Genetic analysis of retinal development in zebrafish

Chapter 1 presents the analysis of retinal patterning in a previously isolated mutant. In cyclops, two retinas are fused in the midline and cell pattern is disrupted. At the center of the fusion, photoreceptors form rosettes; in the periphery they form a lamina continuous across the midline with the laminae of the lateral eyefields. In the ventral region, a complete set of laminae is present where the optic nerve would normally develop. We suggest the cellular changes we describe are triggered by abnormal positional relationships imposed on retinal cells near the midline by the fusion. To explore the possibility of producing novel mutants by prescreening transgenic zebrafish, I injected LacZ constructs into the cytoplasm of one-cell embryos and used a live assay for expression (Chapter 2). Both localized and diffuse fluorescence patterns were seen. The initial screens of F1's indicated a high rate of germline transmission (11/42, or 26%), but most lacked junction fragments and failed to transmit the foreign DNA to F2's, suggesting extrachromosomal transmission. In three lines the gene trap construct appeared to have integrated and was transmitted to the F2 generation. Eight lines in which the founder has been injected with LacZ DNA were bred to homozygosity and screened for recessive phenotypes in F2's. Three novel phenotypes and one non-specific embryonic lethal were found. The bloodless mutation was found in a line with stable integration and transmission but appears to segregate independently from the insertion. It blocks hematopoiesis in the embryo. The sluggish mutation affects motility and pigmentation. Screwtail mutants have small spinal cords, enlarged notochords and reduced numbers of catecholaminergic cells in the retina. The latter mutations were found in lines with extrachromosomal transmission.