Chapter 2 - Dissecting Mechanisms of Myelinated Axon Formation Using Zebrafish
Introduction
The myelin sheath is a plasma membrane extension of specialized glial cells that is “wrapped” around axons. The glial cells that make myelin are called Schwann cells in the peripheral nervous system (PNS) and oligodendrocytes in the central nervous system (CNS) (Fig. 1) (Sherman and Brophy, 2005). A single segment of myelin is called an internode, and, as the name suggests, consecutive internodes are flanked on both sides by nodes (of Ranvier), which are short unmyelinated gaps along the length of the axon where voltage-gated sodium channels are clustered and where the saltatory action potential is propagated (Sherman and Brophy, 2005). The presence of myelin sheaths along axons facilitates rapid, energy-efficient transmission of electrical impulses over long distances, and the unique properties of the myelin sheath and myelinated axons have facilitated the evolution of large and complex nervous systems such as our own (Hartline and Colman, 2007). Disruption to the myelin sheath or myelinated axons, therefore, is extremely detrimental and contributes to numerous neurological disorders and human diseases including multiple sclerosis (MS) (Franklin and ffrench-Constant, 2008) and Charcot–Marie–Tooth neuropathies (Shy, 2004). Demyelinated axons are prone to defects in axonal transport and susceptible to axonal degeneration (Edgar and Nave, 2009), phenotypes that often precede cell death in debilitating neurodegenerative diseases such as motor neuron disease, Alzheimer's disease, and Parkinson's disease (De Vos et al., 2008). There is also increasing evidence that the potentially lifelong regulation of myelination through differential neuronal activity might represent an understudied but important form of nervous system functional plasticity (Fields, 2005).
The stereotypical myelinated axon with nodes of Ranvier is a vertebrate-specific adaptation. The fact that invertebrate model organisms such as Caenorhabditis elegans and Drosophila melanogaster do not possess myelinated axons means that the zebrafish is the simplest model for discovery and functional analysis of genes required for this process. Since the turn of the 21st century, the contribution of zebrafish to this field has become significant, and it now serves as an excellent complement to analyses in higher vertebrate models. This chapter is intended to give an overview of the development of myelinated axons in zebrafish and to review the reagents and techniques currently available for their study.
Schwann cells derive from the neural crest (Jessen et al., 2008), which is specified at the transition between neural ectoderm and nonneural ectoderm during neurulation (Le Douarin and Dupin, 2003, Woodhoo and Sommer, 2008). Once specified, neural crest cells delaminate and migrate throughout the body to generate a multitude of cell types (Le Douarin and Dupin, 2003). A subset of neural crest cells associates with axons during their migration and begins to express markers of the immature Schwann cell precursor stage (Jessen et al., 2008). Fig. 2 illustrates the steps of Schwann cell development. The transcription factor Sox10 is expressed in immature Schwann cells and is required for the transition from the neural crest to immature Schwann cell precursor state (Gilmour et al., 2002). Sox10 is also required for the specification of additional nonectomesenchymal neural crest cell fates, including melanocytes (Dutton et al., 2001). Previous studies of peripheral nerve development in zebrafish have shown that axonal growth cones migrate just ahead of Schwann cells and can direct their migration (Gilmour et al., 2002). In mutants with aberrantly projecting peripheral axons, Schwann cells follow the inappropriate route taken by axons (Gilmour et al., 2002). In a forward genetic screen that aimed to identify mutations that disrupted myelinated axon formation (Pogoda et al., 2006), mutations in the genes that encode the heterodimeric tyrosine kinase receptors ErbB2 and ErbB3 were isolated (Lyons et al., 2005). Documentation of erbb3 expression in Schwann cells, characterization of erbb2 and erbb3 mutant phenotypes, and conditional inactivation of ErbB signaling via a small molecule inhibitor revealed that ErbB signaling is essential for Schwann cell comigration along axons in zebrafish in addition to early proliferation and survival (Lyons et al., 2005). These results are in keeping with knockout studies in mice, where disruption of either erbb2 or erbb3 leads to peripheral nerves that lack Schwann cells (Garratt et al., 2000, Riethmacher et al., 1997).
