Forward Genetic Analysis of Visual Behavior in Zebrafish

Abstract

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.

How to make a teleost adenohypophysis: Molecular pathways of pituitary development in zebrafish

“…Since then, many additional screens using more specific or sophisticated screening assays greatly contributed to today's collection of available mutants (e.g. (Muto et al, 2005, Pogoda et al, 2006a, Sun et al, 2004). At the same time, genomic tools have enormously improved, facilitating the cloning of randomly induced mutations (Dahm and Geisler, 2006, Sivasubbu et al, 2007.…”

Analysis of the retina in the zebrafish model

“…Genetic screening is feasible because adult zebrafish are highly fecund and are easily maintained in large numbers in a fairly small laboratory space. Screens performed in on the zebrafish so far identified hundreds of visual system mutants Fadool et al, 1997;Malicki et al, 1996;Muto et al, 2005;Neuhauss et al, 1999). While designing a genetic screen, one has to consider three important variables: the type of mutagen to be used, the design of the breeding scheme, and mutant defect recognition criteria.…”

“…Several screens based on behavioral criteria have been performed in recent years, leading to the isolation of interesting developmental defects Li and Dowling, 1997;Muto et al, 2005;Neuhauss et al, 1999). Behavioral screens allow one to detect subtle functional defects of the retina that might evade other search criteria.…”

“…Identification of direct and indirect protein binding partners Omori et al (2008) Chemical screens Screens of small-molecule libraries Identification of chemicals that affect a developmental process Kitambi et al (2008) Genetic screens Behavioral Detection of mutant phenotypes by behavioral tests Muto et al (2005), Neuhauss et al (1999) Histological Detection of mutant phenotypes via histological analysis of sections Mohideen et al (2003) Marker/tracer labeling Detection of mutant phenotypes via staining with antibodies, RNA probes, or lipophilic tracers Baier et al (1996), Guo et al (1999) Morphological Detection of mutant phenotypes by morphological criteria Malicki et al (1996) Transgene guided Detection of mutant phenotypes in transgenic lines expressing fluorescent proteins in specific cell populations Xiao et al (2005) In this Kikuchi et al (1997), , Covance PRB-276P rx1 (zrx1) RNA probe Anterior neural keel, optic primordia (≤11 hpf) Chuang et al (1999), Pujic and Malicki (2001) rx2 (zrx2) RNA probe Anterior neural keel, optic primordia (≤11 hpf) Chuang et al (1999), Pujic and Malicki (2001) rx3 (zrx3) RNA probe Anterior neural plate (≤9 hpf); optic primordia (≤12 hpf) Chuang et al (1999), Pujic and Malicki (2001) six3a (six3) RNA probe Neural keel, optic primordia (≤11 hpf) …”

Zebrafish mutants as models for congenital ocular disorders in humans

“…The OKR consists of a smooth pursuit movement of a visual stimulus, followed by a quick saccade to reset the eye position in preparation for another stimulus . Several groups have used the OKR as a behavioral screen with great success to identify dozens of mutants affecting processes throughout the visual system (Brockerhoff et al, 1995;Brockerhoff et al, 1998;Neuhauss et al, 1999;Muto et al, 2005). As the visual response originates in the photoreceptors, many of these mutants exclusively affect photoreceptor biochemistry or physiology.…”