Foxn4 is a temporal identity factor conferring mid/late-early retinal competence and involved in retinal synaptogenesis.

During development, neural progenitors change their competence states over time to sequentially generate different types of neurons and glia. Several cascades of temporal transcription factors (tTFs) have been discovered in Drosophila to control the temporal identity of neuroblasts, but the temporal regulation mechanism is poorly understood in vertebrates. Mammalian retinal progenitor cells (RPCs) give rise to several types of neuronal and glial cells following a sequential yet overlapping temporal order. Here, by temporal cluster analysis, RNA-sequencing analysis, and loss-of-function and gain-of-function studies, we show that the Fox domain TF Foxn4 functions as a tTF during retinogenesis to confer RPCs with the competence to generate the mid/late-early cell types: amacrine, horizontal, cone, and rod cells, while suppressing the competence of generating the immediate-early cell type: retinal ganglion cells (RGCs). In early embryonic retinas, Foxn4 inactivation causes down-regulation of photoreceptor marker genes and decreased photoreceptor generation but increased RGC production, whereas its overexpression has the opposite effect. Just as in Drosophila, Foxn4 appears to positively regulate its downstream tTF Casz1 while negatively regulating its upstream tTF Ikzf1. Moreover, retina-specific ablation of Foxn4 reveals that it may be indirectly involved in the synaptogenesis, establishment of laminar structure, visual signal transmission, and long-term maintenance of the retina. Together, our data provide evidence that Foxn4 acts as a tTF to bias RPCs toward the mid/late-early cell fates and identify a missing member of the tTF cascade that controls RPC temporal identities to ensure the generation of proper neuronal diversity in the retina.

Cone photoreceptor classification in the living human eye from photostimulation-induced phase dynamics.

Human color vision is achieved by mixing neural signals from cone photoreceptors sensitive to different wavelengths of light. The spatial arrangement and proportion of these spectral types in the retina set fundamental limits on color perception, and abnormal or missing types are responsible for color vision loss. Imaging provides the most direct and quantitative means to study these photoreceptor properties at the cellular scale in the living human retina, but remains challenging. Current methods rely on retinal densitometry to distinguish cone types, a prohibitively slow process. Here, we show that photostimulation-induced optical phase changes occur in cone cells and carry substantial information about spectral type, enabling cones to be differentiated with unprecedented accuracy and efficiency. Moreover, these phase dynamics arise from physiological activity occurring on dramatically different timescales (from milliseconds to seconds) inside the cone outer segment, thus exposing the phototransduction cascade and subsequent downstream effects. We captured these dynamics in cones of subjects with normal color vision and a deuteranope, and at different macular locations by: (i) marrying adaptive optics to phase-sensitive optical coherence tomography to avoid optical blurring of the eye, (ii) acquiring images at high speed that samples phase dynamics at up to 3 KHz, and (iii) localizing phase changes to the cone outer segment, where photoactivation occurs. Our method should have broad appeal for color vision applications in which the underlying neural processing of photoreceptors is sought and for investigations of retinal diseases that affect cone function.

Thyroid hormone signaling specifies cone subtypes in human retinal organoids.

The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.

Spectral and spatial selectivity of luminance vision in reef fish.

Luminance vision has high spatial resolution and is used for form vision and texture discrimination. In humans, birds and bees luminance channel is spectrally selective-it depends on the signals of the long-wavelength sensitive photoreceptors (bees) or on the sum of long- and middle-wavelength sensitive cones (humans), but not on the signal of the short-wavelength sensitive (blue) photoreceptors. The reasons of such selectivity are not fully understood. The aim of this study is to reveal the inputs of cone signals to high resolution luminance vision in reef fish. Sixteen freshly caught damselfish, Pomacentrus amboinensis, were trained to discriminate stimuli differing either in their color or in their fine patterns (stripes vs. cheques). Three colors ("bright green", "dark green" and "blue") were used to create two sets of color and two sets of pattern stimuli. The "bright green" and "dark green" were similar in their chromatic properties for fish, but differed in their lightness; the "dark green" differed from "blue" in the signal for the blue cone, but yielded similar signals in the long-wavelength and middle-wavelength cones. Fish easily learned to discriminate "bright green" from "dark green" and "dark green" from "blue" stimuli. Fish also could discriminate the fine patterns created from "dark green" and "bright green". However, fish failed to discriminate fine patterns created from "blue" and "dark green" colors, i.e., the colors that provided contrast for the blue-sensitive photoreceptor, but not for the long-wavelength sensitive one. High resolution luminance vision in damselfish, Pomacentrus amboinensis, does not have input from the blue-sensitive cone, which may indicate that the spectral selectivity of luminance channel is a general feature of visual processing in both aquatic and terrestrial animals.

