As in mammals, pMN progenitors in zebrafish initially generate motor neurons, then switch to producing OPCs in a process tightly regulated by a collection of transcription factors, signalling molecules and extracellular cues. For example, various transcription factors including Olig2 and Nkx2.2, and signalling pathways including Hedgehog have all been shown to have important conserved roles in OPC specification in zebrafish and mammals (Lu et al., 2000, 2002; Park et al., 2002; Kirby et al., 2006; Sun et al., 2006; Li et al., 2007; Kucenas et al., 2008; Lyons and Talbot, 2015) (Supplementary Table 1). More recently, mechanisms of Sonic hedgehog signalling regulation were investigated by Scott et al. (2020) who explored the effects of transcriptional repressor prdm8 on OPC specification. By live in vivo imaging they show that pMN cells in prdm8 mutant embryos have elevated Sonic hedgehog signalling and prematurely switch from motor neuron to OPC production (Scott et al., 2020). Intriguingly, Prdm8 was also shown to influence the fate of oligodendrocyte lineage cells, as prdm8 mutant embryos have increased numbers of oligodendrocytes but lack sufficient OPCs. Together, this suggests that the morphogen Sonic hedgehog plays an important role in controlling the timing of OPC specification in zebrafish (Ravanelli and Appel, 2015; Scott et al., 2020). Similarly, the Notch canonical signalling pathway also influences the timing of the pMN domain progenitor differentiation switch from motor neurons to OPCs. This has been shown in zebrafish where reduced Notch activity induced excess formation of primary motor neurons and reduced numbers of OPCs (Appel et al., 2001; Supplementary Table 1). In contrast, constitutive Notch activity promoted OPC production (Appel et al., 2001; Supplementary Table 1). Complementing this study, Snyder et al. (2012) found that disrupting fbxw7, a ubiquitin ligase that targets Notch for degradation, results in excess OPC production (Supplementary Table 1). Taken together, these data suggest that Notch signalling tightly regulates the timing of the pMN progenitor differentiation switch, and therefore plays a central role in OPC specification.

Concomitant with their specification, OPCs in zebrafish and mammals delaminate from the neuroepithelium and migrate towards their axonal targets. One interesting candidate which has been identified in zebrafish to affect the timing of neuroepithelial delamination to OPCs in the zebrafish is the microRNA mir-29, which can regulate the expression of the polarity genes pard3 and prkci (Hudish et al., 2013; Supplementary Table 1). How OPCs are specified in other regions of the neuraxis and at later stages, will require ongoing analyses, and in particular the way in which radial-glial-like progenitors present in zebrafish can give rise to OPCs following injury or demyelination awaits in-depth analyses ahead of comparison with mammals.

Following delamination from the neuroepithelium OPCs in mammals proliferate extensively and migrate widely to take up positions throughout the parenchyma in which they both continue to proliferate and also adopt specialised functions, including the generation of myelinating oligodendrocytes (Figure 3). In contrast, at the stages and regions principally studied to date in zebrafish, OPCs have a relatively small volume to colonise. Indeed, the total distance that OPCs migrate from the pMN domain in the ventral spinal cord to nearby ventral axonal tracts where oligodendrocytes first differentiate is on the order tens of microns with dorsal regions of the hindbrain or spinal cord remaining only 100–200 μm from the pMN domain. These small distances mean that the OPC population doesn’t undergo a huge degree of expansion through proliferation and migration at early embryonic stages. To what extent principles of OPC population expansion and migration observed in mammals, e.g., whereby OPCs follow the brain vasculature to migrate over long distances (Tsai et al., 2016), are conserved in fish will require analyses at later stages and in other regions of the CNS when and where migratory routes are longer and, likely more complex. Nonetheless, several cell-cell interactions, as well as specific molecular factors have been identified that influence both OPC proliferation and migration in the developing zebrafish (Supplementary Table 1).

