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Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss-and gain-of-function experiments show that L-opsin expression requires trftë activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.

Abstract Vertebrates use cone cells in the retina for color vision and rod cells to see in dim light. Many deep-sea fishes have adapted to their environment to have only rod cells in the retina, while both rod and cone genes are still preserved in their genomes. As deep-sea fish larvae start their lives in the shallow, and only later submerge to the depth, they have to cope with diverse environmental conditions during ontogeny. Using a comparative transcriptomic approach in 20 deep-sea fish species from eight teleost orders, we report on a developmental cone-to-rod switch. While adults mostly rely on rod opsin (RH1) for vision in dim light, larvae almost exclusively express middle-wavelength-sensitive (“green”) cone opsins (RH2) in their retinas. The phototransduction cascade genes follow a similar ontogenetic pattern of cone—followed by rod-specific gene expression in most species, except for the pearleye and sabretooth (Aulopiformes), in which the cone cascade remains dominant throughout development, casting doubts on the photoreceptor cell identity. By inspecting the whole genomes of five deep-sea species (four of them sequenced within this study: Idiacanthus fasciola, Chauliodus sloani; Stomiiformes; Coccorella atlantica, and Scopelarchus michaelsarsi; Aulopiformes), we found that they possess one or two copies of the rod RH1 opsin gene, and up to seven copies of the cone RH2 opsin genes in their genomes, while other cone opsin classes have been mostly lost. Our findings hence provide molecular evidence for a limited opsin gene repertoire in deep-sea fishes and a conserved vertebrate pattern whereby cone photoreceptors develop first and rod photoreceptors are added only at later developmental stages.

Color vision is widespread in marine vertebrates but is notably lacking in whales, dolphins, seals, and apparently also sharks. All sharks studied to date possess only a single spectral class of cone and are thus potentially totally color blind. The reason why sharks lack color vision is unclear, but as the visual pigments of only a handful of this large and ecologically diverse taxon have been studied, more data are required to address this question. Here, we assembled the retinal transcriptomes of 9 species from 7 families and 3 orders within the superorder Galeomorphii to screen for visual opsin and phototransduction genes. We reveal that cone monochromacy is widespread in galeomorph sharks, but the type of cone opsin expressed varies, with lamniform and orectolobiform sharks expressing a long-wavelength-sensitive (LWS) opsin, and carcharhiniform and heterodontiform sharks expressing a rhodopsin-like 2 (RH2) opsin. Cone monochromacy has evolved from a dichromatic ancestral state at least 4 times, implying strong selection pressure to prioritize achromatic over chromatic vision. While all species express the GRK1A and GRK7 isoforms of G protein-coupled receptor kinase, only sharks with the LWS cone opsin express the GRK1B isoform, which suggests that nonspectral functions of photoreception may have influenced, or result from, the opsin complement in the shark retina. Finally, we show that the shark rod (RH1) opsin gene shows evidence of positive selection at sites known to influence pigment kinetics (i.e. metarhodopsin II stability) and that the rate of retinal release likely differs substantially between species in ways that reflect their physiology and ecology.

Abstract Cichlid visual systems can evolve rapidly during adaptive radiations. The Bermin crater lake species flock in Cameroon, comprising 13 (nine valid and four undescribed) Coptodon species, offers an ideal model to investigate visual adaptation to the deep-water light environments. Here, we examine visual opsin genes sequences and expression using 109 retina transcriptomes, focusing on interspecific variation with habitat depth, as well as on seasonal changes in the migratory species between depths. All species possess a multichromatic system with at least five cone opsins. While opsin coding sequences show limited divergence—consistent with the flock's recent origin—opsin expression profiles vary substantially. Deep-water species showed reduced sws1 and sws2b expression, in line with lower UV and violet light availability in deeper waters. Unexpectedly, proportional expression of the red-sensitive lws opsin gene increases with depth, contrasting with patterns in other lacustrine cichlids. In the seasonally migrating species Coptodon imbrifernus, opsin expression is plastic, with decreased sws2b levels in deeper-dwelling dry-season individuals. To contextualize our findings, we compared Bermin cichlids to the older Barombi Mbo crater lake radiation. While single cone adaptations to the depth were convergent (loss of UV/violet sensitivity, enhanced blue sensitivity), double cone response diverged: lws expression was lost in Barombi Mbo while increased in Bermin deep-water species. Our findings suggest that plasticity in opsin expression plays a crucial role at the onset of sensory evolution, potentially paving the way for future genetic change. This study underscores the power of young systems like Bermin for uncovering the mechanisms driving early visual system diversification.

