Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye

• Published in: Experimental eye research Vol. 93; no. 4; pp. 460 - 474
• Main Authors: Murphy, Taylor R., Vihtelic, Thomas S., Ile, Kristina E., Watson, Corey T., Willer, Gregory B., Gregg, Ronald G., Bankaitis, Vytas A., Hyde, David R.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-10-2011
• Subjects: Animals; Apoptosis; cataract; Cataract - genetics; CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase - physiology; cell death; Cell Survival - physiology; Danio rerio; DNA Primers - chemistry; Epithelial Cells - cytology; Epithelial Cells - enzymology; Fluorescent Antibody Technique, Indirect; Freshwater; Genotype; Immunoblotting; In Situ Nick-End Labeling; lens epithelial cell; lens fiber cell; Lens, Crystalline - cytology; Lens, Crystalline - enzymology; Membrane Proteins - physiology; MicroRNAs - genetics; Mutation, Missense; phosphatidylinositol synthase; photoreceptor; Photoreceptor Cells, Vertebrate - cytology; Photoreceptor Cells, Vertebrate - enzymology; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish; Zebrafish Proteins - physiology; lens fiber cell; lens epithelial cell; cell death; cataract; phosphatidylinositol synthase; photoreceptor
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2011.06.010

The zebrafish
lens opaque (
lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the
lop phenotype results from a defect in the
cdipt (
phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the
lop mutant contained a missense mutation in the
lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the
cdipt-encoded PI synthase protein phenocopied the
cdipt
lop/lop
mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of
in vitro transcribed, wild-type
cdipt mRNA into 1–4 cell stage
cdipt
lop/lop
embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a
cdipt retroviral-insertion allele,
cdipt
hi559
, exhibited similar lens and retinal abnormalities and failed to complement the
cdipt
lop
mutant phenotype.
To determine the initial cellular defects associated with the
cdipt mutant, we examined homozygous
cdipt
hi559/hi559
mutants prior to gross lens opacification at 6 dpf. The
cdipt
hi559/hi559
mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the
cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum revealed that the PI synthase protein was reduced in amount in both the
cdipt
lop/lop
and
cdipt
hi559/hi559
mutants. Furthermore, we used a heterologous yeast phenotypic complementation assay to confirm that the wild-type zebrafish
cdipt allele encodes functional PI synthase activity. Taken together, the
cdipt-encoded PI synthase is required for survival of photoreceptor cells and lens epithelial and secondary cortical fiber cells. These zebrafish
cdipt alleles represent excellent
in vivo genetic tools to study the role of phosphatidylinositol and its phosphorylated derivatives in lens and photoreceptor development and maintenance.
► The lens opaque mutation is a missense change in PI (phosphatidylinositol) synthase. ► PI synthase is required for photoreceptor cell viability. ► PI synthase is necessary to maintain lens epithelial cells. ► PI synthase is required for lens cortical secondary fiber differentiation.