For example, OPC proliferation appears to be responsive to the density of axons fated for myelination in the embryonic zebrafish CNS, with OPC proliferation reduced in a mutant that lacked axons fated for myelination in the posterior spinal cord, Almeida and Lyons (2016). One set of axonal signals known to regulate many aspects of the oligodendrocyte lineage in mammals, including OPC proliferation, are those associated with neuronal activity (Geraghty et al., 2019). Indeed, a recent study has indicated that neuronal activity also influences OPC proliferation in the zebrafish where a pharmacologically-induced increase in neural activity resulted in increased OPC proliferation (Marisca et al., 2020). Although the axonal signals (activity-related or not) that regulate OPC proliferation in zebrafish await identification, receptors on oligodendrocytes that influence proliferation have been uncovered (Supplementary Table 1). In addition to proliferation, neuronal activity has also been implicated in regulating OPC migration in mammals (Harlow et al., 2015) and recently it was found that disruption to the AMPA receptor subunit GluR4A (encoded by gria4a) expressed on OPCs impaired migration in zebrafish (Piller et al., 2021) (Supplementary Table 1).

Following migration to their target regions, OPCs undergo a set of cell-cell interactions to demarcate the space in which they will ultimately reside. During this tiling behaviour OPCs continuously extend and retract processes to sample their environment and appear to be highly influenced by contact with surrounding OPCs, whereby contact between distinct OPCs appears to induce repulsion of contacting processes (Kirby et al., 2006). Indeed, when a single or small group of OPCs are ablated, nearby OPCs increase cell division to replenish numbers, fill the space, and reinitiate tiling behaviours (Kirby et al., 2006). This appears to be a largely conserved mechanism between species, given that similar observations were made in the cortex of mature mice, where neighbouring OPC processes exhibited contact-mediated withdrawal, and increased proliferation following damage to neighbouring OPCs (Hughes et al., 2013). Recently, in a mouse model, the molecular mechanism for this OPC-OPC interaction was proposed (Chavali et al., 2020), conservation of which remains to be tested in zebrafish, but appears likely, given the remarkable similarity in associated cell behaviour across species.

The broadly accepted view of OPCs is that they are principally sources of myelinating oligodendrocytes. However, it is now becoming clear that OPCs can exhibit a diverse array of functions beyond their roles in making myelinating oligodendrocytes (Harrington et al., 2020; Akay et al., 2021; Clayton and Tesar, 2021) (Figure 3).

The existence of OPCs with potentially distinct fates was first investigated in zebrafish by Kucenas et al. (2008), who showed that OPCs that express the transcription factor Nkx2.2a, an ortholog of rodent Nkx2.2, typically differentiate into myelinating oligodendrocytes, whereas OPCs that do not express nkx2.2a mostly remain as non-myelinating OPCs. The fate of distinct zebrafish OPCs was recently investigated further in a study that combined fate mapping of individual OPC clones, morphological characterisation of their dynamic behaviour by time-lapse microscopy, and associated single cell RNA sequencing (Marisca et al., 2020). This study confirmed the existence of two discrete groups of OPCs in the zebrafish spinal cord, those with cell bodies located within axonal tracts and those with cell bodies surrounded by the cell bodies of other cells, principally neurons (Marisca et al., 2020). Interestingly, OPCs with cell bodies located in the axon-dense areas tended to directly generate myelinating oligodendrocytes, whereas those with cell bodies in neuron/soma-rich areas tended to self-renew and remain relatively stable, although these cells could also generate the OPCs that moved to axon-rich areas. These two OPC types displayed both unique branching morphologies and very different patterns of calcium activity and interactions with axons. Interestingly, and perhaps surprisingly, OPCs with their cell bodies within the neuron-rich areas had higher levels of calcium activity, but yet infrequently differentiated, despite having extensive processes that made very stable contact with axons. Interestingly and correspondingly, these OPCs had a molecular signature of being responsive to neuronal activity, and it was these cells whose proliferation was responsive to changes in activity noted earlier (Marisca et al., 2020). This suggests that although neuronal activity can regulate OPC proliferation, it may not, directly at least, influence oligodendrocyte differentiation.