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision. Activating the spared neurons downstream of rods and cones is a potential therapeutic approach for retinal degeneration, but has been limited by the characteristics of the opsins available. Here, the authors use medium wavelength cone opsin which has faster kinetics than others and show that it resolves some of these difficulties in a mouse model.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.

Cartilaginous fishes (e.g., sharks, rays, and skates) cannot see blue or violet light, potentially because they lack the shortwave-sensitive cone opsin gene ( sws ). Widespread gene loss can occur during evolution, but the evolutionary mechanisms underlying sws loss remains unclear. Here, we construct whole-genome assemblies of Okamejei kenojei (skate) and Prionace glauca (blue shark). We then analyze the distribution characteristics and intragroup differences of opsin-related genes in cartilaginous fishes. Using a zebrafish model with sws deleted we infer that in the presence of SWS1 and SWS2, blue and violet light respectively, can induce cell aging. This is followed by photoreceptor layer thinning, demonstrating, sws loss aids in preventing shortwave light damage to the eye. In the retinas of numerous cartilaginous fishes, the tapetum lucidum strongly reflects light. Therefore, in cartilaginous fish, the existence of tapetum lucidum in the retina and loss of sws may be interdependent; in other words, this adaptive gene loss may increase cartilaginous fish fitness. Cartilaginous fishes lost the short-wave-sensitive ( sws ) opsin genes at some point in their evolutionary history. Here, the authors present genome assemblies of Okamejei kenojei (skate) and Prionace glauca (blue shark) that confirm sws loss, together with experimental evidence that sws loss reduces shortwave light damage to the eye.

Color vision is mediated by the expression of different major visual pigment proteins (opsins) on retinal photoreceptors. Vertebrates have four classes of cone opsins that are most sensitive to different wavelengths of light: short wavelength sensitive 1 (SWS1), short wavelength sensitive 2 (SWS2), medium wavelength sensitive (RH2), and long wavelength sensitive (LWS). UV wavelengths play important roles in foraging and communication. However, direct evidence provide links between sws1 and first feeding is lacking. Here, CRISPR/Cas9 technology was performed to generate mutant zebrafish lines with sws1 deletion. sws1 mutant zebrafish larvae exhibited decreased sws1, rh2-2, and lws1 expression, and increased rod gene (rho and gnat1) expression. Furthermore, the sws1-deficient larvae exhibited significantly reduced food intake, and the orexigenic genes npy and agrp signaling were upregulated at 6 days postfertilization (dpf). The transcription expression of sws1 and rh2-3 genes decreased in sws1−/− adults compared to wild type. Surprisingly, the results of feeding at the adult stage were not the same with larvae. sws1 deficiency did not affect food intake and appetite gene expression at adult stages. These results reveal a role for sws1 in normal cone development and first feeding in larval zebrafish.

L-cone opsin expression by gene therapy is a promising treatment for blue cone monochromacy (BCM) caused by congenital lack of long- and middle-wavelength-sensitive (L/M) cone function. Eight patients with BCM and confirmed pathogenic variants at the OPN1LW/OPN1MW gene cluster participated. Optical coherence tomography (OCT), chromatic perimetry, chromatic microperimetry, chromatic visual acuity (VA), and chromaticity thresholds were performed with unmodified commercial equipment and/or methods available in the public domain. Adaptive optics scanning laser ophthalmoscope (AOSLO) imaging was performed in a subset of patients. Outer retinal changes were detectable by OCT with an age-related effect on the foveal disease stage. Rod and short-wavelength-sensitive (S) cone functions were relatively retained by perimetry, although likely impacted by age-related increases in the pre-retinal absorption of short-wavelength lights. The central macula showed a large loss of red sensitivity on dark-adapted microperimetry. Chromatic VAs with high-contrast red gratings on a blue background were not detectable. Color vision was severely deficient. AOSLO imaging showed reduced total cone density with majority of the population being non-waveguiding. This study developed and evaluated specialized outcomes that will be needed for the determination of efficacy and safety in human clinical trials. Dark-adapted microperimetry with a red stimulus sampling the central macula would be a key endpoint to evaluate the light sensitivity improvements. VA changes specific to L-opsin can be measured with red gratings on a bright blue background and should also be considered as outcome measures in future interventional trials.