After the completion of migration, Schwann cells undergo proliferation to regulate their number with respect to axons, which is also regulated by ErbB signaling (Lyons et al., 2005). During this time, individual Schwann cells extend processes into axon bundles and associate with just one axon (Fig. 2) (Jessen et al., 2008). This process, called radial sorting, has been extensively studied in mammals (Chan, 2007), and studies in zebrafish show that both proliferation and ErbB signaling are essential for its completion (Raphael et al., 2011). After radial sorting is complete, Schwann cells initiate myelination. The onset and extent of myelination in the PNS is related to axonal thickness (caliber), such that axons that have a cross-sectional area greater than ∼1 μm2 tend to be myelinated and the amount of myelin surrounding an axon correlates positively with caliber (Jessen et al., 2008, Nave and Salzer, 2006, Sherman and Brophy, 2005). Studies in mammals have shown that large caliber myelinated axons tend to express high levels of the growth factor neuregulin 1 type III (Nrg1-III) (Taveggia et al., 2005), an ErbB receptor ligand (Nave and Salzer, 2006), whereas smaller axons that are normally not myelinated express less Nrg1-III (Taveggia et al., 2005). Remarkably, overexpression of Nrg1-III in small-caliber axons that are not normally myelinated is sufficient to induce myelination, which suggests that Nrg1-III is a key regulator of the decision to myelinate (Taveggia et al., 2005). Conditional abrogation of ErbB receptor signaling in zebrafish after Schwann cell migration is complete shows that this pathway is also required for myelination in zebrafish (Lyons et al., 2005), although the requirement for Nrg1-III remains to be demonstrated.
The expression and function of key transcription factors during the development of Schwann cells in zebrafish also reflects that of mammals. The transcription factors oct6 and krox20 are expressed in zebrafish Schwann cells (Levavasseur et al., 1998, Pogoda et al., 2006) just prior to the onset of myelination, as in mammals (Blanchard et al., 1996, Ghazvini et al., 2002, Jaegle et al., 2003, Murphy et al., 1996, Topilko et al., 1994, Zorick et al., 1996). The stage when Schwann cells have completed radial sorting but not yet initiated myelination is called the promyelinating stage (Fig. 2). krox20 mutant zebrafish are arrested at the promyelinating stage (Monk et al., 2009), as are krox20 knockout mice (Zorick et al., 1996). Following a forward genetic screen (Pogoda et al., 2006), Monk et al. (2009) identified a novel key regulator of PNS myelination, a member of the adhesion family of G protein–coupled receptors, Gpr126. In gpr126 mutants, Schwann cells do not progress through the promyelinating stage and therefore fail to express myelin-specific genes, such as myelin basic protein (MBP), and fail to initiate myelination. Further characterization showed that gpr126 acts in Schwann cells, upstream of both oct6 and krox20 transcription factors, and that mutant phenotypes can be rescued by forskolin, which elevates cyclic AMP levels, placing gpr126 upstream of the cAMP cascade (Monk et al., 2009). It will be very interesting to identify the Gpr126 ligand and to determine its cellular locus of activity. Following the initiation of myelination, a small number of molecules have been implicated in controlling the extent of myelination including interactions between dDiscs large homolog 1 (Dlg1) and PTEN (Cotter et al., 2010), and Nrg1-III signaling (Michailov et al., 2004).
Oligodendrocytes, in contrast to Schwann cells, are neuroectodermal in origin. The first oligodendrocyte precursor cells (OPCs) arise from the motor neuron progenitor (pMN) domains of the ventral CNS, but at later stages additional sources of oligodendrocytes emerge in both the dorsal spinal cord and forebrain (Richardson et al., 2006). The transcriptional control of oligodendrocyte development has been very well characterized in mammals and has been reviewed extensively elsewhere (Li et al., 2009). In general, the roles of known transcription factors during zebrafish oligodendrocyte development appear well conserved with mammals. The basic helix–loop–helix transcription factor Olig2, for example, is expressed from the early oligodendrocyte progenitor cell stage through migration, differentiation, and myelination, and is essential for the generation of oligodendrocytes in both mammals and zebrafish (Park et al., 2002, Zhou and Anderson, 2002, Zhou et al., 2001). The related transcription factor Olig1 is expressed in cells of the oligodendrocyte lineage at a slightly later stage of differentiation (Schebesta and Serluca, 2009, Zhou and Anderson, 2002) and acts, together with Sox10, to activate the mbp promoter in both fish and mammals (Li et al., 2007). Sox10 mutant mice do not express MBP or myelinate CNS axons (Stolt et al., 2002), and studies in zebrafish suggest that this may be due to induction cell death shortly after the onset of axon–glial contact (Takada et al., 2010). Some differences between fish and mammalian oligodendrocytes do exist, such as the apparent absence of Olig2 binding to Sox10 in fish oligodendrocytes (Li et al., 2007). Furthermore, the major myelin protein, myelin protein zero (MPZ or P0), is expressed in myelinating oligodendrocytes in fish (Brosamle and Halpern, 2002) but not in mammals. The bases of such differences are not yet clear.
