The myelin sheath allows axons to rapidly conduct action potentials in the vertebrate nervous system. Incompletely understood axonal signals activate specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cAMP can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we report that Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20, and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.During peripheral nervous system (PNS) development, promyelinating Schwann cells associate with one segment of an axon and differentiate into myelinating Schwann cells that iteratively wrap their membrane around an axonal segment to form the myelin sheath (1). Axonal signals transiently activate the expression of the transcription factor Oct6 in Schwann cells that will form myelin, and cAMP can mimic axonal contact in vitro (2,3). Oct6 regulates Krox20 expression (4), and both transcription factors are required for Schwann cells to initiate myelination (5-7). Neuregulin signals and their ErbB receptors are involved in regulation of Oct6 and Krox20 (8), but the signaling pathways in Schwann cells that regulate myelination are not well understood.In a genetic screen for zebrafish mutants with abnormalities in myelinated axons, we previously identified two allelic mutations, st49 and st63, in which Myelin basic protein (Mbp) expression was not observed in peripheral nerves (9). Central nervous system (CNS) Mbp expression and PNS axonal marker expression were unaffected (9; Fig. S1). Except for an enlargement of the ear that was evident at 5 days post fertilization (dpf), st49 homozygous mutant larvae were morphologically indistinguishable from wild-type and heterozygous siblings (Fig. S2). High-resolution mapping experiments placed the st49 mutation in a region of Linkage Group 20 (LG20) that contains g-protein coupled receptor 126 (gpr126), which encodes a member of the adhesion G-protein coupled receptor (GPCR)

Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action.

In peripheral nerves, Schwann cells form myelin, which facilitates the rapid conduction of action potentials along axons in the vertebrate nervous system. Myelinating Schwann cells are derived from neural crest progenitors in a step-wise process that is regulated by extracellular signals and transcription factors. In addition to forming the myelin sheath, Schwann cells orchestrate much of the regenerative response that occurs after injury to peripheral nerves. In response to injury, myelinating Schwann cells dedifferentiate into repair cells that are essential for axonal regeneration, and then redifferentiate into myelinating Schwann cells to restore nerve function. Although this remarkable plasticity has long been recognized, many questions remain unanswered regarding the signaling pathways regulating both myelination and the Schwann cell response to injury.

SUMMARYIn peripheral nerves, Schwann cells form the myelin sheath, which allows the efficient propagation of action potentials along axons. The transcription factor Krox20 regulates the initiation of myelination in Schwann cells and is also required to maintain mature myelin. The adhesion G protein-coupled receptor (GPCR) Gpr126 is essential for Schwann cells to initiate myelination, but previous studies have not addressed the role of Gpr126 signaling in myelin maturation and maintenance. Through analysis of Gpr126 in zebrafish, we define two distinct mechanisms controlling the initiation and maturation of myelin. We show that gpr126 mutant Schwann cells elaborate mature myelin sheaths and maintain krox20 expression for months, provided that the early signaling defect is bypassed by transient elevation of cAMP. At the onset of myelination, Gpr126 and protein kinase A (PKA) function as a switch that allows Schwann cells to initiate krox20 expression and myelination. After myelination is initiated, krox20 expression is maintained and myelin maturation proceeds independently of Gpr126 signaling. Transgenic analysis indicates that the Krox20 cis-regulatory myelinating Schwann cell element (MSE) becomes active at the onset of myelination and that this activity is dependent on Gpr126 signaling. Activity of the MSE declines after initiation, suggesting that other elements are responsible for maintaining krox20 expression in mature nerves. We also show that elevated cAMP does not initiate myelination in the absence of functional Neuregulin 1 (Nrg1) signaling. These results indicate that the mechanisms regulating the initiation of myelination are distinct from those mediating the maturation and maintenance of myelin.

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