1 zebrafish myelinatio a transparent model for remyelination 脱髓鞘

斑马鱼髓鞘化：髓鞘为透明模式？

DMM

Disease Models & Mechanisms1, 221-228 (2008) doi:10.1242/dmm.001248

PERSPECTIVE

Zebrafish myelination: a transparent model forremyelination?

Clare E. Buckley1, Paul Goldsmith2and Robin J. M. Franklin1,3,*

There is currently an unmet need for a therapy that promotes the regenerative process of remyelination in centralnervous system diseases, notably multiple sclerosis (MS). A high-throughput model is, therefore, required to screenpotential therapeutic drugs and to refine genomic and proteomic data from MS lesions. Here, we review the value ofthe zebrafish (Danio rerio) larva as a model of the developmental process of myelination, describing the powerfulapplications of zebrafish for genetic manipulation and genetic screens, as well as some of the exciting imaging

capabilities of this model. Finally, we discuss how a model of zebrafish myelination can be used as a high-throughputscreening model to predict the effect of compounds on remyelination. We conclude that zebrafish provide a highlyversatile myelination model. As more complex transgenic zebrafish lines are developed, it might soon be possible tovisualise myelination, or even remyelination, in real time. However, experimental outputs must be designed carefully forsuch visual and temporal techniques.

INTRODUCTION

Multiple sclerosis (MS) is one of the most common diseases of thehuman central nervous system (CNS), affecting over 1.1 millionpeople worldwide (Zamvil and Steinman, 2003). There are bimodalpeaks in disease prevalence; the highest number of cases occurs inyoung adults and middle-aged people, but MS can occur at anyage.It is an inflammatory autoimmune disease that is initiated bya combination of genetic susceptibility and environmental triggersthat cause myelin sheath breakdown through recurrent immuneattacks on the CNS (Compston and Coles, 2002). This myelinbreakdown is probably one of the reasons why axons eventuallydegenerate, causing most of the disability associated with theprogressive stages of MS. There are two broad ways in which MScould be therapeutically targeted. First, the inflammatory immuneresponse could be suppressed. This is the basis of current MStherapies. The second approach is to attempt to halt diseaseprogression by developing therapies aimed at maintaining axonalsurvival, for example, by promoting the process of remyelination(Franklin and ffrench-Constant, 2008). Zebrafish larvae couldprovide a high-throughput in vivo vertebrate model for testingpotential remyelination therapies because of their small size,external development, transparency and homology withmammalian myelin biology.

PROMOTING REMYELINATION IS AN IMPORTANT OBJECTIVE FORFUTURE MS THERAPY

Remyelination occurs naturally in the early stages of MS but oftenfails during the later progressive phases. Therefore, in order to

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combat MS effectively, there is a need to devise therapies that canpromote the regeneration of myelin sheaths around demyelinatedaxons in the CNS (remyelination; see Box 1) (Dubois-Dalcq et al.,2005; Dubois-Dalcq et al., 2008; Franklin and ffrench-Constant,2008; Irvine and Blakemore, 2008).

Several anti-inflammatory and immunomodulatory therapiesare currently in routine clinical use. However, these are only partlyeffective and may not have a significant effect on overall diseaseprogression (DeAngelis and Lublin, 2008b). New therapies arebeing developed, including novel immunomodulatory agents thatare aimed at inhibiting specific aspects of the immune system,thereby reducing the autoimmune attack on oligodendrocytes andmyelin. For example, alemtuzumab is an antibody that targetslymphocytes and monocytes and has generated promising resultsin clinical trials (Coles et al., 2008). These new therapies arereviewed elsewhere (DeAngelis and Lublin, 2008a; DeAngelis andLublin, 2008b; Kieseier et al., 2007).

Although these therapies might help to reduce initialinflammation and/or the autoimmune attack on myelin, andtherefore reduce the number of relapses in early stages of MS, nonespecifically target the process of remyelination. In fact, althoughcurrent MS therapies try to reduce the inflammatory response,aspects of this response such as macrophage-mediated removal ofmyelin debris are important in creating an environment thatsupports OPC differentiation, a process which is necessary forremyelination (Foote and Blakemore, 2005; Kotter et al., 2006; Setzuet al., 2006).