In vivo time-lapse microscopy in zebrafish has revealed that OPCs extend dynamic growth cone-like processes during migration. Fig. 3 summarizes the steps of oligodendrocyte differentiation following completion of cellular migration.
Toward the end of migration, cells of the oligodendrocyte lineage withdraw their processes when they contact one another (Kirby et al., 2006), which suggests that repulsive cell–cell interactions might regulate the spacing of oligodendrocytes along axons. Interestingly, when individual cells were ablated, their neighbors moved into the vacant territory until they came into contact with one another, whereupon they again exhibited mutually repulsive behaviors to establish their positions (Kirby et al., 2006). The cellular behavior during the subsequent initiation of myelination, however, remains unclear, as do the molecular bases of axon–oligodendrocyte interactions that might control CNS myelination. Nrg1-III ErbB signaling, for instance, plays a negligible role in CNS myelination (Brinkmann et al., 2008, Taveggia et al., 2008), and Gpr126 also appears superfluous for CNS myelination (Monk et al., 2009). Further molecular analyses in zebrafish are likely to help reveal the genetic basis of CNS myelination.
The compaction of the myelin sheath is mediated by the coordinated expression of myelin-specific proteins and their localization to specific regions in the myelin membrane. Major myelin proteins that are essential for membrane compaction in the PNS and/or CNS include MBP, the proteolipid protein (PLP) and its splice variant DM20 (Baumann and Pham-Dinh, 2001), and MPZ or P0 (Hartline and Colman, 2007) (see Fig. 1). MBP is a small and strongly cationic protein that localizes to the cytoplasmic side of the myelin membrane and is implicated in bringing the intracellular membranes into tight apposition (Boggs, 2006). The shiverer mouse, a naturally occurring mutant in mbp, almost completely lacks compact CNS myelin (Popko et al., 1987, Readhead et al., 1987). The differential targeting of mbp mRNA to distal processes of myelinating glia was documented nearly 30 years ago (Colman et al., 1982), but how or why this occurs remained unknown. Using zebrafish with a mutation in the gene encoding the zebrafish kinesin motor Kif1b, it was found that this motor was required autonomously in oligodendrocytes for the normal localization of mbp mRNA to myelinating oligodendrocyte processes (Lyons et al., 2009). Ultrastructural analyses of myelinated axons in kif1b mutant zebrafish revealed a striking phenotype, namely, the ectopic appearance of myelin-like membrane along part of oligodendrocyte processes not wrapping axons (Lyons et al., 2009). This observation suggested the hypothesis that specific localization of mRNAs (including those that encode MBP) prevents deleterious effects of their protein products elsewhere in the cell. Zebrafish with disruption of the tubulin alpha 8-like 3a gene also exhibit mislocalization of mbp mRNA in oligodendrocytes (Larson et al., 2010), which suggests that this microtubule subunit may be a component of the glial cytoskeleton that mediates the transport of myelin constituents. Further cell-type-specific analyses remain to be carried out to document that this factor functions autonomously in oligodendrocytes during mRNA localization.
The trafficking of essential components of the myelin sheath in vivo is not well characterized, but the zebrafish is poised to make a significant contribution in this area given its amenability to live cell imaging.
The identification of factors required for the correct localization of essential proteins to the node of Ranvier (e.g., voltage-gated sodium channels) has been successfully pursued in mammals (Rosenbluth, 2009, Salzer et al., 2008). Genetic analyses using zebrafish, have, however, also contributed to this important area of research. For example, zebrafish with mutations in the gene encoding N-ethylmaleimide-sensitive factor (NSF, a protein essential for membrane fusion at synaptic termini; Kawasaki et al., 1998, Tolar and Pallanck, 1998) completely fail to cluster voltage-gated sodium channels at nodes of Ranvier (Woods et al., 2006), which suggests that NSF may be required for the fusion of vesicles containing node components to the axonal membrane. Testing this hypothesis would, in part, require the visualization of nodal components in live axons, which should be possible in zebrafish. Another protein with a newly defined role in the formation of normal nodes is the cytoskeletal-associated protein alpha II spectrin (Voas et al., 2007). Alpha II spectrin is expressed at the node and paranodal domains during myelinated axon development (Voas et al., 2007), where it likely interacts with either beta II or beta IV spectrin, as suggested by the localization of these factors in myelinated axons (Susuki and Rasband, 2008, Voas et al., 2007).
