Six6 and Six7 coordinately regulate expression of middle-wavelength opsins in zebrafish.

Color discrimination in the vertebrate retina is mediated by a combination of spectrally distinct cone photoreceptors, each expressing one of multiple cone opsins. The opsin genes diverged early in vertebrate evolution into four classes maximally sensitive to varying wavelengths of light: UV (SWS1), blue (SWS2), green (RH2), and red (LWS) opsins. Although the tetrachromatic cone system is retained in most nonmammalian vertebrate lineages, the transcriptional mechanism underlying gene expression of the cone opsins remains elusive, particularly for SWS2 and RH2 opsins, both of which have been lost in the mammalian lineage. In zebrafish, which have all four cone subtypes, rh2 opsin gene expression depends on a homeobox transcription factor, sine oculis homeobox 7 (Six7). However, the six7 gene is found only in the ray-finned fish lineage, suggesting the existence of another evolutionarily conserved transcriptional factor(s) controlling rh2 opsin expression in vertebrates. Here, we found that the reduced rh2 expression caused by six7 deficiency was rescued by forced expression of six6b, which is a six7-related transcription factor conserved widely among vertebrates. The compensatory role of six6b was reinforced by ChIP-sequencing analysis, which revealed a similar pattern of Six6b- and Six7-binding sites within and near the cone opsin genes. TAL effector nuclease-induced genetic ablation of six6b and six7 revealed that they coordinately regulate SWS2 opsin gene expression. Mutant larvae deficient for these transcription factors showed severely impaired visually driven foraging behavior. These results demonstrate that in zebrafish, six6b and six7 govern expression of the SWS2 and RH2 opsins responsible for middle-wavelength sensitivity, which would be physiologically important for daylight vision.

Homeobox transcription factor Six7 governs expression of green opsin genes in 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.

Identification of nonvisual photomotor response cells in the vertebrate hindbrain.

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. Using in vivo calcium 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.

Stealth Omicron: A Novel SARS-CoV-2 Variant That Is Insensitive to RT-qPCR Using the N1 and N2 Primer-Probes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is causing a worldwide pandemic since the spring of 2020. In Osaka, the second biggest prefecture in Japan, we identified a novel SARS-CoV-2 variant from a coronavirus disease 2019 (COVID-19) patient that was detected by polymerase chain reaction (PCR) using E primers, but not by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the N1 and N2 primer-probe sets recommended by CDC. We analyzed the S and N gene regions by reverse-transcription and nested PCR using the S and N specific primers, and DNA sequencing, and found that this BA.5 variant contained point mutations in the probe sequences of both the N1 and N2 primer-probe regions. This finding led us to affirm the importance of monitoring the genome sequence of the SARS-CoV-2 variants continuously.

Foxq2 determines blue cone identity in zebrafish.

Most vertebrate lineages retain a tetrachromatic visual system, which is supported by a functional combination of spectrally distinct multiple cone photoreceptors, ultraviolet (UV), blue, green, and red cones. The blue cone identity is ensured by selective expression of blue (sws2) opsin, and the mechanism is poorly understood because sws2 gene has been lost in mammalian species such as mouse, whose visual system has been extensively studied. Here, we pursued loss-of-function studies on transcription factors expressed predominantly in zebrafish cone photoreceptors and identified Foxq2 as a blue cone­specific factor driving sws2 gene expression. Foxq2 has dual functions acting as an activator of sws2 transcription and as a suppressor of UV (sws1) opsin transcription in blue cones. A wide range of vertebrate species retain both foxq2 and sws2 genes. We propose that Foxq2-dependent sws2 expression is a prevalent regulatory mechanism that was acquired at the early stage of vertebrate evolution.

Light-induced body color change in developing zebrafish.

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.

Mechanistic Study on Allylic Arylation in Water with Linear Polystyrene-Stabilized Pd and PdO Nanoparticles.

The catalytic cycle of allylic arylation in water catalyzed by linear polystyrene-stabilized Pd or PdO nanoparticles (PS-PdNPs or PS-PdONPs) was investigated. Stoichiometric stepwise reactions indicated that the reaction did not proceed stepwise on the surface of the catalyst. In the case of the reaction with PS-PdNPs, the leached Pd species is the catalytically active species and the reaction takes place through a similar reaction pathway accepted in the case of a complex catalyst. In contrast, allylic arylation using PS-PdONPs as a catalyst occurs via a Pd(II) catalytic cycle.

A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish.

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.