REMYELINATION THERAPIES – WHERE TO START?

The regenerative process of remyelination occurs in a similar wayto the developmental process of myelination, proceeding throughthe processes of OPC activation, proliferation, recruitment anddifferentiation (Box 1) (Franklin and ffrench-Constant, 2008; Millerand Mi, 2007). Therefore, remyelination in MS may fail eitherbecause no OPCs are present in the lesion, or because the OPCs

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Department of Veterinary Medicine, University of Cambridge, Cambridge,CB30ES, UK2

Department of Neurology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP,UK3

Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, CB2 0PY,UK

*Author for correspondence (e-mail: rjf1000@cam.ac.uk)Disease Models & Mechanisms

斑马鱼髓鞘化：髓鞘为透明模式？

Zebrafish myelination: a model

Box 1. Myelin, myelination and remyelination

Myelin is an insulating membranous sheath, made up of around 70% lipid and30% protein. It is produced by myelinating oligodendrocyte cells in the CNSand by Schwann cells in the peripheral nervous system (PNS). Myelin

surrounds nerve axons, allowing saltatory nerve conduction and maintenanceof the axon at a long distance from the cell body (Griffiths et al., 1998; Lappe-Siefke et al., 2003; Nave and Trapp, 2008).

In the CNS, myelinating oligodendrocytes develop from a population ofcells called oligodendrocyte precursor cells (OPCs). These cells are sometimesreferred to as oligodendrocyte progenitor cells. During development, OPCs arenormally restricted to an oligodendrocyte fate; however, recent evidencesuggests that adult OPCs are multipotent, especially following injury.

Consequently, it has been suggested that the adult OPC should be regarded asan ‘adult neural precursor cell’ (reviewed by Zawadzka and Franklin, 2007;Zhao et al., 2008). For the sake of simplicity, we will retain the term OPC in thisreview. When OPCs are activated, they proliferate and are recruited to

unmyelinated (development) or demyelinated (regeneration) axons. As theOPCs differentiate, they extend multiple processes, each wrapping an axon.When the OPCs have fully differentiated into myelinating oligodendrocytes,these processes compact to form myelin sheaths around the axons.

Although the processes of developmental myelination and regenerativeremyelination are very similar, differences do occur. One important distinctionis that remyelination occurs in a pathological environment, whereasmyelination occurs in a normal environment (Franklin, 2002b). However,

spontaneous remyelination in response to injury has been demonstrated andshown to correlate with functional recovery of axons and axonal preservation(Irvine and Blakemore, 2008; Kornek et al., 2000; Liebetanz and Merkler, 2006).This suggests that the function of the remyelination sheaths is essentiallysimilar to that of the myelin sheaths of myelination.

present are unable to differentiate into myelin-producingoligodendrocytes. By studying circumstances where remyelinationis impaired, such as in older animals, it appears that both OPC

recruitment and differentiation are important in maintainingremyelination efficiency. OPC colonisation of demyelinated lesions(recruitment) was delayed in older rats following ethidium bromide(EB)-induced demyelination, as was the time interval betweenequivalent OPC marker expression and differentiatedoligodendrocyte marker expression (differentiation) (Sim et al.,2002). However, when the process of OPC recruitment intodemyelinated focal lesions was artificially increased byoverexpressing platelet-derived growth factor A (PDGF-A) intransgenic mice, there was no change in remyelination efficiency(Woodruff et al., 2004). A recent study also illustrated that in oldermice brains, recruitment of histone deacetylase 1 (HDAC1) and,therefore, the ability to downregulate oligodendrocytedifferentiation inhibitors is reduced, causing remyelinationimpairment. In further support of this result, defectiveremyelination and oligodendrocyte differentiation were induced invivo and in vitro, respectively, by the application of HDAC inhibitors(Shen et al., 2008). These studies suggest that, although bothprocesses become less efficient in older animals, the failure of OPCdifferentiation, rather than that of recruitment, might bepredominantly responsible for inhibiting remyelination.