To determine the role of Schwann cells in the clustering of nodal components, Voas and colleagues made use of sox10 and erbb mutants that lack Schwann cells along peripheral nerves. Surprisingly, they found that numerous components of mature nodes were capable of coclustering in the complete absence of Schwann cells (Voas et al., 2009). However, gpr126 mutants, which have Schwann cells in association with axons but fail to initiate myelination, also fail to cluster proteins at putative nodes. gpr126 mutants treated with an inhibitor of ErbB signaling to block Schwann cell development can, however, cluster components to the node, which shows that Grp126 is not required for clustering of nodal proteins per se. This suggests that immature Schwann cells disrupt the early formation and clustering of nodal components at inappropriate positions (Voas et al., 2009). These data also suggest that the initiation of myelination is essential for node of Ranvier formation in the PNS.
In the postgenomic era, the genetic bases of numerous human diseases are being elucidated. For example, an intronic SNP in the gene encoding human KIF1B has been associated with susceptibility for MS (Aulchenko et al., 2008), which raised the possibility that analysis of Kif1b function in zebrafish may reveal aspects of disease susceptibility or progression. However, the association reported in the initial genome-wide association study (GWAS) has been called into question (Booth et al., 2010). MS results from a multitude of genetic and environmental factors, each of which is likely to contribute in varying degrees to disease susceptibility (Handel et al., 2010, Oksenberg et al., 2008, Sawcer, 2008). Therefore, mutant zebrafish that disrupt individual genes implicated in MS susceptibility will, most likely, be informative with respect to the basic biological function of the gene but will not serve as general models for such a complex, multifactorial disease. In other cases, where the genetic bases of diseases are purely Mendelian such as in Charcot-Marie-Tooth neuropathies and Leukodystrophies (Shy, 2004, Boespflug-Tanguy et al., 2008), zebrafish models are potentially much more informative. For example, a mutation in the gene encoding zebrafish kinesin-binding protein (KBP; Lyons et al., 2008) may serve as a useful model for certain aspects of the rare human neurological disorder, Goldberg-Shprintzen syndrome, caused by homozygous null mutations in human kbp (Brooks et al., 2005, Murphy et al., 2006).
In conclusion, zebrafish have contributed significantly to our growing understanding of the basis of formation of myelinated axons. In the rest of this chapter, we review some of the available reagents, techniques, and protocols that have facilitated such studies. We also preview new technologies that will ensure that the zebrafish remains a major contributor to future advances in the study of myelin formation, its disruption, and repair.
Section snippets
Raising Animals for Analysis of Myelinated Axons
Numerous factors are important to consider when raising animals for analysis at postembryonic stages (Parichy et al., 2009). The chemical PTU is often used to prevent pigment formation during embryonic development (Wang et al., 2004), but previous studies have found that it can cause developmental defects (Elsalini and Rohr, 2003). As an alternative to PTU, one can instead use mutant zebrafish lines in which pigment formation is disrupted, such as golden, which disrupts a cation exchanger
Summary
The study of myelinated axons in zebrafish is a growing field. To date the most significant contributions from zebrafish research have arisen from two of the main strengths of the system, namely, high-resolution imaging of cell behavior in vivo and forward genetic screens. Given current advances in microscopy and the growing number of reagents to visualize cells, subcellular structures, and molecules in vivo, we expect live imaging in zebrafish to be at the forefront of efforts to better
Acknowledgments
We would like to acknowledge the work of Drs. Kelly Monk, Matt Voas, and Joann Buchanan, who contributed to the development of the protocol that allows visualization of zebrafish myelin by TEM. We thank Dr. Robert Hartley and Theo Hirst for deconvolving confocal images. We would like to acknowledge Prof. Charles ffrench-Constant for support, and we are grateful to the Euan MacDonald Centre for Motor Neurone Disease Research and Crerar Hotels for providing free access to a Zeiss LSM 710 confocal
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