase (CST) is responsible for converting GALC to sulfatide [200,201]. Both CST and CGT mutant animals showed a regionally specific loss of myelin stability [200]. Thus, GALC and sulfatide have a pivotal role in the long-term maintenance of myelin, with the GALC being more crucial for myelin development than its assembly [200,201]. Additionally, peroxisomal metabolism also influences myelin survival [202]. For example, a peroxisomal transmembrane protein responsible for very long-chain fatty metabolism is encoded by the ABCD1 gene and is key in maintaining myelin stability [164,203]. Lastly, the age-dependent changes of TMEM10 might be linked with its action in maintaining CNS myelin [204].

Myelin Formation after Infancy
Although myelinogenesis has been described in the nascent developmental years, myelination does naturally occur for the duration of a person's life to promote learning and memory through brain circuit plasticity [205], or as remyelination after an injury [206]. The synaptic plasticity has been studied in depth; however, a newly discovered form of brain plasticity, namely myelin plasticity or myelin remodelling, is under intensive investigation [205]. Extrinsic factors can influence, either positively or negatively, this remodelling in the toddler, adolescent, and adult brain. For example, since myelin formation is sensitive to experience, sensory stimulation may upregulate myelination, while sensory or social deprivation can potentially downregulate axon ensheathment [205,207]. Myelin remodelling initiates when pre-existing OPCs recruit or directly differentiate into newly-formed mature OLs, whereas existing OLs have the ability to engage in plasticity [205]. The principal cues for this "adaptive" myelination should not be different from the ones we scrutinize in this review.
The regenerative process following injury also presents many similarities with specific steps of myelinogenesis [208]. The neonatal OPCs are maintained in a resting, quiescent state through adulthood, and they are referred to as adult aOPCs, constituting~6% of all cells in the CNS [206]. Interestingly, aOPCs have a transcriptome similar to mature OLs. After injury, the innate immune response activates aOPCs, transforming them into a neonatal-like transcriptome [209]. The activation of aOPCs is followed by their proliferation, migration, and final differentiation into mature OLs. Older literature describes these aOPCs as the primary remyelinating cells [210]. Nevertheless, newer research has suggested that neural progenitors in SVZ, Schwann cells, and surviving mature OLs are also implicated in the remyelinating process [211][212][213].
The myelination efficiency is age-dependent, as the impairment of aged OPCs to recruit and differentiate into mature OLs leads to decreased remodelling and remyelination [214]. The nutrient support of OLs is highly compromised in aging due to the presence of senescent astrocytes, leading to decreased cholesterol biosynthesis which in turn weighs in the impaired OLs membrane development [15,215,216]. This age-related energy depletion that decreases the myelination efficiency is further fed from the accumulation of DNA damage while rendering the neurons vulnerable to oxidative stress through free radicals [15,217]. Additionally, the ineffectiveness of microglia, which translates to aged phagocytes to clear out impaired myelin, is a potential aetiology for the downregulation of remyelination [218]. Taken all together, the detailed investigation of cues that drives de novo myelination could be a crucial point for revisiting them in demyelination and remyelination of the adult CNS, a concept that is discussed briefly in the following section.

Myelinogenesis in Disease and Beyond
Although aging is a natural process that leads to a decreased turnover of functional OLs and diminished myelin formation, the integrity of myelinogenesis can be highly compromised in pathological situations such as demyelination, characterized by extensive myelin loss [219]. This condition has to be distinguished from dysmyelination, which is a genetic-based anomaly affecting basic myelin proteins and leads to uneven/not properly compacted myelin sheaths [219]. Demyelinating diseases could be divided into many categories; according to their pathogenesis mechanism, which mostly implicate environmental factors, nutritional deficits, presence of myelinotoxic agents, or virus-mediated impairments. In quite frequent cases, immune system mediators are deregulated, leading to autoimmune inflammatory demyelination [219,220]. Among the three most prevalent inflammatory demyelinating diseases are multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), and acute disseminated encephalomyelitis (ADEM).
In this review, we summarized all the potential molecules responsible for the long-term maintenance of myelin along the axoglial junction (see Sections 2.2.5 and 2.2.6), serving simultaneously as key factors in demyelinating disease sequelae. Recent data revealed that impaired mitochondrial function and oxidative stress are also candidate pathophysiology mechanisms for demyelinating diseases [221]. Berghoff et al. demonstrated that disruption of cholesterol metabolism alters brain lipid metabolism in CNS and is associated with neurological diseases such as autoimmune inflammatory conditions, including MS [222]. Nonetheless, under such circumstances, an autoimmune attack generates myelin debris from damaged myelin [223]. These components impair the CNS remyelination by obstructing OPCs and OLs functionality while triggering additional deleterious immune responses, also known as epitope spreading [213,223,224]. The clearance of myelin debris is crucial for rearrangement since recent studies suggest that the failure of myelin clearance leads to inefficient remyelination [225,226].
Remyelination can be spontaneous or in an experimental setup, achieved by the providence of an exogenous source of neural precursor cells (NPCs) with myelinating potential [227,228]. In various transplantation paradigms, it is shown that these cells can either exert an in situ myelinating effect, as seen and applied successfully in spinal cord injury (SCI) cases [229,230], or by instructing and enhancing the capacity of endogenous cells to remyelinate, documented in experimental autoimmune encephalomyelitis (EAE) [208,231,232] (324). Proposed mechanisms of action also underline immunomodulatory effects rather than direct cell replacement [233,234]. Nevertheless, the scarce population of surviving mature OLs after demyelination is shown to be less effective in comparison to newly created ones [213,[235][236][237]. Towards this trajectory, which is a fully functional recruitment of aOPCs to form myelinating OLs [208,238] (324, 325), it is extremely important to comprehend the developmental molecular cues and factors governing the process of myelinogenesis (see Section 2.2.1, Section 2.2.2, Section 2.2.3, Section 2.2.4), since these same molecules can be candidate targets for therapeutic intervention in demyelinating diseases.

Conclusions
Through this comprehensive review, we attempt to list and categorize the genes and proteins that act as developmental morphogens to the CNS development and, more specifically, those that are activated in the process of oligodendrogenesis. The fully functional OLs, originating from unspecialized stem cells, are able to identify newly-formed axons which emanate and branch in regions that need fast conduction early in life, completing their task of myelinogenesis. Some of these cells persist in adult life in an intermediate, dormant phenotype scattered or organized around the primordial niches. An argument that was intended to bring into attention is how the powerful dynamics that shape myelination, which is naturally declining as we age, can be sustained, or even re-engaged after an injury or demyelinating disease. In the current era, transcriptomic profiling or metabolomic data can potentially give an answer as to which of the enlisted molecules, drivers, and regulators should be prioritized.

Conflicts of Interest:
The authors declare no conflict of interest. C-X-C motif chemokine ligand 1 CXCL12

Appendix A
The following appendix contains all the genes and relevant chromosomal loci from proteins involved in myelinogenesis in relation to their biological role. This data is supplemental to the main text based on the current knowledge and literature on cues driving oligodendrocyte development, ensheathment and myelin maintenance.          [200,201] * Data are retrieved from "The Human Protein Atlas" [285].