This conclusion mirrors clinical findings, where it is commonto see MS plaques containing OPCs with no evidence ofremyelination (Chang et al., 2000; Chang et al., 2002; Kuhlmannet al., 2008; Wolswijk, 2002). Therefore, potential endogenousremyelination therapies, and consequently remyelination screeningmodels, must concentrate on manipulating the processes of OPC

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recruitment and, in particular, OPC differentiation into myelinatingoligodendrocytes (Dubois-Dalcq et al., 2005; Franklin and ffrench-Constant, 2008).

Transplantation of myelinating cells is one approach fortherapeutic remyelination. There are many studies showing repairof focal lesions by direct cell transplantation (Franklin, 2002a).Recent studies have also shown amelioration of experimentalautoimmune encephalomyelitis (EAE) using systemic celltransplantation (Einstein et al., 2006; Pluchino et al., 2003). However,the findings from the EAE studies are thought to be the result ofneural stem cell immunosuppression of T cells, rather than anincreased capacity for remyelination (Einstein et al., 2007; Pluchinoet al., 2005). Tissue incompatibility and cell delivery problems alsomake cell transplantation a challenging option. Therefore, a moreattractive approach for promoting remyelination is throughpharmacological promotion of endogenous remyelination.

The signalling systems that are responsible for remyelination arecomplex (for a review, see Franklin and ffrench-Constant, 2008).This makes elucidation of tractable remyelination targets difficult,especially when extrapolating from in vitro to in vivo models. Themost widely used in vivo model of MS is EAE, in which theprocesses of demyelination and remyelination occursimultaneously. As the inflammatory process of EAE is damagingto oligodendrocytes, this lack of temporal separation makes itchallenging to distinguish between an effect that is enhancingremyelination and one that is ameliorating the inflammatoryresponse to EAE and, therefore, enabling the normal process ofremyelination to occur. Some types of EAE cause inflammatorydisease without extensive demyelination and so do not provideeasily interpretable models of the neurobiological aspects of MS.The benefits and drawbacks of using EAE models to recapitulateMS are reviewed elsewhere (Altmann and Boyton, 2004; Dubois-Dalcq et al., 2005; Friese et al., 2006; Gold et al., 2006).

More reductionist in vivorodentmodels based on the use oftoxins such as EB and lysolethicin allow a clear temporal separationbetween the myelin insult and its subsequent regeneration.However, a higher throughput system would enable rapid drugscreening for potential remyelination enhancement. Ideally, alogical hierarchy of models could be built, starting with a high-throughput model to identify potential therapeutic agents. In vitrocell culture models are not appropriate for studying myelinationunless axons are present. Recently, in vitro myelinating co-culturesof dorsal root ganglion (DRG) neurons and oligodendrocytes havebeen used to investigate processes such as the effect of growthfactors on myelination (Chan et al., 2004; Wang et al., 2007c). Thissystem allows for specific investigation of myelination byoligodendrocytes in the absence of any other cell types such asastrocytes. However, myelinating co-culture systems are time-consuming to set up, with each experiment taking several weeks,and are therefore not amenable to high-throughput screeningprojects. It is also technically challenging to carry out manipulationssuch as gene knockdown in vitro. Zebrafish can potentially fulfilthe requirement for a high-throughput myelination model,combining the speed of an in vitro model with the context of anin vivo vertebrate system (Fig. 1, discussed later), and are very easyto genetically manipulate. Zebrafish myelination can be assessedafter only a few days postfertilisation. However, owing to the manycell types present in a whole animal, interpretation of results might

斑马鱼髓鞘化：髓鞘为透明模式？

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Zebrafish myelination: a model

PERSPECTIVE

Fig. 1. Zebrafish myelination: high-throughput drug screening.

