Investigation of Phosphatidylinositol Synthase in the Zebrafish Larval Lens and Photoreceptors

The zebrafish lens opaque (lop) mutant was identified in a mutagenesis screen for eye morphological phenotypes, and exhibits lens opacity at 7 days post fertilization due to unregulated lens epithelial cell proliferation and fiber cell degeneration. The lop mutation was shown to be a missense mutation (A331T) in the cdipt (CDP-diacylglycerol--inositol 3-phosphatidyltransferase; phosphatidylinositol (PI) synthase) gene, resulting in an S111C substitution in the encoded protein. A second allele, cdipthi559, was previously described as possessing a viral insertion in the first intron and failed to complement cdiptlop. Morpholino-mediated knockdown of PI synthase and cdipt mRNA micro-injection rescue confirmed the gene identity. To examine the initial cellular defects associated with the cdipt mutant, PCR-based genotyping to identified homozygous cdipthi559/hi559 mutants prior to lens opacification. The cdipthi559/hi559 mutant exhibited photoreceptor disruption and cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. Anti-zebrafish PI synthase polyclonal antiserum localized the protein throughout the developing wild-type eye, including the photoreceptor layer and lens cortical secondary fiber cells, but the protein was reduced in both the cdiptlop/lop and cdipthi559/hi559 mutants. Moreover, tetrad analysis of a heterozygous PIS1- diploid yeast strain transformed with either wild-type or cdiptlop zebrafish cDNAs demonstrated that both proteins possessed PI synthase activity. To identify signaling pathways that may be disrupted in the cdipt mutant eye, global gene expression changes were analyzed by comparative microarray hybridization using RNA extracted from cdiptlop/lop mutant and wild-type eyes. In a complimentary approach, protein was isolated from cdiptlop/lop mutant and wild-type eyes and analyzed by fluorescent 2D difference gel electrophoresis, followed by mass spectrometry of differentially expressed proteins. The resulting proteomic data complemented the transcriptome analysis, with both approaches identifying components in the PI 3-kinase signaling pathway. Additionally, differentially regulated genes were identified that correlated with genes previously identified with human anterior subcapsular cataracts. Through phenotypic, transcriptomic and proteomic analyses, the zebrafish cdipt alleles represent an excellent in vivo model to investigate the contribution of phosphoinositide-associated signaling pathways in maintaining lens clarity and photoreceptor cell integrity.