Peripheral axons of the adult zebrafish maxillary barbel extensively remyelinate during sensory appendage regeneration

Myelination is a cellular adaptation of vertebrate nervous systems allowing rapid electrical conduction along axons. Myelination occurs in both the central nervous system (CNS) and the peripheral nervous system (PNS) when glial cells—oligodendrocytes and Schwann cells, respectively—wrap thin cytoplasmic processes around developing axons. Although oligodendrocytes and Schwann cells establish myelin during embryogenesis and maintain it during maturity, these cells are also studied for their ability to inhibit or enhance myelin repair. In mammals, oligodendrocytes inhibit CNS remyelination (Chen et al., 2002), whereas Schwann cells promote PNS remyelination (Gordon and Gordon, 2010; Hall, 1989). Strategies to enhance myelination include promoting the endogenous repair mechanisms of Schwann cells in the PNS, or introducing Schwann cells into the injured CNS (for review see Stangel and Hartung, 2002). Understanding the repertoire of Schwann cell behavior in multiple contexts is thus important for both CNS and PNS myelination. Although much has been learned about the molecular basis of remyelination, many regulatory processes remain to be studied. The zebrafish is a popular species in which to investigate these processes, many of which are evolutionarily conserved (Avila et al., 2007; Buckley et al., 2008; Dubois-Dalcq et al., 2008). Zebrafish axons are myelinated by the same glial cell types as in mammals (Buckley et al., 2008; Jeserich et al., 2008), and the embryonic origins and migrations of these cell types are understood in detail (Woodhoo and Sommer, 2008). The spatial and temporal expression of myelin in early embryos and larvae has been mapped (Br€osamle and Halpern, 2002; Buckley et al., 2010), and the molecular similarities and differences between zebrafish and mammalian myelin are increasingly well characterized (Avila et al., 2007; Bai et al., 2011; Buckley et al., 2008; Jeserich et al., 2008; Schaefer and Br€osamle, 2009). Although there are species-specific differences in myelin composition, the function of myelin is highly conserved, as is the network of transcription factors regulating glial cell differentiation and myelin production (Levavasseur et al., 1998; Li et al., 2007). Recently, zebrafish mutagenesis screens have identified specific genes required for myelinating the CNS and/or PNS (Kazakova et al., 2006; Larson et al., 2010; Lyons et al., 2005; Monk and Talbot, 2009; Pogoda et al., 2006), for developing oligodendrocytes (Takada and Appel, 2010; Zhao et al., 2010), and for establishing the nodes of Ranvier (Woods et al., 2006). Transgenic lines expressing fluorescent proteins allow zebrafish axons (Goldman et al., 2001), oligodendrocyte lineages (Park et al., 2007; Yoshida and Macklin, 2005), and/or transcripts of myelin basic protein (Jung et al., 2010) to be easily observed in vivo. Finally, the rapid embryonic development of zebrafish facilitates whole-organism assays of behaviors, such as larval escape swimming, that depend on fast conduction of action potentials (Fetcho, 2007). Most studies of zebrafish myelination address the embryonic stages, up to 4-5 days postfertilization. Less is known, however, about how myelination is regulated in zebrafish adults. An exception is the zebrafish optic nerve, which, in contrast to mammals, both regenerates and remyelinates after injury (for review see Matsukawa et al., 2004; Nona et al., 1992, 2000). Similarly, the adult zebrafish caudal fin regenerates myelinated axons of the lateral line (Dufourcq et al., 2006). These adult systems are useful for comparing how developmental myelination that occurs in the intact embryo is distinct from remyelination occurring in the context of injury, inflammation, and scarring. The identification of genes that are expressed in adult regenerating tissues, but not in the same tissues embryonically, has led to the notion of “injury-induced molecular programs” with distinct regulatory controls (for review see Kizil et al., 2011). This idea is further supported by experiments in vivo in which embryonically important genes were knocked down, with no effect on adult myelin maintenance or regeneration (Atanasoski et al., 2006; for review see Taveggia et al., 2010). These data suggest that multiple, intersecting molecular pathways regulate myelin at different stages of the life cycle, highlighting the need for further studies of this phenomenon. We have introduced the zebrafish maxillary barbel (ZMB) as a novel system in which to investigate nerve, skin, and vascular regeneration (LeClair and Topczewski, 2009, 2010). Barbels first develop in zebrafish juveniles 30-40 days postfertilization and remain optically transparent throughout the life cycle. There are no intrinsic muscles and few other cell types in the barbel, making the neural architecture of these appendages exceptionally visible. The larger maxillary barbel is innervated by many small-diameter axons descending from the facial nerve (cranial nerve VII). Most of these axons project ventrally to innervate taste buds; other neural cell types in the barbel include solitary chemosensory cells (SCCs) and a diffuse epithelial nerve net. After a proximal amputation in which more than 85% of the maxillary barbel is removed, the appendage regenerates all of these structures within 2-3 weeks at 28°C (Fig. 1; see also Fig. 10 of LeClair and Topczewski, 2010). Interestingly, the central axons of the barbel grow out several millimeters despite extensive scarring of the appendage and a permanent disorganization of mesodermal cells and associated matrix. This makes the ZMB a convenient structure in which to study how peripheral axons navigate the extracellular environment and establish connections to distal sensory targets. Figure 1 Anatomical overview of adult zebrafish maxillary barbel (ZMB) regeneration. A: Morphological sequence of ZMB regeneration from 10 to 28 days postamputation. Proximal amputation (dotted line) induces regeneration of a new appendage (black). Barbel regeneration ... Having previously documented robust regrowth of peripheral axons into regenerating barbel tissue, we wished to determine whether these axons were originally myelinated and whether, after regeneration, myelination was restored. Our experimental goals were 1) to assess the myelination state of adult, developing, and regenerating ZMB axons; 2) to observe the time course of remyelination during barbel regeneration; 3) to determine whether regenerated axons were similar to normal axons in size, fiber type, and myelin thickness; and 4) to test whether regenerating barbel tissue expresses conserved transcription factors, including sox10, pou3f1 (=oct6), and egr2a/b (=krox20a/b), all characteristic of myelinating peripheral Schwann cells (Kawasaki et al., 2003; Mande-makers et al., 1999; Monk et al., 2009; Monk and Talbot, 2009; Svaren and Meijer, 2008). Although intensively studied in embryos, the expression of these genes in zebrafish adults is not well mapped. Zebrafish pou3f1, for example, has curated expression data only up to larval day 5 (www.zfin.org). We therefore sought to determine whether these transcripts were active in regenerating adult barbels, indicating reactivation of the embryonic myelinating regulatory program. If demonstrated, these features would make the ZMB an attractive context in which to study the cellular and molecular basis of remyelination in the adult zebrafish PNS, enhancing the utility of this popular model organism.