The function of OPCs beyond their generation of oligodendrocytes has been hard to experimentally disentangle, but has recently been investigated in zebrafish, by taking advantage of the presence of abundant OPCs in a brain area, the optic tectum, that does not become myelinated in the developing larva. The optic tectum is the largest retino-recipient brain structure in zebrafish, receiving input from a large proportion of retinal ganglion cell axons and is analogous to the superior colliculus in mammals. Upon arrival at the tectum, distinct retinal ganglion cell axons enter specific tectal layers where they make precise connections with defined tectal neuron dendrites (Erskine and Herrera, 2007). To investigate a potential role for OPCs in connectivity with target neurons, Xiao et al. (2021) ablated tectal OPCs and found that this resulted in enlarged axonal arbors in the tectum and branching of retinal ganglion cell axons beyond the tectal neuropil where target dendrites are located. Indeed, live imaging implicated OPCs in regulating axonal pruning/remodelling in vivo, which had previously been predicted by in vitro studies in mammals (Goldberg et al., 2004). Very interestingly, ablation of tectal OPCs in zebrafish also resulted in impairments to visual function. Recent investigation in rodent models suggest that OPCs can engulf axons in mammals (Buchanan et al., 2021), and even directly modulate neuronal activity (Sakry et al., 2014). Given the availability of reporters to label OPCs as well as synaptic and neuronal activity in zebrafish, it will be possible to directly and more deeply interrogate how OPCs affect both neuronal structure and function in vivo using zebrafish. As well as determining the currently unrecognised roles of OPCs in larval zebrafish, another exciting area of future study will be determining the functions of OPCs in the adult zebrafish brain (Tsata et al., 2020), both in health and disease.

The process of oligodendrocyte differentiation from OPCs is one that can occur by default in the absence of axonal signals (Zeller et al., 1985; Lee et al., 2012; Bechler et al., 2018), upon the activation of gene expression programmes that ultimately converge on building the myelin sheath [reviewed in Czopka and Lyons (2011), Bergles and Richardson (2016), and D’Rozario et al. (2017)]. This default differentiation programme appears to be conserved in vivo in zebrafish, as evidence by relatively unaltered oligodendrocyte differentiation observed in mutants with a large reduction in axons fated for myelination (Almeida and Lyons, 2016). As observed in mammals, there appears to be a critical period following differentiation in which oligodendrocytes either commit to myelination, or undergo cell death (Takada and Appel, 2010; Almeida and Lyons, 2016). In general, the molecular regulation of oligodendrocyte differentiation and initiation of myelin gene expression appears well conserved with mammals (Supplementary Table 1). For example, the lysosomal transcription factor Tfeb was recently identified through a forward genetic screen in zebrafish as a negative regulator of myelination, whereby its abrogation leads to premature and excess myelination (Meireles et al., 2018). In parallel, mice lacking TFEB in oligodendrocytes were also found to exhibit precocious and excessive myelination (Sun et al., 2018). In the mouse this was found to be mediated, at least in part, by reduced cell death of newly generated oligodendrocytes. It remains to be determined precisely to what extent the molecular control of oligodendrocyte survival and myelination are distinct or overlap downstream of Tfeb, and how Tfeb and other major transcription factors co-operate to control oligodendrocyte differentiation.

Similar to other vertebrates, zebrafish oligodendrocytes make multiple myelin sheaths on many axons upon differentiation (Figure 3), and although some differences in components have been noted the overall molecular composition and ultrastructural nature of myelin in zebrafish and mammals appears broadly similar (Jahn et al., 2009, 2020; Czopka and Lyons, 2011; Preston and Macklin, 2015; Czopka, 2016; Siems et al., 2021) (Figure 3). By live imaging of oligodendrocytes in zebrafish over time using transgenic reporters, Czopka et al. (2013) found that individual cells initiate formation of essentially all of their sheaths within a matter of hours following the initiation of the first sheath, pointing to a restricted period in which oligodendrocytes have to select axons for myelination. Following the selection of axons, the process of myelination continues via wrapping of the innermost layer of the myelinating process around and along the axon in a spiralling manner (Snaidero et al., 2014; Nawaz et al., 2015). Following the initial dynamic period of their formation, most sheaths are maintained over time, except for occasional sheath retractions (Czopka et al., 2013; Auer et al., 2018). Whereas initial myelin sheath formation occurs over a matter of hours, imaging studies indicated that subsequent wrapping-based sheath growth occurs over several days, before slowing down and growing in step with the overall growth of the tissue (Auer et al., 2018).

