Biochemistry and physiology of zebrafish photoreceptors.

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade.

Introduction

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones. Rod photoreceptors are characterized by higher light sensitivity and slower kinetics, mainly mediating monochromatic low-light vision [191, 50, 57, 105]. Cone photoreceptors on the other hand function under bright light, conveying luminance and color information. In vertebrates, they come in up to four different subtypes, depending on their peak absorption. Both photoreceptor types share a peculiar morphology with a large outer segment comprised of an ordered stack of discs, which contain the proteins of the visual transduction cascade. This biochemical pathway transforms the physical stimulus of light into a biological signal. Outer segments are modified primary cilia that are connected via an axoneme to the mitochondrium-rich inner segment [84]. Synapses of the photoreceptors are among the most complex synapses in the vertebrate brain, featuring ribbons that are thought to enable tonic glutamate release into the synapse [162, 175].

Photoreceptors have been intensively studied in different model organisms. Biochemists favor large bovine eyes for their large yield of proteins. Electrophysiologists favor the amphibians for their comparatively large photoreceptors and geneticists have mainly focused on rodent eyes due to the genetic amenities available in these systems.

More recently, the small tropical teleost zebrafish (Danio rerio) joined the ranks of model system for retinal research. Besides their favorable biological properties, such as small body size, easy maintenance, and large number of offspring, there are several properties of their visual system that have endeared this model system to visual scientists [157]. Unlike the rod-dominant amphibian or rodent retina, the majority of photoreceptors in zebrafish are cones, with about 92% in zebrafish larvae and about 60% in the adult [49, 2, 222]. The larval retina also serves as a model for the primate fovea, featuring a cone-rich acute zone responsible for prey detection [212]. Moreover, more than 70% of human genes have direct orthologues in the zebrafish genome [69], making zebrafish an ideal model to study eye or more specifically cone diseases in humans [61, 11, 113]. The genetic toolbox to manipulate zebrafish has massively expanded during the past decade, including DNA insertion, precisely controlled transgene expression, and CRISPR/Cas genome editing [131]. Because the zebrafish retina starts to transmit visual information at very early stages (3 days post fertilization (dpf)), the function of the visual system can be assessed at early larval stages. Finally, zebrafish larvae are transparent, making them well suited for live imaging (e.g., [135, 222]).

Zebrafish retina signaling with related ocular and retinal diseases have been reviewed recently [8, 123, 113, 126, 18, 11]. In this review, we will provide an overview of biochemical and physiological processes in zebrafish photoreceptors with a focus on the visual transduction cascade, the very first step of image-forming vision.

Zebrafish outer retina

The zebrafish retina possesses one rod type and four morphologically and spectrally distinct cone subtypes, namely short single cones (ultraviolet (UV)-sensitive), long single cones (blue-sensitive), double cone accessory members (green-sensitive), and double-cone principle members (red-sensitive). Double cones exist in most vertebrates, but are absent in most placental mammals, elasmobranches, and catfish [44]. Zebrafish photoreceptors are coupled by gap junction, mainly mediated by Connexin 35 (the zebrafish homologue of mammalian Cx36) [111]. Fish photoreceptor coupling is regulated by the circadian clock, with cone-cone and rod-cone coupling being increased during nighttime [151, 111].

In the absence of pupillary reflexes, many lower vertebrates developed retinomotor movements to adapt to changes in light conditions. In darkness, a mobile part of photoreceptor inner segment, called the myoid, drives cones to elongate and rods to contract [124, 67]. Meanwhile, pigment granules (melanosomes) of the retinal pigment epithelium (RPE) concentrate at the basal part of the RPE. In this way, cone outer segments are buried deeply inside basal RPE while rod outer segments are optimally exposed to incoming light, by being situated far from pigment granules. During light adaptation, cones contract while rods elongate concomitant to pigment granule translocation towards the apical part of the RPE. Therefore, cone outer segments are exposed to light and the rod outer segments are protected by the RPE, akin to sunglasses [1]. The zebrafish retina shows adult-like retinomotor movement from 28 dpf on. Pigment granules take about an hour to migrate to fully light adapted position, while double cone outer segment contraction finishes in about 20 minutes [124, 67]

Longitudinal sections of adult retina demonstrate that different photoreceptors are organized into different layers in zebrafish [153, 20] (Fig. 1a, b). The nuclei of rods are located distal to all cone nuclei. The nuclei of UV cones, blue cones, and double cones are located in the distal, medial, and proximal zones of the outer retina, respectively. Cone photoreceptors in the adult zebrafish retina are orderly arranged into a row mosaic pattern (Fig. 1c), in which a red cone neighbors a blue cone while a green cone neighbors a UV cone [2, 107]. Rods project into a square shape around each UV cone to form an integral photoreceptor mosaic [49].