(A)Anatomy of a transparent 3 d.p.f. zebrafish larva. (B) Several zebrafish larvaewere placed in each well of a 96-well plate, with different drugs in each well.(C)Live sagittal image of a 3 d.p.f. olig2:EGPF zebrafish larva spinal cord.Dorsally migrated cells were counted in response to different drugs. Greendots in the middle of the magnified region indicate some of these cells.Examples of fainter, elongated migrating cells can be seen below the greenstar. The green arrow indicates a cell on the border of the pMN. (D)An

automated system was used to count olig2-positive cells. Larvae (the arrowindicates one larva) were aspirated in methylcellulose into a capillary tube. Thecapillary was inserted through a water chamber (blue square) to reducerefraction of light, then anchored onto a movable stage (arrowhead) andremotely positioned until the larvae were orientated sagittally above aninverted microscope (outer circle). A z-stack of images was taken and

combined into one collapsed image. The number of dorsally migrated olig2-positive cells was automatically counted. (E)Mbp immunohistochemistry of atransverse hindbrain section from a 5 d.p.f. zebrafish larva. Arrows indicate themedial longitudinal fascicle and arrowheads indicate the ventral commissure.(F) Electron micrograph of a transverse spinal cord section from a 10 d.p.f.zebrafish larva. Arrows indicate the large Mauthner axons. These aresurrounded ventrally by axons with a smaller diameter.

be more challenging than in the co-culture system. The most usefulposition for the zebrafish model in a hierarchy of myelinationmodels might therefore be alongside in vitromyelinating co-cultures, and as an alternative to myelinating slice cultures(Notterpek et al., 1993).

Disease Models & Mechanisms

ZEBRAFISH MODELS: APPLICATIONS FOR UNDERSTANDINGMYELINATION

Zebrafish provide an ideal in vivo model for high-throughputexperiments. Their larvae are very small (only a few millimetreslong) and transparent (Fig. 1A). Embryogenesis occurs ex vivo andis complete by 3 days post fertilisation (d.p.f.) (Berger and Currie,2007; Kimmel, 1989; Kimmel et al., 1995). This allows for easyphenotypic assessment. Each pair of fish produces approximately100 to 300 embryos per week and maintenance costs are 1000-foldlower than for mice(Goldsmith and Solari, 2003; Kari et al., 2007).

Zebrafish and mammalian myelin: a comparison

The molecular and cellular organisation of zebrafish is remarkablysimilar to that of humans, and homologues for most human genescan be found and studied in zebrafish (Barbazuk et al., 2000;Postlethwait et al., 2000). However, owing to genome duplicationafter the divergence of the tetrapods, there are often severalzebrafish genes for each mammalian gene, as in the case for thegenes encoding one of the major myelin-associated proteins,proteolipid protein (Plp), and its splice variant Dm20(Schweitzeret al., 2006). The genetic sequences of the major myelin proteinsare also diverse among different species offish, whereas they areoften highly conserved among mammals (Geltner et al., 1998). Inaddition, there are physiological differences between zebrafish andmammals (reviewed by Lieschke and Currie, 2007).

The major biochemical difference between zebrafish andmammalian myelin is the presence of protein zero (P0) as a majorCNS myelin protein in zebrafish, rather than PLP in mammals(Table 1) (Jeserich et al., 2008; Waehneldt et al., 1986). Thissuggests an evolutionary neuroprotective change in myelinchemistry between aquatic and terrestrial vertebrates, which issupported by the degenerative phenotype observed in transgenicmice that express P0instead of Plpin their CNS (Yin et al., 2006).Although there is sequence conservation between zebrafish andmammalian P0(Schweitzer et al., 2003), the zebrafish p0gene showsgreater promoter region sequence conservation with themammalian Plpgene rather than mammalian P0(Jeserich et al.,2008; Jeserich et al., 1997). In keeping with this, a zebrafishtransgenic line, which expresses enhanced green fluorescent protein(EGFP) under the regulation of a mouse Plppromoter, exhibitsstrong EGFP expression in oligodendrocytes and their precursors,although it is not clear whether this promoter regulates zebrafishdm20or p0genes (Yoshida and Macklin, 2005). This suggeststhatthere is not a clear biochemical distinction betweenoligodendrocytes and Schwann cells in the zebrafish (Jeserich etal., 2008).

