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.

A mechanism for red coloration in vertebrates.

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at the sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, the homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Partitioning of gene expression among zebrafish photoreceptor subtypes.

Vertebrate photoreceptors are categorized into two broad classes, rods and cones, responsible for dim- and bright-light vision, respectively. While many molecular features that distinguish rods and cones are known, gene expression differences among cone subtypes remain poorly understood. Teleost fishes are renowned for the diversity of their photoreceptor systems. Here, we used single-cell RNA-seq to profile adult photoreceptors in zebrafish, a teleost. We found that in addition to the four canonical zebrafish cone types, there exist subpopulations of green and red cones (previously shown to be located in the ventral retina) that express red-shifted opsin paralogs (opn1mw4 or opn1lw1) as well as a unique combination of cone phototransduction genes. Furthermore, the expression of many paralogous phototransduction genes is partitioned among cone subtypes, analogous to the partitioning of the phototransduction paralogs between rods and cones seen across vertebrates. The partitioned cone-gene pairs arose via the teleost-specific whole-genome duplication or later clade-specific gene duplications. We also discovered that cone subtypes express distinct transcriptional regulators, including many factors not previously implicated in photoreceptor development or differentiation. Overall, our work suggests that partitioning of paralogous gene expression via the action of differentially expressed transcriptional regulators enables diversification of cone subtypes in teleosts.

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.