Unsupervised learning of cone spectral classes from natural images.

The first step in the evolution of primate trichromatic color vision was the expression of a third cone class not present in ancestral mammals. This observation motivates a fundamental question about the evolution of any sensory system: how is it possible to detect and exploit the presence of a novel sensory class? We explore this question in the context of primate color vision. We present an unsupervised learning algorithm capable of both detecting the number of spectral cone classes in a retinal mosaic and learning the class of each cone using the inter-cone correlations obtained in response to natural image input. The algorithm's ability to classify cones is in broad agreement with experimental evidence about functional color vision for a wide range of mosaic parameters, including those characterizing dichromacy, typical trichromacy, anomalous trichromacy, and possible tetrachromacy.

Transcriptional co-regulation of evolutionarily conserved microRNA/cone opsin gene pairs: implications for photoreceptor subtype specification.

The vertebrate retina contains two types of photoreceptor cells, rods and cones, which use distinct types of opsins and phototransduction proteins. Cones can be further divided into several subtypes with differing wavelength sensitivity and morphology. Although photoreceptor development has been extensively studied in a variety of vertebrate species, the mechanism by which photoreceptor subtypes are established is still largely unknown. Here we report two microRNAs (miRNAs), miR-726 and miR-729, which are potentially involved in photoreceptor subtype specification. In the medaka Oryzias latipes, the genes encoding miR-726 and miR-729 are located upstream of the red-sensitive opsin gene LWS-A and the UV-sensitive opsin gene SWS1, respectively, and are transcribed in the opposite direction from the respective opsin genes. The miR-726/LWS pair is conserved between teleosts and tetrapods, and the miR-729/SWS1 pair is conserved among teleosts. in situ hybridization analyses and fluorescence reporter assays suggest that these miRNAs are co-expressed with the respective opsins in specific cone subtypes. Potential targets of miR-726 and miR-729 predicted in silico include several transcription factors that regulate photoreceptor development. Functional analyses of cis-regulatory sequences in vivo suggest that transcription of the paired microRNA and opsin genes is co-regulated by common cis-regulatory modules. We propose an evolutionarily conserved mechanism that controls photoreceptor subtype identity through coupling between transcriptional and post-transcriptional regulations.

Sensory experience shapes the development of the visual system's first synapse.

Specific connectivity patterns among neurons create the basic architecture underlying parallel processing in our nervous system. Here we focus on the visual system's first synapse to examine the structural and functional consequences of sensory deprivation on the establishment of parallel circuits. Dark rearing reduces synaptic strength between cones and cone bipolar cells, a previously unappreciated effect of sensory deprivation. In contrast, rod bipolar cells, which utilize the same glutamate receptor to contact rods, are unaffected by dark rearing. Underlying the physiological changes, we find the localization of metabotropic glutamate receptors within cone bipolar, but not rod bipolar, cell dendrites is a light-dependent process. Furthermore, although cone bipolar cells share common cone partners, each bipolar cell type that we examined depends differentially on sensory input to achieve mature connectivity. Thus, visual experience differentially affects maturation of rod versus cone pathways and of cell types within the cone pathway.

A tale of two retinal domains: near-optimal sampling of achromatic contrasts in natural scenes through asymmetric photoreceptor distribution.