Nevertheless, the structural properties and cell lineagerelationship of oligodendrocytes is highly comparable betweenzebrafish and mammals (Jeserich et al., 2008; Jeserich andStratmann, 1992; Jeserich and Waehneldt, 1986; Sivron et al., 1990).Also, orthologous genes for all of the major mammalian myelin-associated genes have been found in zebrafish (dm20, mbp andp0)(Brosamle and Halpern, 2002). Although there is some variationbetween zebrafish and mammals in the expression pattern andsequence of these genes, there is enough conservation of predictedprotein properties to suggest that the zebrafish orthologuesfunction in a comparable way to the mammalian proteins (see Table1). Further, the coexpression of all three major myelin-associated

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斑马鱼髓鞘化：髓鞘为透明模式？

Zebrafish myelination: a model

Table 1. Comparison of the major myelin-associated proteins between mammals and zebrafish

Mammals

Zebrafish

PNSNo

CNS

A pair of orthologues ispresent in zebrafish,Dm1 and Dm2;Dm2 is the closesthomolog to themammalian gene;coexpressed inoligodendrocyteswith P0 and MbpYes, major myelincomponent;coexpressed inoligodendrocyteswith Dm20 and P0Yes, prominent

myelination protein;coexpressed inoligodendrocyteswith Dm20 and Mbp

PNSNo

Protein

PLP/DM20

Description

Myelin structural protein,necessary to compactand maintain myelinstructure; highly

hydrophobic tetraspanprotein

CNS

Yes, major myelinsplice variantsdiffering by 35amino acids

Homology between zebrafish

and mammals?

51% of identical amino acidsequences to mouse; fourhydrophobic stretches inzebrafish Dm20 couldcorrespond to the fourmammalian PLP

transmembrane domainsused to compact andmaintain myelin structure40% of identical amino acidsequences to mouse; overallpredicted protein propertiessuggest similar functions46% of identical amino acidsequences to mouse; highestsequence conservation atextracellular Ig-like domain

MBP

Myelin adhesion protein;small cationic molecule

Yes, majormyelin

componentYes, majormyelin

componentYes, majormyelin

component

P0

Cell adhesion

immunoglobulin;member of the Igsuperfamily of

recognition molecules;contains a signalpeptide, single Igdomain,

transmembranesegment and anintracellular domain

No

Yes, majormyelin

component

Yes, but lessprominentand doesnot

function asa myelinadhesionprotein inthe PNS

(References: Brosamle and Halpern, 2002; Jessen and Richardson, 2001; Schweitzer et al., 2003; Schweitzer et al., 2006.

genes in zebrafish oligodendrocytes precedes the appearance ofcompact myelin in the zebrafish brain by about 2 days, providingfurther evidence for a comparable function in myelination(Brosamle and Halpern, 2002).

Feasible genetic manipulation and analysis

The zebrafish genome sequence is nearing completion and mostgenetic manipulation techniques can be carried out efficiently inzebrafish owing to the high number of offspring that can beproduced, the transparency of larval stages and their fast, externalembryonic development. Although some individual geneticmanipulation techniques may not be more rapid than in otherorganisms, zebrafish are superior in efficiency and ease oftechnique.

External embryonic development enables straightforwardmicroinjection technique. This allows transgenic lines to begenerated through the injection of large insert clones such as P1artificial chromosomes (PACs) or bacterial artificial chromosomes(BACs) (Lee et al., 2001; Shin et al., 2003). The recent developmentof the Tol2 and Sleeping Beauty (SB) transposon systems allowsanother method of transgenic generation and produces muchhigher germline transmission efficiencies (Kawakami, 2005). Thedevelopment of the GAL4-UAS system in zebrafish is especiallyversatile as it allows gene expression to be targeted both spatiallyand temporally (Scheer and Campos-Ortega, 1999; Scheer et al.,2001). Homologous recombination of DNA into embryonic stemcells and the subsequent transplantation of these cells into anembryo has allowed targeted gene deactivation in mice and chicks.So far, it has not been possible to knockout a specific zebrafishgene in this way because zebrafish embryonic stem cell cultures