But how are specific axons selected for myelination and why do oligodendrocytes avoid other axons, and indeed other cellular targets for myelination? Almeida et al. (2018) recently found that the relative abundance of oligodendrocytes and axons fated for myelination influences targeting, given that in mutants with fewer axons fated for myelination or in animals with excess oligodendrocytes, oligodendrocytes made myelin around inappropriate targets including neuronal cell bodies. In contrast to myelinating neuronal cell bodies, oligodendrocytes in animals with fewer axons fated for myelination do not myelinate incorrect axons, suggesting more stringent mechanisms preventing their myelination, likely a combination of typically being of smaller axon calibre and expressing inhibitory signals (Klingseisen and Lyons, 2018). Following a forward genetic screen in zebrafish, it was subsequently found that the cell adhesion molecule Neurofascin functions in oligodendrocytes to prevent inappropriate myelination of cell bodies, a mechanism conserved in rodents (Klingseisen et al., 2019). This finding was also made in a parallel study, which implicated the neuronal partner of oligodendrocyte Neurofascin, Contactin1, in mediating correct myelin targeting (Djannatian et al., 2019). Studies in zebrafish have also indicated that certain axons can positively influence myelination (Almeida et al., 2011; Nelson et al., 2020), with many studies now demonstrating that neuronal activity can influence myelination (Nave and Werner, 2014; Baraban et al., 2016; Swire and ffrench-Constant, 2018; Williamson and Lyons, 2018). Studies in zebrafish have contributed to this, with evidence that blocking synaptic vesicle release from all neurons reduces the number of myelin sheaths made by oligodendrocytes and increasing activity increases sheath number (Mensch et al., 2015). Correspondingly, experiments in which the synaptic vesicle release of only some axons was affected indicated that myelination is biased towards higher activity axons (Hines et al., 2015; Koudelka et al., 2016), but interestingly that this only remains true of certain neuronal subtypes (Koudelka et al., 2016), a feature that has more recently also been shown to be true in rodents (Yang et al., 2020).

The mechanisms by which vesicle release regulates myelination was recently investigated by live-cell imaging a transgenic reporter (SypHy) that allows assessment of vesicular release along axons together with a reporter that allows assessment of myelination along the same axons. Somewhat surprisingly, it was found that axonal synaptic vesicle fusion increases upon myelination, and in fact requires myelination. Upon myelination, axonal vesicular fusion becomes enriched adjacent to sites of myelin sheath formation, where it becomes increased upon increasing neuronal activity, which in turn promotes myelination. This led to a feedforward model of activity-regulated myelination, whereby myelination stimulates the localised axonal vesicular release that in turn consolidates myelin sheath growth along axons (Almeida et al., 2021). Although the various molecular mechanisms that regulate the axonal vesicular release remain to be determined, the cell adhesion molecule N-cadherin has been proposed to mediate the effects of neuronal activity on myelination in zebrafish (Chen et al., 2017), and may do so by influencing vesicular recycling, as has been shown in mammals (Van Stegen et al., 2017). Recent data has also indicated that axons employ mechanisms that consolidate synapse formation in myelination. This was evidenced by observations that oligodendrocytes in zebrafish can localise the postsynaptic scaffold protein Psd95 to myelin sheaths, and that myelination can be dysregulated by interfering with molecules involved in synapse organisation (Hughes and Appel, 2019; Supplementary Table 1), also seen in mammals (Elazar et al., 2019). In addition to direct signalling between axons and myelinating processes, recent studies have also shown that neuronal activity can influence myelination indirectly, including through regulation of endothelin signalling from the vasculature, which influences myelin sheath production by oligodendrocytes in an Endothelin receptor B dependent manner in both fish and mice (Swire et al., 2019). Future studies will be required to elucidate the potentially multiple activity related axonal signals that influence myelination and the receptors on myelinating processes or on intermediate cells that mediate these effects.

