Academic journal article Genetics

Identification of Zebrafish Insertional Mutants with Defects in Visual System Development and Function

Academic journal article Genetics

Identification of Zebrafish Insertional Mutants with Defects in Visual System Development and Function

Article excerpt

ABSTRACT

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

THE zebrafish has been an important model through which genes necessary for visual system development and function have been identified (reviewed in EASTER and MALICKI 2002 and NEUHAUSS 2003). Zebrafish eyes are large, easily accessible, and structurally similar to the human eye. Eye formation in zebrafish is analogous to that observed in other vertebrate embryos, thus providing an excellent model system with which the understanding of vertebrate eye development can be advanced. Additionally, many disrupted genes and pathways identified as integral to the formation of the zebrafish eye produce phenotypes that resemble disorders of the human visual system. Thus, characterization of the molecular mechanisms of eye development in zebrafish should facilitate a better understanding of these human pathologies (GOLDSMITH and HARRIS 2003).

Eye development in zebrafish first becomes morphologically obvious at the 6 somite stage (SS), the time at which the optic lobes evaginate from the diencephalon (SCHMITT and BOWLING 1994). Thereafter, eye development proceeds rapidly with lens induction occurring around the 14-15 SS and morphological distinction between the retina and retinal pigment epithelium (RPE) apparent by the 18-19 SS. The first postmitotic neurons of the retina are generated at 28 hr postfertilization (hpf) and by 72 hpf the retina is functional (EASTER and NICOLA 1996; Hu and EASTER 1999; SCHMITT and BOWLING 1999). Retinas of many fish and amphibians also possess a specialized region at their margins, termed peripheral or ciliary marginal zones, that perpetually adds cells to the retina during the lifetime of the animal (JOHNS 1977).

Several generations of chemically based forward genetic screens have been undertaken in zebrafish (DRIEVER et al. 1996; HAFFTER et al. 1996; MATSUDA and MISHINA 2004), some of which have focused on eye development and function (MALICKI et al. 1996; FADOOL et al. 1997; NEUHAUSS et al. 1999). While these chemically based screens have been instrumental in generating interesting mutant phenotypes, positional cloning of these mutations is still quite laborious, despite the genomic advances of the last few years. Retrovirus-mediated insertional mutagenesis provides an attractive alternative to chemical mutagenesis techniques since the affected gene can be rapidly identified using the proviral insert as a molecular tag to localize the site of insertion in the genome and thereby to identify the mutated gene (GAiANO et al. 1996; AMSTERDAM et al 1999; AMSTERDAM 2003). Indeed, a large-scale insertional mutagenesis screen performed over the last 6 years has generated >500 insertional mutants of which 315 different affected loci have been identified (COLLING et al. …

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