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were not able to contribute to germ cell lineage. However, recentattempts at generating gene knockout zebrafish are promising (Maet al., 2001; Wang et al., 2007b) and the development of thesetechniques will make the zebrafish even more tractable as anexperimental model. In the meantime, insertional mutagenesis inzebrafish is possible through transposon or retroviral systems(Ellingsen et al., 2005; Sivasubbu et al., 2007; Wang et al., 2007a),and target-induced local lesions in genomes (TILLING) (McCallumet al., 2000) have allowed the production of gene-specific mutations(Wienholds et al., 2002). Zebrafish embryos also allow fastinvestigation of in vivo gene function at early embryonic stagesthrough RNA overexpression and morpholino gene knockdownexperiments, because transgenic or mutant generation is notnecessary, and gene alteration occurs within a few hours ofmicroinjection (Nasevicius and Ekker, 2000). For example, in amorpholino study, hdac1was shown to be essential in allowing theexpression of oligodendrocyte-lineage-specific genes, such as olig2and sox10, whilst suppressing the expression of neural progenitordeterminants such as nkx2.2 and her6(Cunliffe and Casaccia-Bonnefil, 2006).

High mutagenesis rates can be achieved in zebrafish because oftheir resistance to N-ethyl-N-nitrosourea (ENU) toxicity andconsequent survival of mutated embryos. The first use of thismethod of mutagenesis in zebrafish was in genome-wide forwardgenetic screens (Driever et al., 1996; Haffter et al., 1996; Lieschkeand Currie, 2007). For example, screening for defects in myelin basicprotein (Mbp) expression demonstrated that N-ethylmaleimidesensitive factor (Nsf), which is essential for membrane fusion(Wilson et al., 1989), also plays a role in myelination and theorganisation of the nodes of Ranvier(Woods et al., 2006). Similar

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Zebrafish myelination: a model

PERSPECTIVE

Fig. 2. A hierarchy for identifying remyelination-enhancing therapies.Here, we indicate where zebrafish fit among other experimental models. Invitro myelinating co-cultures and in vivo zebrafish larval models ofmyelination should be used alongside each other to efficiently refinecandidate drugs or genes of interest. These should then be investigatedfurther in vivo in mammalian systems before moving to clinical trials. Thishierarchy provides a resource-effective method of developing a therapy thattargets remyelination.

forward genetic screens have identified several other myelin-related genes (Kazakova et al., 2006; Pogoda et al., 2006).

Imaging techniques

Labelling and imaging techniques can be carried out in wholezebrafish larvae owing to their small size. For example, whole-mount immunohistochemistry (IHC) staining of zebrafish axonswith myelin, axonal and nodal markers was used to determinesodium channel disruption in response to mutations (Voas et al.,2007; Woods et al., 2006). Such clear staining of nodes could alsobe used as a way of defining whether an axon is myelinated or not,as nodes are only formed in myelinated axons. In vivoretrogradefluorescent labelling of individual neurons is another way in whichneurons can be visualised in whole larvae. This can be performedjust 5-10 hours after injection of a fluorescent dye, such as dextran,into the spinal cord (Gahtan and O’Malley, 2001; Gahtan andO’Malley, 2003; Hale et al., 2001). However, the damage caused tothe axons through this procedure may confound results (Gahtanand O’Malley, 2001). The ability to trace the lineage, and fate, ofindividual cells through fluorescent labelling in vivo is possibleinzebrafish larvae. For example, the establishment of normal sodiumchannel clusters in α-spectrinmutant zebrafish larvae, whichwould otherwise have abnormal clustering, was achieved bytransferring dextran-labelled wild-type cells into the mutants (Voaset al., 2007). This technology could have potential applications inmany fields, such as cancer research (Lee et al., 2005).

