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D (5? days post-fertilization, dpf), by which time myelination has been initiated D (5? days post-fertilization, dpf), by which time myelination has been initiated in wild-type fish. Studies of remyelination mechanisms and therapies also would be enabled by mutants with myelination defects arising principally during later development, as such phenotypes could parallel some human disorders. In this study, we report on the zebrafish puma mutant, which was recovered in a screen for mutations affecting the adult pigment pattern (Parichy and Turner, 2003; Parichy et al.,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptDev Biol. Author manuscript; available in PMC 2011 October 15.Larson et al.Page2003). puma mutants have a normal complement of neural crest-derived embryonic and early larval pigment cells, including melanin-containing melanophores. During the larval-toadult transformation, however, these fish develop markedly fewer "metamorphic" melanophores than the wild-type, resulting in gross perturbations to the normal pigment pattern of adult stripes. During these later stages, puma mutants also have a reduced complement of Schwann cells and exhibit defasciculation of peripheral nerves. Here, we examine the onset of myelination defects in the PNS and also uncover defects in adult craniofacial morphology and swimming behavior. We then map the puma mutant phenotype, identify a mutation in the alpha tubulin-encoding gene tuba8l3a, and establish the orthology of this gene relative to other alpha tubulin loci. We show that tuba8l3a is expressed widely in the early embryo expression, whereas expression becomes apparent in the CNS during the larval-to-adult transformation. This observation led us to test if PNS myelination defects are paralleled by CNS defects in oligodendrocyte specification or myelination. While early oligodendrocytes develop relatively normally, we find a gross reduction in CNS myelination and the numbers of differentiated oligodendrocytes, both during the larval-to-adult transformation and in the adult. Together, these analyses link demyelination, pigment pattern, and craniofacial defects to an alpha tubulin mutation, and identify the puma mutant as a potentially valuable model for future studies of demyelination as well as tests of therapeutic remyelination strategies.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMaterials and MethodsFish rearing, staging, genetic stocks, genetic mapping, and genotyping Fish were reared at 28?9 , 1410D. Embryonic staging followed (Kimmel et al., 1995) and post-embryonic staging used standardized standard length (SSL) measurements following (Parichy et al., 2009). The pumaj115e1 allele was isolated in an early pressure gynogenetic screen for mutations induced by N-ethyl-N-nitrosourea mutagenesis in the SJD background. puma was subsequently introgressed into ABwp, an inbred line used for genetic mapping, and map crosses were generated by crossing homozygous puma mutants to the inbred wik genetic background, then backcrossing the resulting F1s to puma mutants. A wild-type sox10GFP reporter line was generously provided by R. N. Kelsh. For genotyping fish in experiments, we amplified a 944 bp product that included the tuba8l3a lesion (see text) and we distinguished wild-type and puma mutant haplotypes by differential cutting with restriction enzymes Dde-I, Rsa-I, or Nla-IV. In situ hybridization In situ hybridization followed standard procedures. For some analyses, fish were sectioned by vibratome at 200?50 m prior to hyb.