Fig. 1

Adult zebrafish retina and photoreceptor mosaic. Dark-adapted adult zebrafish retina section (a) and light-adapted section (b) are organized into different cellular layers. The nuclei of rod and cone photoreceptors are located in the outer nuclear layer. During light adaptation, photoreceptor myoid drives cones to contract and rods to elongate to protect rods from over-bleaching, known as retinomotor movement. UV opsin (sws1) is labeled by in situ hybridization. Arrowhead denotes double cone. Arrow denotes blue cone. Star denotes cell body of rod. Schematic of the zebrafish photoreceptor planar mosaic arrangement (c) [153, 2, 49]. UV, UV cone; R, red cone; G, green cone; B, blue cone

The situation is different in the larval retina, where photoreceptors are anisotropically distributed [222]. All cone types are concentrated at the horizon and lower visual field, which may mediate color vision. UV cone density shows a peak at around 30° above the horizon, which is essential for visual prey hunting [212]. The upper visual field is dominated by rods, supporting the effectively achromatic vision towards the sky. This anisotropic distribution is adapted to the spectral content in the natural visual environment serving behavioral demands.

Visual transduction cascade

The main function of photoreceptors is the capture of photons of visual light and the subsequent transformation of this physical stimulus into a biological signal, ultimately modifying the release of the neurotransmitter glutamate by the photoreceptor synapse [50, 105, 57, 22]. The visual transduction cascade and its regulation are among the best-understood trimeric G protein signaling pathways. All reactions take place in the outer segments of photoreceptors, most of them associated with the membrane (Fig. 2). Zebrafish genes involved in visual transduction cascade are summarized in Table 1.

Fig. 2

Cone photoreceptor morphology (a). Schematic representation of vertebrate visual transduction cascade and Ca2+-regulated deactivation processes (b). Photon absorption activates R. R* then triggers the exchange of GTP for GDP on the Tα. Tα-GTP binds to cyclic nucleotide PDE. Stimulated PDE hydrolyzes free cyclic guanosine monophosphate (cGMP). In darkness, CNGC allows an influx of Na+ and Ca2+, while during illumination CNGC is shut off by cGMP decrease. NCKX is not affected by light, which results in a light-induced intracellular Ca2+ concentration decline. Rcv modulates phosphorylation of R* via GRK in a Ca2+-dependent manner. Phosphorylated R then is fully deactivated by the binding of Arr. R, visual pigment (inactive); R*, light-activated visual pigment; Tα, transducin α subunit; Tβγ, transducin β and γ subunits; PDE, phosphodiesterase (inactive); PDE*, PDE-transducin α complex: NCKX, Na+/Ca2+, K+ exchanger; Arr, arrestin; GRK, G protein–coupled receptor kinase; Rcv, Recoverin; CNGC, cyclic nucleotide–gated ion channel; P, phosphorylation; M, CNG-modulin; GC, guanylate cyclase; GCAP, guanylate cyclase activating protein. Figure was drawn using Inkscape. Inkscape http://www.inkscape.org/. Reproduced with permission from Zang and Neuhauss [217]

