Forward Genetic Analysis of Visual Behavior in Zebrafish

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

The <i>darl</i> Mutant Shows Retinotectal Mapping Deficits

<p>(A and B) The nasal-dorsal quadrant of the retina was labeled with DiO (green), and the temporal-ventral quadrant was labeled with DiD (magenta). In <i>darl^s327,</i> the ventral branch of the optic tract is missing (arrow). Scale bar is 100 μm.</p> <p>(C and D) Dorsal view of the tectum in the same larvae as in A and B. The ventral half of the <i>darl^s327</i> tectum is not innervated by the dorsal-nasal RGC axons. Anterior is to the left and ventral is to the bottom. Tectal neuropil is demarcated by the dotted line, based on DAPI counterstaining (blue). Scale bar is 50 μm.</p

Example of a Mutant with Abnormal Morphology of Cone Photoreceptors

<p>Photoreceptors in a retinal section stained with DAPI (A and B) and a marker for double cones, zpr1 (C and D) at 7 dpf in WT larva (A, C, and E) and <i>yoi^s121</i> mutant retina (B, D, and F). Merged images of DAPI (in green) and zpr1 (in magenta) are also shown (E and F). Both zpr1-positive and zpr1-negative cone photoreceptors in the mutant are “stumpy” when compared to those in the control retina (arrows). B, bipolar cells; C, cone photoreceptor cells; H, horizontal cells; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar is 10 μm.</p

Example of a Mutant with a Potential Defect in Light Adaptation

<p>OKR is plotted at several time points before and after dark treatment for 45 min. WT sibling larvae (<i>n</i> = 6) recover quickly from the dark pulse, while <i>nki^s136</i> mutants (<i>n</i> = 6) show reduced responsiveness for several minutes after return to the light. Average number of saccades to a constant motion stimulus is shown for each time point. Error bars indicate standard deviation.</p

Examples of Retinofugal Projection Mutants

<p>(A and B) Sections of WT and <i>boj^s307</i> retina stained with DAPI. The mutant retina has a thinner RGC layer (arrow).</p> <p>(C and D) Dorsal views of RGC axons from the right eye of a WT and a <i>boj^s307</i> mutant labeled with DiO, showing mutant axons in the ipsilateral tectum (arrow). To show that there is no ipsilateral projection in WT, the image is overexposed.</p> <p>(E–J) Lateral views of RGC axons labeled with DiO after removal of the eye. Anterior is to the left, dorsal to the top. In WT, the tectum and other retinorecipient areas are clearly visible (E). The arrow indicates AF-4. In <i>darl^s327,</i> the ventral branch of the optic tract is missing (arrow), and only dorsal tectum is innervated (F). In <i>walk^s536,</i> innervation of AF-4 (arrow) is disorderly (G). In <i>exa^s174,</i> the posterior tectum (arrow) appears to be incompletely innervated, while AF-4 is larger than in WT (H). In <i>miss^s522,</i> AF-4 (arrow) is reduced in size (I). In <i>mich^s314,</i> there is an ectopic arborization (arrow) at the root of the optic tract (J). Scale bars are 100 μm.</p

Behavioral Screening Assays

<p>(A) OMR. WT larvae in the racetrack reflexively swim in the same direction as a moving stimulus (top). Mutant larvae (for example, <i>dln^s393</i>) with an OMR index of 0 fail to respond (bottom). A contrast-enhanced image outlining the fish is shown in the lower image. In this experiment, WT fish larvae were driven all the way to the right end of the racetrack, which differs slightly from our screening assay [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010066#pgen-0010066-b016" target="_blank">16</a>].</p> <p>(B) OKR. Eye positions (angles shown by white arrows, far left image) were plotted over time during optokinetic stimulation in one direction. The OKR has a sawtooth profile, consisting of alternating quick and slow phases. OKR mutants show slowed eye movements (for example, <i>nebo^s342</i>), absence of the OKR <i>(lim^s382),</i> or no eye movements <i>(flan^s513)</i>. Corresponding OKR indices are given in parentheses.</p> <p>(C) VBA. WT (VBA index = 1) shows fully contracted melanophores in bright illumination. Mutants <i>(edpo^s371, ymj^s392,</i> and <i>amj^s391)</i> show three gradations of darker pigmentation, due to enhanced melanin dispersal. Scale bar is 1 mm.</p> <p>(D) SSA. Movies of six fish per rectangular well, taken at 0.5 frame per second for 20 min, were subtracted frame by frame and projected into a single image to show the locomotor behavior over time. Blind mutants, such as <i>mti^s113</i> (OKR and OMR indices = 0), may show normal spontaneous activity (SSA index = 1). The <i>mti</i> mutants are also darker (VBA = 0.3), resulting in a higher-contrast image than WT. The <i>walk^s536</i> mutants (OKR = 0.8; OMR = 0) show less activity, with some circling (SSA = 0.7), which could explain part of their OMR defect. In <i>beat^s348</i> mutants, locomotion is severely compromised (SSA = 0.1). SSA-defective mutants were not systematically kept.</p