Although the mechanisms of activity-regulated myelination remain to be fully disentangled, two parallel studies in zebrafish indicated that they may influence specific codes of Ca2+ activity in myelin sheaths, that in turn affect myelination. By live imaging individual myelin sheaths, distinct modes of Ca2+ activity were found to prefigure either myelin sheath retractions or myelin sheath elongation (Baraban et al., 2018; Krasnow et al., 2018). Global inhibition of neuronal activity indicated that roughly half of the calcium transients in myelin sheaths were regulated by neuronal firing (Krasnow et al., 2018). A causal role for localised myelin Ca2+ in mediating myelination was evidenced by the finding that localised Ca2+ dependent Calpain protease activity was responsible for driving myelin sheath retractions, Baraban et al. (2018) and that manipulating free Ca2+ levels influenced sheath elongation (Krasnow et al., 2018). How the firing patterns of individual neurons influences distinct Ca2+ activities in myelin sheaths remains to be determined, but is likely to be experimentally tractable in zebrafish by combining optogenetic control of neuronal activity with analyses of corresponding myelin responses. Another mechanism that has been proposed to influence myelination downstream of neuronal activity is that of the local translation of proteins, including myelin structural proteins such as myelin basic protein at the axon-myelin interface (Wake et al., 2011). It has been known for some time that oligodendrocytes transport mRNAs encoding myelin proteins to their distal myelinating processes (Colman et al., 1982), and a forward genetic screen in zebrafish revealed that such mRNA transport in oligodendrocytes is dependent on the kinesin motor protein Kif1b (Lyons et al., 2009). A more recent study has shown that neuronal activity can affect the regulation of mRNAs within myelin sheaths in zebrafish, and that specific 3′UTR elements in mbp mRNA regulate its localisation and local translation (Torvund-Jensen et al., 2018). To further investigate the mechanisms that regulate mRNA localisation to myelin sheaths, Yergert et al. (2019) tracked mRNAs as they were transported in vivo in the zebrafish. In doing so, distinct 3′UTR elements were identified which direct mRNA to areas of active sheath growth. This study also showed that additional mRNAs have conserved 3′UTR sequences, which appear to drive their localisation to myelin sheaths, including the mRNA encoding the RNA binding fragile X mental retardation protein FMRP, fmr1 (Yergert et al., 2019). The importance of fmr1 in directing sheath growth has also been demonstrated in zebrafish, whereby oligodendrocytes lacking fmr1 have shorter myelin sheaths (Doll et al., 2020), a phenotype that will be important to pursue in the context of understanding circuit function and relevance to developmental disorders.

In addition to mechanisms potentially related to activity-regulated myelination, studies in zebrafish have also provided insight into fundamental cell biology of myelination by oligodendrocyte in the CNS. Live imaging studies in zebrafish placed actin as a major regulator of sheath wrapping, as in mammals (Nawaz et al., 2015; Zuchero et al., 2015), and more recently this has been shown to be influenced by the p21-activated kinase 1 (Pak1) in zebrafish (Brown et al., 2021). In parallel to its role in influencing myelin targeting, oligodendrocyte Neurofascin has been shown to support the growth of myelin sheaths along axons, likely through its interaction with axonal Caspr (Klingseisen et al., 2019). In addition to the regulation of the growth of individual myelin sheaths, the total amount of myelin made by oligodendrocytes is an important factor, and one that has been shown to be regulated by the akt-mtor pathway (Mathews and Appel, 2016a), under the influence of the ubiquitin ligase Fbxw7 (Kearns et al., 2015), a factor that when absent can lead to Schwann cells in the peripheral nervous system myelinating multiple axons like oligodendrocyte through an unknown mechanism independent of its control of Mtor (Harty et al., 2019). Perhaps surprisingly, it remains unclear how the overall production of myelin is regulated over time, an area important for investigation in the future.