For efficient coding, sensory systems need to adapt to the distribution of signals to which they are exposed. In vision, natural scenes above and below the horizon differ in the distribution of chromatic and achromatic features. Consequently, many species differentially sample light in the sky and on the ground using an asymmetric retinal arrangement of short- (S, "blue") and medium- (M, "green") wavelength-sensitive photoreceptor types. Here, we show that in mice this photoreceptor arrangement provides for near-optimal sampling of natural achromatic contrasts. Two-photon population imaging of light-driven calcium signals in the synaptic terminals of cone-photoreceptors expressing a calcium biosensor revealed that S, but not M cones, preferred dark over bright stimuli, in agreement with the predominance of dark contrasts in the sky but not on the ground. Therefore, the different cone types do not only form the basis of "color vision," but in addition represent distinct (achromatic) contrast-selective channels.

Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors.

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 ß2 (trß2) 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 trß2 activity before cone differentiation. Ectopic expression of trß2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trß2 expression could explain why some species have mixed, and others have pure, cone types.

What has driven the evolution of multiple cone classes in visual systems: object contrast enhancement or light flicker elimination?

BACKGROUND: Two competing theories have been advanced to explain the evolution of multiple cone classes in vertebrate eyes. These two theories have important, but different, implications for our understanding of the design and tuning of vertebrate visual systems. The 'contrast theory' proposes that multiple cone classes evolved in shallow-water fish to maximize the visual contrast of objects against diverse backgrounds. The competing 'flicker theory' states that multiple cone classes evolved to eliminate the light flicker inherent in shallow-water environments through antagonistic neural interactions, thereby enhancing object detection. However, the selective pressures that have driven the evolution of multiple cone classes remain largely obscure. RESULTS: We show that two critical assumptions of the flicker theory are violated. We found that the amplitude and temporal frequency of flicker vary over the visible spectrum, precluding its cancellation by simple antagonistic interactions between the output signals of cones. Moreover, we found that the temporal frequency of flicker matches the frequency where sensitivity is maximal in a wide range of fish taxa, suggesting that the flicker may actually enhance the detection of objects. Finally, using modeling of the chromatic contrast between fish pattern and background under flickering illumination, we found that the spectral sensitivity of cones in a cichlid focal species is optimally tuned to maximize the visual contrast between fish pattern and background, instead of to produce a flicker-free visual signal. CONCLUSIONS: The violation of its two critical assumptions substantially undermines support for the flicker theory as originally formulated. While this alone does not support the contrast theory, comparison of the contrast and flicker theories revealed that the visual system of our focal species was tuned as predicted by the contrast theory rather than by the flicker theory (or by some combination of the two). Thus, these findings challenge key assumptions of the flicker theory, leaving the contrast theory as the most parsimonious and tenable account of the evolution of multiple cone classes.

[Current views on vision of mammals].

In the review, research data are presented on mammals' vision including visual pigments, color and contrast vision, and visual behaviour in different species. It is shown that in course of evolution mammals were gradually losing the elements of daylight cone vision system that are typical of other vertebrates. In monotremes, visual pigments SWS2 (cone blue-sensitive 2) and MWS/LWS (green/red-sensitive) are still present, as well as rod RH1. Theria, except some primates, also have two cone visual pigments: SWS1 (ultraviolet/violet or blue-sensitive 1) and MWS/LWS along with rod RH1. Humans and some other higher primates evolved the new visual pigment, MWS, and acquired trichromatic vision. Marine mammals (cetaceans and pinnipeds) and some species of other orders have lost also the visual pigment SWS1, probably due to specificity of processing the information received by these cones. Current view on mammals' vision with two cone pigments and rods is presented. Data on maximum spectral sensitivity of visual pigments in different species and orders are given along with data on spatial contrast sensation. High visual acuity has been acquired by ungulates, artiodactyls, and primates, while the highest one--by humans with their specialized fovea.