One of the most dramatic imaging methods used in zebrafish isfluorescence transgenesis (Gong et al., 2001; Udvadia and Linney,2003). There are several transgenic zebrafish lines that are directlyrelevant for studying myelin biology, including the plp:EGPF andthe olig2:EGFP lines, both of which express EGFP in cells belonging

Disease Models & Mechanisms

to the oligodendrocyte lineage. The olig2:EGFP transgenic line hasbeen used as an oligodendrocyte and neuronal marker to investigateseveral myelination-related pathways; for example, the control ofcell cycle exit, primary neurogenesis and maintenance of precursorpopulations by Delta-Notch was shown to occur through theregulation of cyclin-dependent kinase inhibitor 1C(cdkn1c)expression, which is itself necessary for oligodendrocytespecification (Park et al., 2005). These experiments used Notchmutants, inducible constitutive expressers of Notchand cdkn1cmorpholinos in combination with IHC co-labelling and in situstudies. Clonal analysis of olig2:EGFP motor neuron precursordomain (pMN) cells has also been used to show that, rather thatbeing binary, olig2precursors can produce several types ofinterneuron as well as motor neurons and oligodendrocytes. Thefate of these olig2precursors is under the control of Hedgehogsignalling (Park et al., 2004).

More relevant to remyelination, olig2:EGFP larvae were used withanother transgenic line that labels a subset of OPCs, nkx2.2a:EGFP,to illustrate the density-dependent regulation of oligodendrocytemigration and division in response to the laser ablation of OPCs.The extension and retraction of filopodium-like processes wasimaged through real-time in vivo movies (Kirby et al., 2006). Thesame model could be used to determine the time it takes for OPCsto repopulate a lesioned area in response to mutation or drugtreatment, therefore, providing a measurement of OPC recruitmentduring remyelination. However, laser ablation might cause non-specific damage to the tissue surrounding the OPCs.

REMYELINATION SCREENING USING ZEBRAFISH MODELS

The examples discussed above illustrate the tractability of zebrafishmodels in the elucidation of a wide range of oligodendrocyte- andmyelination-related pathways. If such flexibility in an animal systemcan be harnessed for screening purposes, it has the potential fornot only screening the effects of drugs, but also clarifying the greatquantities of genomic and proteomic information generated byrecent microarray studies (Arnett et al., 2003; Han et al., 2008; Locket al., 2002). The key to a successful screen for remyelination is touse an appropriate model at each screening stage. The use of larval(rather than adult) stages of the zebrafish allows a rapid andrelatively low-cost method of in vivo vertebrate analysis. Manysuccessful high-throughput zebrafish larvae screens have beenestablished, both for forward genetic screens and for identifyingand optimising lead drugs (for reviews, see Berger and Currie, 2007;Kari et al., 2007; Lieschke and Currie, 2007; Rubinstein, 2003).Using a model of myelination rather than remyelination allowsfor much faster screening because it does not require myelin injuryand can be assessed at a single time point. For example, Fig. 1illustrates the methods we used to screen drug libraries for theirpotential enhancement of myelination. olig2:EGFP transgenic larvaewere placed into 96-well plates and treated with different drugsduring oligodendrocyte specification (from 1 d.p.f. to 3 d.p.f.) (Fig.1B). The drugs used were either part of reprofiled drug libraries oridentified in the literature as being relevant to myelination.Oligodendrocyte recruitment was assessed in response to each drugby counting the number of olig2-positive cells that had dorsallymigrated from the pMN in the spinal cord (oligodendrocyte-lineage-specific) (Fig. 1C). The olig2:EGFP transgenic line had previously beenused to find a zebrafish gene which increased the number of olig2-225

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Zebrafish myelination: a model

positive cells (Bruce Appel, personal communication). Therefore, weattempted to find a drug that replicated this result. An automated

counting system was developed that allowed us to screenapproximately 80 drugs per week (Fig. 1D). Drugs that produced aconcentration-dependent change in olig2-positivecell number weretested for their effects on OPC differentiation by measuring relativelevels of larval mbpmRNA using real-time PCR. Drugs that alteredrecruitment and/or differentiation of OPCs are currently beingvalidated through IHC co-labelling with neuron and oligodendrocytemarkers to ensure that their effect is specific to the oligodendrocytelineage. IHC is also being used in combination with electronmicroscopy to assess whether there is a visual effect of these drugson myelin (Fig. 1E,F) (Buckley et al., 2007). Any drugs that specificallyincrease the number of oligodendrocyte lineage cells, withoutdepleting other cell types such as neurons, or that cause an increasein OPC differentiation and/or zebrafish myelination could then betransferred to an appropriate mammalian remyelination model, suchas those described previously (Ibanez et al., 2004; Penderis et al., 2003)(Fig. 2).

