Abstract
Abstract: :
Purpose: During development, retinal progenitor cells pass through various stages of competence and cell fate choices in order to generate each of the specific retinal cell types. These progenitor cells are influenced by a number of intrinsic and extrinsic factors. Little is known, however, about the signaling pathways responsible for generating retinal cell types, specifically rod photoreceptors. The zebrafish is a powerful genetic model organism and mutagenesis screens for defects in retinal development have yielded a number of mutants with abnormal eye morphology. These screens, however, relied on crude observational techniques and likely missed more subtle mutations affecting single cell types or changes in gene expression. We have conduced a novel genetic screen using transgenic zebrafish to identify more subtle mutations affecting eye development and rod photoreceptor differentiation in living animals. Methods: Zebrafish expressing a GFP transgene in rods were bred to F1 mutagenized fish to generate transgenic F2 families that were heterozygous for a number of point mutations. F2 sibling crosses uncovered recessive mutations in 25% of the F3 progeny. Using a fluorescent dissecting microscope, fish were screened at 4 days post fertilization (dpf) and 6 dpf for changes in eye morphology and GFP expression, indicating changes in rod development. Histology of mutant retinas was done to study retinal anatomy and retinal patterning was assessed with cell type specific markers. The expression of a number of genes involved in retinogenesis, such as rx2, pax6, six3, six6, and sonic hedgehog (shh), was examined by in situ hybridization. Results: By screening over 50 F2 families, we have identified 3 major classes of mutations: 1) mutants with reduced eye size and little to no GFP expression; 2) mutants with reduced eye size and some GFP expression; and 3) embryos with normal eye morphology but no GFP expression. The retinas of one particular mutation retain all the major cell types but the eyes are significantly smaller at 3 dpf and rx2 expression is dramatically reduced at 24 hours post fertilization (hpf). These fish also undergo retinal degeneration during development but remain otherwise healthy to at least 10 dpf. Conclusions: These data show that we can identify new mutations affecting eye development and rod differentiation. We conclude that some genes required for regulating eye size are not essential for proper rod differentiation and maturation. Further characterization and the eventual identification of the underlying mutations will provide information on the key signaling pathways required for normal retinal development.
Keywords: retinal development • transgenics/knock-outs • photoreceptors