Tomoya Shiraki

A transcriptional regulator Foxq2 controls blue-type cone opsin gene in vertebrate species having tetrachromatic visual system.

The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We found that the Tg(ubb:SpCas9,u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.

Colour discrimination in vertebrates requires cone photoreceptor cells in the retina, and high-acuity colour vision is endowed by a set of four cone subtypes expressing UV-, blue-, green- and red-sensitive opsins. Previous studies identified transcription factors governing cone photoreceptor development in mice, although loss of blue and green opsin genes in the evolution of mammals make it difficult to understand how high-acuity colour vision was organized during evolution and development. Zebrafish ( Danio rerio ) represents a valuable vertebrate model for studying colour vision as it retains all the four ancestral vertebrate cone subtypes. Here, by RT-qPCR and in situ hybridization analysis, we found that sine oculis homeobox homolog 7 ( six7 ), a transcription factor widely conserved in ray-finned fish, is expressed predominantly in the cone photoreceptors in zebrafish at both the larval and the adult stages. TAL effector nuclease-based six7 knock-out revealed its roles in expression of green, red and blue cone opsin genes. Most prominently, the six7 deficiency caused a loss of expression of all the green opsins at both the larval and adult stages. six7 is indispensable for the development and/or maintenance of the green cones.

Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light but does not require the eyes, pineal gland, or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical nonvisual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Usingin vivocalcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.

Non‐technical summary  When rod and cone photoreceptors in the eye respond to light, they need to recover, and the first step in recovery involves a protein called G‐protein receptor kinase (GRK). Rods, which underlie night vision, employ a variant called GRK1, whereas cones, which mediate day vision, typically employ a variant called GRK7. We have engineered rod cells in the zebrafish retina that additionally express the cone variant, GRK7. By recording electrically from these modified rods, we have found that they are less sensitive to light than normal rods, in that regard mimicking cones. We have also found evidence to suggest that the size of the cell's response to a single photon (the smallest particle of light) is normal when recovery is mediated by GRK1, but is small (and hence somewhat cone‐like) when mediated by GRK7. These results help us understand the differences between rod and cone photoreceptors.

In response to ambient light levels, many lower vertebrates darken or lighten their body colors by regulating dispersion or aggregation, respectively, of melanin granules (melanosomes) in the melanophore. This physiological reaction is mediated by photoreception in the eyes, the pineal gland, the deep brain and the melanophores themselves, depending on species and their developmental stages. In this study, we established a method for quantitative measurement of the light-induced body color change in zebrafish larvae. From 2 days post-fertilization (dpf), the dermal melanophores responded to light illumination, but the response patterns and temporal profiles changed across the developmental stages. At 2 dpf, light illumination on larvae induced a relatively fast dispersion of the pigments in the melanophores, whereas continuous illumination additionally caused a delayed pigment aggregation at 3 dpf or later stages. Removal of the eyes abolished the light-dependent pigment aggregation but not the pigment dispersion at 5 dpf, while the pigment dispersion at 2 dpf was retained even in the isolated tail. These results suggest that the pigment dispersion is triggered by photoreception intrinsic to the melanophores and that the pigment aggregation is mediated by photoreception in the eyes. The monitoring system developed in this study will be useful to understand the neural mechanisms underlying the body color change depending on the ocular system. We also discussed the putative role(s) of opsin-type photoreceptive molecules in the light-induced body color change of the larval zebrafish.