Abstract
Purpose: :
Phosphorylated derivatives of phosphatidylinositol (PI) participate in a number of signaling pathways that can affect broad cellular processes. We wanted to examine the contribution of these phosphoinositides to zebrafish ocular development. We identified two independent zebrafish cdipt (Phosphatidylinositol synthase) mutants that exhibit larval cataracts due to irregular lens epithelial cell proliferation and fiber cell deterioration. The following studies examined the extent and onset of ocular defects within cdipt mutants and identified potential signaling pathways that may be affected.
Methods: :
Histological analysis of PCR-genotyped cdipthi559/hi559 mutants, relative to wild-type developing retinas, was used to study the onset and the progression of ocular defects within cdipt mutants. To identify signaling pathways that may be disrupted in cdipt mutant eye tissues, comparative microarray hybridization was performed using RNA extracted from cdiptlop/lop mutant and wild-type eyes at 7 dpf. In addition, protein was extracted from 7 dpf cdiptlop/lop mutant and wild-type eyes for fluorescent 2D differential gel electrophoresis. A number of differentially expressed spots were analyzed by mass spectrometry and selected proteins were immunolocalized in mutant eye tissues.
Results: :
Examination of cdipthi559/hi559 mutants revealed retinal photoreceptor cell defects as early as 3 dpf, followed by cell apoptosis that resulted in the loss of photoreceptors at 5 dpf. In addition, lens fiber cell dismorphogenesis and lens epithelial cell accumulation were first observed at 5 dpf. Lastly, the resulting proteomic and transcriptomic data sets were complimentary and identified several proteins that are involved in the phosphatidylinositol cycle and previous reports of human anterior subcapsular cataracts. Further investigation of select proteins revealed discrepancies in expression pattern within the mutant eye tissues relative to controls.
Conclusions: :
Through ocular phenotypic, transcriptomic and proteomic analysis, the zebrafish cdipt alleles may reveal the contribution of phosphoinositide metabolism and associated signaling pathways in maintaining lens clarity and photoreceptor cell integrity.
Keywords: cataract • lipids • development