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The zebrafish runzel mutant results in a novel muscular dystrophy phenotype due to a titin mutation

ABSTRACT: The zebrafish runzel (ruz) carries a mutation in the titin gene resulting in muscle degeneration and embryonic lethality. At the onset of the visible phenotype (3-3.5 days post fertilization), RNA expression of the tail and trunk (primarily skeletal muscle) was compared between ruz mutants and wildtype siblings using the Affymetrix Zebrafish Gene Chip. Mutant RNA was matched with RNA of wildtype siblings from the same clutch. 3 separate clutches were used (n=3). RNA was labeled, sheered, and hybridized as in Shephard et al. (Shepard et al., 2005) and the data analyzed as in Choe et al. and Weber et al. (Choe et al., 2005; Weber et al., 2005). Results suggest a novel mechanism of titin muscular dystrophy pathogenesis including upregulation of heat shock proteins. Keywords: disease state analysis

ORGANISM(S): Danio rerio

PROVIDER: GSE4585 | GEO | 2006/07/30

SECONDARY ACCESSION(S): PRJNA94433

REPOSITORIES: GEO

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Project description:In order to detect transcriptional differences between primitive and definitive hematopoietic stem and progenitor cells during regular development in the zebrafish embryo, gata1-GFP+/+(18 somites), lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf) cells from transgenic embryos were individually separated from GFP-/- cells by flow cytometry at the indicated stages. For each individual population, pools of 600 - 1500 transgenic embryos were collected. After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays. Individual experiments were performed with 2 or 3 biological replicates. Experiment Overall Design: gata1-GFP+/+(18 somites), lmo2-GFP+/+ (18 somites and 35 hpf)1 and cd41-GFP+/+ (35 hpf)2 cells were separated from GFP-/- cells Experiment Overall Design: by flow cytometry at the indicated stages. Sorting was based on propidium iodide exclusion, forward scatter, and GFP Experiment Overall Design: fluorescence, using a FACSVantage flow cytometer (Beckton Dickinson). Sorted cell populations were run twice to optimize cell Experiment Overall Design: purity. Total RNA from cell-sorted populations was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy resins Experiment Overall Design: (Qiagen) according to the manufacturerâs recommendations. Total RNA was subjected to two rounds of linear amplification Experiment Overall Design: (Ambion) and hybridized to Affymetrix zebrafish Gene Chips, according to Affymetrix guidelines. After staining with a Experiment Overall Design: streptavidin-phycoerythrin conjugate (Molecular Probes), the fluorescence of bound RNA was quantitated by using a Gene Chip Experiment Overall Design: scanner (Affymetrix). The raw expression data were calculated and, after pairing of GFP+/+ and GFP-/- samples, statistically Experiment Overall Design: analyzed using methods implemented in Bioconductorâs âaffyâ package and available custom scripts 3,4. Experiment Overall Design: references: Experiment Overall Design: 1. Zhu, H. et al. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev Biol 281, Experiment Overall Design: 256-69 (2005). Experiment Overall Design: 2. Lin, H. F. et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106, 3803-10 (2005). Experiment Overall Design: 3. Choe, S. E., Boutros, M., Michelson, A. M., Church, G. M. & Halfon, M. S. Preferred analysis methods for Affymetrix Experiment Overall Design: GeneChips revealed by a wholly defined control dataset. Genome Biol 6, R16 (2005). Experiment Overall Design: 4. Weber, G. J. et al. Mutant-specific gene programs in the zebrafish. Blood 106, 521-30 (2005).

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The zebrafish runzel mutant results in a novel muscular dystrophy phenotype due to a titin mutation

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