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

Kctd12 and Ulk2 partner to regulate dendritogenesis and behavior in the habenular nuclei.

The habenular nuclei of the limbic system regulate responses, such as anxiety, to aversive stimuli in the environment. The habenulae receive inputs from the telencephalon via elaborate dendrites that form in the center of the nuclei. The kinase Ulk2 positively regulates dendritogenesis on habenular neurons, and in turn is negatively regulated by the cytoplasmic protein Kctd12. Given that the habenulae are a nexus in the aversive response circuit, we suspected that incomplete habenular dendritogenesis would have profound implications for behavior. We find that Ulk2, which interacts with Kctd12 proteins via a small proline-serine rich domain, promotes branching and elaboration of dendrites. Loss of Kctd12 results in increased branching/elaboration and decreased anxiety. We conclude that fine-tuning of habenular dendritogenesis during development is essential for appropriate behavioral responses to negative stimuli

Leptin system loss of function in the absence of obesity in zebrafish

The leptin system plays a crucial role in the regulation of appetite and energy homeostasis in vertebrates. While the phenotype of morbid obesity due to leptin (LEP) or leptin receptor (LEPR) loss of function is well established in mammals, evidence in fish is controversial, questioning the role of leptin as the vertebrate adipostat. Here we report on three (Lepr) loss of function (LOF) and one leptin loss of function alleles in zebrafish. In order to demonstrate that the Lepr LOF alleles cannot transduce a leptin signal, we measured socs3a transcription after i.p. leptin which is abolished by Lepr LOF. None of the Lepr/Lepa LOF alleles leads to obesity/a body growth phenotype. We explore possible reasons leading to the difference in published results and find that even slight changes in background genetics such as inbreeding siblings and cousins can lead to significant variance in growth

Ulk2 morphants have a decreased extension/retraction ratio.

<p><b>A. and B.</b> Habenular neurons were scatter labeled and imaged for one hour. Red arrows point to dendrites being tracked over the 20-minute period displayed. Green arrows point to an additional dendrite tracked during the same time-lapse. Dendrites in the WT neurons are maintained for longer than in Ulk2 morphants. Scale bar is 10 µm. <b>C. and D.</b> The number of extension (p = 1) and retraction (p = 0.1095) events per dendrite per minute was similar between WT (n = 16) and Ulk2 morphants (n = 16), but <b>E.</b> the ratio of extension events to retraction events per dendrite was significantly reduced (**p = 0.00488, ANOVA) in Ulk2 morphants.</p

Kctd12 mutation causes a decrease in thigmotactic behavior.

<p><b>A.</b> Each well of a 6-well plate is divided into halves with dark (gray) or clear (blue) bottoms. The well is further divided into center (light) and edge (dark). The position of the fish is recorded every half second to measure scototaxis or thigmotaxis. The center is defined as 60% of the area of the circle (inner) and the edge as the remaining 40% (outer). Double heterozygous larvae were used as controls <b>B.</b> Scototaxis. No change in scototaxis is detected between genotypes. All genotypes prefer the light. Dotted lines are ns. Thigmotaxis. Preference for the center increases in Kctd12 single and double mutants (solid line: p<0.05, double line: p<0.01, 2-tailed t-test). Pooling mutant animals for a comparison to double heterozygote controls showed a significant effect of genotype (white vs. black * p<0.01). kctd12.1/2+/− n = 19; kctd12.1/2−/− n = 9; kctd12.1−/−, kctd12.2+/− n = 11; kctd12.1+/−, kctd12.2−/− n = 7.</p