A distinct contribution of short-wavelength-sensitive cones to light-evoked activity in the mouse pretectal olivary nucleus.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) combine inputs from outer-retinal rod/cone photoreceptors with their intrinsic phototransduction machinery to drive a wide range of so-called non-image-forming (NIF) responses to light. Defining the contribution of each photoreceptor class to evoked responses is vital for determining the degree to which our sensory capabilities depend on melanopsin and for optimizing NIF responses to benefit human health. We addressed this problem by recording electrophysiological responses in the mouse pretectal olivary nucleus (PON) (a target of ipRGCs and origin of the pupil light reflex) to a range of gradual and abrupt changes in light intensity. Dim stimuli drove minimal changes in PON activity, suggesting that rods contribute little under these conditions. To separate cone from melanopsin influences, we compared responses to short (460 nm) and longer (600/655 nm) wavelengths in mice carrying a red shifted cone population (Opn1mw®) or lacking melanopsin (Opn4⁻/⁻). Our data reveal a surprising difference in the quality of information available from medium- and short-wavelength-sensitive cones. The majority cone population (responsive to 600/655 nm) supported only transient changes in firing and responses to relatively sudden changes in light intensity. In contrast, cones uniquely sensitive to the shorter wavelength (S-cones) were better able to drive responses to gradual changes in illuminance, contributed a distinct off inhibition, and at least partially recapitulated the ability of melanopsin to sustain responses under continuous illumination. These data reveal a new role for S-cones unrelated to color vision and suggest renewed consideration of cone contributions to NIF vision at shorter wavelengths.

Cone photoreceptor contributions to noise and correlations in the retinal output.

Transduction and synaptic noise generated in retinal cone photoreceptors determine the fidelity with which light inputs are encoded, and the readout of cone signals by downstream circuits determines whether this fidelity is used for vision. We examined the effect of cone noise on visual signals by measuring its contribution to correlated noise in primate retinal ganglion cells. Correlated noise was strong in the responses of dissimilar cell types with shared cone inputs. The dynamics of cone noise could account for rapid correlations in ganglion cell activity, and the extent of shared cone input could explain correlation strength. Furthermore, correlated noise limited the fidelity with which visual signals were encoded by populations of ganglion cells. Thus, a simple picture emerges: cone noise, traversing the retina through diverse pathways, accounts for most of the noise and correlations in the retinal output and constrains how higher centers exploit signals carried by parallel visual pathways.

Chromatic bipolar cell pathways in the mouse retina.

Like most mammals, mice feature dichromatic color vision based on short (S) and middle (M) wavelength-sensitive cone types. It is thought that mammals share a retinal circuit that in dichromats compares S- and M-cone output to generate blue/green opponent signals, with bipolar cells (BCs) providing separate chromatic channels. Although S-cone-selective ON-BCs (type 9 in mouse) have been anatomically identified, little is known about their counterparts, the M-cone-selective OFF-BCs. Here, we characterized cone connectivity and light responses of selected mouse BC types using immunohistochemistry and electrophysiology. Our anatomical data indicate that four (types 2, 3a/b, and 4) of the five mouse OFF-BCs indiscriminately contact both cone types, whereas type 1 BCs avoid S-cones. Light responses showed that the chromatic tuning of the BCs strongly depended on their position along the dorsoventral axis because of the coexpression gradient of M- and S-opsin found in mice. In dorsal retina, where coexpression is low, most type 2 cells were green biased, with a fraction of cells (≈ 14%) displaying strongly blue-biased responses, likely reflecting S-cone input. Type 1 cells were also green biased but did not comprise blue-biased "outliers," consistent with type 1 BCs avoiding S-cones. We therefore suggest that type 1 represents the green OFF pathway in mouse. In addition, we confirmed that type 9 BCs display blue-ON responses. In ventral retina, all BC types studied here displayed similar blue-biased responses, suggesting that color vision is hampered in ventral retina. In conclusion, our data support an antagonistically organized blue/green circuit as the common basis for mammalian dichromatic color vision.

The influence of retinal illuminance on L- and M-cone driven electroretinograms.

The electroretinographic response to L- and M-cone isolating stimuli was measured at different luminance levels to study the effect of retinal illuminance on amplitude and phase, and how this may influence estimates of L:M ratios in the retina. It was found that the amplitude of L- and M-cone driven responses increases differently with increasing retinal illuminance: L-cone responses increase more quickly than those of M-cones. The L:M ratio does not change strongly with retinal illuminance. The phase of both L- and M-cone driven responses advances with increasing retinal illuminance. There is considerable interindividual variability in the phase difference between the two, but generally M-cone driven responses are phase advanced.

L- and M-cone ratios and magnocellular sensitivity in reading.

It has been proposed that magnocellular deficits cause the reading problems in dyslexia. However, how magnocellular deficiencies are supposed to cause these problems is unclear. Recently it has been proposed that reading performance is limited by the L-/M-cone inputs to the magnocellular system. However, as explained in this review, this is problematic for a number of reasons. Particularly difficult is the linking of L- and M-cone sensitivity specifically to the magnocellular system.

