May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Zebrafish lens opaque (lop) Mutation Mapping and Gene Identification
Author Affiliations & Notes
  • T. S. Vihtelic
    Biological Sciences and Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana
  • T. R. Murphy
    Biological Sciences and Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana
  • C. T. Watson
    Mutant Zebrafish Mapping Facility, University of Louisville, Louisville, Kentucky
  • G. B. Willer
    Mutant Zebrafish Mapping Facility, University of Louisville, Louisville, Kentucky
  • R. G. Gregg
    Mutant Zebrafish Mapping Facility, University of Louisville, Louisville, Kentucky
  • D. R. Hyde
    Biological Sciences and Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana
  • Footnotes
    Commercial Relationships T.S. Vihtelic, None; T.R. Murphy, None; C.T. Watson, None; G.B. Willer, None; R.G. Gregg, None; D.R. Hyde, None.
  • Footnotes
    Support NIH Grant EY014455
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2447. doi:
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      T. S. Vihtelic, T. R. Murphy, C. T. Watson, G. B. Willer, R. G. Gregg, D. R. Hyde; Zebrafish lens opaque (lop) Mutation Mapping and Gene Identification. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2447.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: The zebrafish lens opaque (lop) mutant was identified in a chemical mutagenesis screen for eye morphological phenotypes. The lop mutant, which appears to develop normally through 4 days post-fertilization (dpf), exhibits lens opacity at 7 dpf due to unregulated lens epithelial cell proliferation. These studies were designed to identify the lop mutant gene.

Methods:: The lop mutation was mapped by linkage to simple sequence length and single nucleotide polymorphisms. Candidate genes in the critical genomic region were evaluated by cDNA sequencing and, in some cases, antisense morpholino knockdown. Complementation analysis was carried out by pair mating lop heterozygotes with cdipthi559 retroviral insertion heterozygotes. RNA rescue experiments were performed by injecting in vitro-synthesized capped cdipt mRNA into 1-2 cell-stage lop embryos. The morphants and rescued embryos were analyzed by stereomicroscopy, histology, and PCNA immunohistochemistry to assess cell proliferation.

Results:: The lop mutation was localized to chromosome 3 between markers Z59420 and Z59380. High resolution mapping identified a genomic region of 200 kb covered by BAC DKEY-183N6, which contains 7 genes, including cdipt (phosphatidylinositol synthase; CDP-diacylglycerol--inositol 3-phosphatidyltransferase). Morpholino-mediated knockdown of CDIPT protein by blocking either mRNA splicing or protein translation resulted in increased proliferation of lens epithelial cells, which was similar to the lop phenotype. A single nucleotide change was identified within the cdipt open reading frame causing a nonconservative amino acid substitution (S25F) within, or very near, a putative transmembrane domain of phosphatidylinositol synthase. Complementation analysis confirmed that lop is a cdipt allele. Finally, injection of cdipt mRNA into lop embryos resulted in a significant reduction in embryos displaying lens opacity.

Conclusions:: The lop mutation lies within the cdipt (phosphatidylinositol synthase) gene. Phosphatidylinositol is the precursor of all phosphorylated derivatives of the phosphatidylinositol cycle; therefore, the zebrafish cdipt alleles represent an excellent in vivo genetic model to further elucidate the roles of phosphatidylinositol metabolism in regulating lens cell proliferation, differentiation and maintenance of optical clarity.

Keywords: positional cloning • cataract • proliferation 
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