Thereare some important critiques for this screening model.First, there was quite a high variation between larvae in thenumbers of dorsally migratedolig2-positive cells. Second, it wasoften difficult to identify which olig2-positive cells to count. Somecells were on the border of the pMN and were, therefore, hard todistinguish from non-oligodendrocyte lineage cells (Fig. 1C). As aresult, it was challenging to automate the counting of these cells.Finally, using microarray technology rather than real-time PCRwould increase the throughput of the differentiation screen,allowing OPC differentiation to be assessed in parallel with OPCrecruitment. This would prevent any compounds that exclusivelyaltered differentiation and not recruitment from being missed.There are also some other caveats of the zebrafish myelinationmodel in general that require consideration. First, high-throughputscreening of drugs is usually accomplished by adding them to themedium in which the larvae swim. Although zebrafish embryosare permeable to small molecules and drugs during organogenesis(Kari et al., 2007), some potentially therapeutic drugs might bemissed owing to a lack of penetration. Second, zebrafish larvae areused in the techniques described here, rather than adults. In otherspecies there is a decrease in remyelination efficiency in olderanimals, which is associated with a decrease in the processes ofrecruitment and differentiation of OPCs, as discussed earlier (Simet al., 2002; Woodruff et al., 2004) (for a review about the effectsof ageing on remyelination, see Rist and Franklin, 2008). Finally,although using a model of the developmental process of myelinationrather than the regenerative process of remyelination allows formuch faster screening, there are differences between myelinationand remyelination (see Box 1).

It is possible that a zebrafish model of remyelination could bedeveloped through the use of laser ablation or expression of therecently developed cyan fluorescent protein and nitroreductase(NTR) (Curado et al., 2007) in oligodendrocytes. As more zebrafish-specific markers and fluorescent lines are created, real-timevisualisation of remyelination is a realistic goal. Also, thedevelopment of a transparent adult zebrafish line (White et al.,2008) means that it might soon be possible to visualiseremyelination in vivo in adult fish. However, zebrafish have a highregenerative capacity when compared with mammals.For example,

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their hearts are able to regenerate completely without scarring, aphenomenon that has lead to the discovery that fibroblast growthfactor (Fgf) and platelet-derived growth factor (Pdgf) signallingpathways positively influence cardiac regeneration (Lepilina et al.,2006; Lien et al., 2006; Poss, 2007). Spinal cord neurons are alsoable to regenerate after spinal cord transection (Becker et al., 1997).Similarly, OPC recruitment in response to injury in zebrafish larvaeoccurs rapidly following a lesion (Kirby et al., 2006). Although anin vivo real-time remyelination model is an exciting concept, itmight be challenging to quantifiably improve this already efficientprocess. If such a visual and temporal technique is used as a screenfor pro-myelination mutations or treatments, a lot of thought mustbe put into designing a realistic output.

CONCLUSION

Zebrafish provide a highly versatile model, both genetically andexperimentally, and their myelination biology is homologous to themammalian system. Therefore, they are very useful models forexploring the process of developmental myelination. High-throughput drug screens have been carried out looking forenchancers of OPC recruitment and differentiation duringmyelination; the drugs obtained from these screens can be testedin mammals for their potential effects on remyelination. Thus,zebrafish provide a realistic and resource-effective starting pointtowards developing a therapy that may eventually target CNSremyelination in MS. As the techniques for transgenic manipulationprogress, zebrafish have the potential to provide the first real-timein vivo imaging models of myelination and remyelination. However,care must be taken when designing realistic experimental outputsfor zebrafish remyelination models. The real power of zebrafishmay be in modelling the developmental, rather than theregenerative, process.

ACKNOWLEDGEMENTS

We acknowledge the support of Summit plc. Also, thank you to Anita Marguerie,Clare Chappell, Heather Wardle, Peter Munday and Helen Reynolds for their workon the drug library screening. Deposited in PMC for immediate PETING INTERESTS

The authors declare no competing financial interests.

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DMM

Zebrafish myelination: a model

PERSPECTIVE

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