Ret-PCP2 colocalizes with protein kinase C in a subset of primate ON cone bipolar cells.

Purkinje cell protein 2 (PCP2), a member of the family of guanine dissociation inhibitors and a strong interactor with the G-protein subunit G alpha(o), localizes to retinal ON bipolar cells. The retina-specific splice variant of PCP2, Ret-PCP2, accelerates the light response of rod bipolar cells by modulating the mGluR6 transduction cascade. All ON cone bipolar cells express mGluR6 and G alpha(o), but only a subset expresses Ret-PCP2. Here we test the hypothesis that Ret-PCP2 contributes to shaping the various temporal bandwidths of ON cone bipolar cells in monkey retina. We found that the retinal splice variants in monkey and mouse are similar and longer than the cerebellar variants. Ret-PCP2 is strongly expressed by diffuse cone bipolar type 4 cells (DB4; marked with anti-PKCalpha) and weakly expressed by midget bipolar dendrites (labeled by antibodies against G alpha(o), G gamma 13, or mGluR6). Ret-PCP2 is absent from diffuse cone bipolar type 6 (DB6; marked with anti-CD15) and blue cone bipolar cells (marked with anti-CCK precursor). Thus, cone bipolar cells that terminate in stratum 3 of the inner plexiform layer (DB4) express more Ret-PCP2 than those that terminate in strata 3 + 4 (midget bipolar cells), and these in turn express more than those that terminate in stratum 5 (DB6 and blue cone bipolar cells). This expression pattern approximates the arborization of ganglion cells (GC) with different temporal bandwidths: parasol GCs stratifying near stratum 3 are faster than midget GCs stratifying in strata 3 + 4, and these are probably faster than the sluggish GCs that arborize in stratum 5.

Avian cone photoreceptors tile the retina as five independent, self-organizing mosaics.

The avian retina possesses one of the most sophisticated cone photoreceptor systems among vertebrates. Birds have five types of cones including four single cones, which support tetrachromatic color vision and a double cone, which is thought to mediate achromatic motion perception. Despite this richness, very little is known about the spatial organization of avian cones and its adaptive significance. Here we show that the five cone types of the chicken independently tile the retina as highly ordered mosaics with a characteristic spacing between cones of the same type. Measures of topological order indicate that double cones are more highly ordered than single cones, possibly reflecting their posited role in motion detection. Although cones show spacing interactions that are cell type-specific, all cone types use the same density-dependent yardstick to measure intercone distance. We propose a simple developmental model that can account for these observations. We also show that a single parameter, the global regularity index, defines the regularity of all five cone mosaics. Lastly, we demonstrate similar cone distributions in three additional avian species, suggesting that these patterning principles are universal among birds. Since regular photoreceptor spacing is critical for uniform sampling of visual space, the cone mosaics of the avian retina represent an elegant example of the emergence of adaptive global patterning secondary to simple local interactions between individual photoreceptors. Our results indicate that the evolutionary pressures that gave rise to the avian retina's various adaptations for enhanced color discrimination also acted to fine-tune its spatial sampling of color and luminance.

Blue-yellow opponency in primate S cone photoreceptors.

The neural coding of human color vision begins in the retina. The outputs of long (L)-, middle (M)-, and short (S)-wavelength-sensitive cone photoreceptors combine antagonistically to produce "red-green" and "blue-yellow" spectrally opponent signals (Hering, 1878; Hurvich and Jameson, 1957). Spectral opponency is well established in primate retinal ganglion cells (Reid and Shapley, 1992; Dacey and Lee, 1994; Dacey et al., 1996), but the retinal circuitry creating the opponency remains uncertain. Here we find, from whole-cell recordings of photoreceptors in macaque monkey, that "blue-yellow" opponency is already present in the center-surround receptive fields of S cones. The inward current evoked by blue light derives from phototransduction within the outer segment of the S cone. The outward current evoked by yellow light is caused by feedback from horizontal cells that are driven by surrounding L and M cones. Stimulation of the surround modulates calcium conductance in the center S cone.