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Alysis uncovers three previously described loci (A to C) and three new loci (G to I) with interesting changes in expression.The downregulated locus D contains a three-gene operon involved in the synthesis of curli. Curli are common in Enterobacteriaceae and are typically involved in attachment to surfaces and biofilm formation (46). Although curli have not previously been described in Yersinia, it is known that Yersinia do not form biofilms at 37 . Therefore, this result makes sense if these curli are involved in biofilm formation rather than host-cell binding. The highly downregulated genes of locus E are all involved in synthesis of the bacterial flagella. It is well established that Y. enterocolitica flagella are downregulated at 37 , likely as an adaptation to avoid being recognized by Toll-like receptor 5 (TLR5) (18). This expected result confirms the utility of our technique in discovering systems that are differentially expressed upon changing environmental conditions. A more unexpected finding was seen in locus F with the upregulation of the glg operon, encoding five genes involved in the synthesis and metabolism of glycogen. Although the role of glycogen has not been studied in the context of a Y. enterocolitica infection, it has been demonstrated that E. coli O157:H7 glg operon mutants have a decreased ability to colonize the intestines of mice (47). It has been hypothesized that during colonization of the intestines, E. coli relies on internal stores of carbon during periods of low carbon availability (47). This is likely the case in Y. enterocolitica as well, with bacteria storing available carbon from the RPMI media as glycogen at 37  in preparation for colonization of the mammalian host. Y. enterocolitica transcriptional changes in response to extracellular infection. Bacteria infecting macrophages would be expected to upregulate virulence factors that aid in colonizationand evasion of the host immune response. We identified 180 genes with a 4-fold change in expression when comparing the transcriptomes of the extracellular bacteria to those in the conditioned RPMI after 120 min of infection (Fig. 3B), and we observed several loci of genes (A to C, G to I) that encode known and putative virulence factors (Fig. 5 and see Table S3 in the supplemental material). Interestingly, we see that all three of the downregulated loci are the same as upregulated loci in the previous comparison (A to C). The fimbria-encoding operons that were upregulated in response to the conditioned RPMI (A and C) are downregulated by bacteria attached to the macrophages (extracellular). This intriguing observation may indicate that the fimbria proteins are very stable once produced so that after the initial upregulation of the genes there is no need to create more fimbriae. Alternatively, they may be facilitating adherence to other cell types or noncellular host surfaces such as the soluble host factors present in the conditioned RPMI. The genes in locus B encode the sucrose uptake system, which was upregulated in the conditioned RPMI, an observation consistent with the hypothesis that the bacteria are storing carbon while in RPMI in preparation for the infection. Once the bacteria come into contact with the macrophages, they likely rely [https://www.medchemexpress.com/Tipifarnib.html Tipifarnib In stock] primarily on the stored glycogen as a carbon source and no longer actively import sugar. The highly upregulated locus G encodes the pH 6 antigen (Psa [Myf in Y.
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D (5? days post-fertilization, dpf), by which time myelination has been initiated
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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.,[https://britishrestaurantawards.org/members/design73stamp/activity/271390/ Title Loaded From File] 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.

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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.,Title Loaded From File 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.

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