Table 1

Summary of zebrafish genes involved in visual transduction cascade

Gene name	Protein encoded	Expression pattern in photoreceptor layer	Phenotype in zebrafish with abnormal gene expression	Mouse homologs	Associated human eye diseases
opn1sw1	UV opsin	UV cones		Opn1sw	Tritan color blindness [200, 201]
opn1sw2	Blue opsin	Blue cones
opn1mw1	Green opsin	Green cones			Achromatopsia [130]
opn1mw2	Green opsin	Green cones
opn1mw3	Green opsin	Green cones
opn1mw4	Green opsin	Green cones
opn1lw1	Red opsin	Red cones		Opn1mw	Achromatopsia [130]
opn1lw2	Red opsin	Red cones		Opn1mw
rho	Rod opsin	Rods	Rod photoreceptor degeneration [218]	Rho	Night blindness [168], retinitis pigmentosa [43]
gnat1	Transducin α subunits	Rods and UV cones		Gnat1	Night blindness [128]
gnb1a	Transducin β subunits	Rods and UV cones		Gnb1
gnb1b	Transducin β subunits	Rods and UV cones		Gnb1
gngt1	Transducin γ subunits	Rods and UV cones		Gngt1
gnat2	Transducin α subunits	Cones	Largely reduced photoresponse [21]	Gnat2	Achromatopsia [91]
gnb3a	Transducin β subunits	Cones		Gnb3
gnb3b	Transducin β subunits	Cones		Gnb3
gngt2a	Transducin γ subunits	Cones		Gngt2
gngt2b	Transducin γ subunits	Cones		Gngt2
pde6a	PDE catalytic α subunit	Rods and UV cones		Pde6a	Autosomal recessive retinitis pigmentosa [72]
pde6b	PDE catalytic β subunit	Rods and UV cones		Pde6b	Autosomal recessive retinitis pigmentosa [122]
pde6ga	PDE inhibitory γ subunit	Rods and UV cones		Pde6g
pde6gb	PDE inhibitory γ subunit	Rods and UV cones		Pde6g
pde6c	PDE catalytic α′ subunit	Cones	Diminished cone ERG and OKR, and cone degeneration [134, 172]	Pde6c	Cone dysfunction and achromatopsia [27, 59, 186]
pde6ha	PDE inhibitory γ′ subunit	Cones		Pde6h
pde6hb	PDE inhibitory γ′ subunit	Cones		Pde6h
pde6i	PDE inhibitory γ′ subunit
cnga1a	CNG channel α1 subunit	Rods		Cnga1	Autosomal recessive retinitis pigmentosa [42]
cnga1b	CNG channel α1 subunit	Rods		Cnga1
cngb1a	CNG channel β1 subunit	Rods		Cngb1	Autosomal recessive retinitis pigmentosa [5, 14, 93]
cngb1b	CNG channel β1 subunit	Rods		Cngb1
cnga3a	CNG channel α3 subunit	Cones		Cnga3
cnga3b	CNG channel α3 subunit	Cones		Cnga3
cngb3.1	CNG channel β3 subunit	Cones		Cngb3
cngb3.2	CNG channel β3 subunit	Cones		Cngb3
grk1a	G protein–coupled receptor kinase 1a	Rods	Overexpression of grk1a in rods shows minor effect [194]	Grk1	Oguchi disease [209]
grk1b	G protein–coupled receptor kinase 1b	Cones	Delayed ERG response recovery and reduced temporal contrast sensitivity[31]	Grk1
grk7a	G protein–coupled receptor kinase 7a	Cones	grk7a knockdown [152], grk7a knockout [31], ectopic expression of grk7a in rods [194]
grk7b	G protein–coupled receptor kinase 7b	UV cones
arrSa	ArrestinSa	Rods and UV cones		Arr1	Oguchi disease [58]
arrSb	ArrestinSb	Rods and UV cones		Arr1
arr3a	Arrestin3a	Double cones	Delayed ERG response recovery and decreased temporal contrast sensitivity [148]	Arr3
arr3b	Arrestin3b	Blue and UV cones		Arr3
rgs9a	Regulators of G protein signaling 9a	Cones		Rgs9	Bradyoposia [133]
rgs9b	Regulators of G protein signaling 9b	Rods		Rgs9
gucy2e	Guanylate cyclase E	Rods and UV cones	Outer segment loss and shortening, OMR defects [176]	Gucy2e	Leber congenital amaurosis 1 [142]
gucy2f	Guanylate cyclase F	Rods and UV cones		Gucy2f
gucy2d	Guanylate cyclase D	Cones	OKR and OMR impairments [127], PDE6c downregulation [79]	Gucy2d
slc24a1	Na+/Ca2+, K+ exchanger 1			Slc24a1	Congenital stationary night blindness [150]
slc24a2	Na+/Ca2+, K+ exchanger 2			Slc24a2
rcv1a	Recoverin 1a	Rods and UV cones	Accelerates photoresponse recovery [215]	Rcv1
rcv1b	Recoverin 1b	Cones
rcv2a	Recoverin 2a	Cones	Accelerates photoresponse recovery [215]
rcv2b	Recoverin 2b	Cones	Accelerates photoresponse recovery [215]
gcap1	Guanylate cyclase activation protein 1	Rods and UV cones		Guca1a	Autosomal dominant cone dystrophy [140]
gcap2	Guanylate cyclase activation protein 2	Rods and UV cones		Guca2b	Autosomal dominant retinal dystrophies [160]
gcap3	Guanylate cyclase activation protein 3	Cones	Prolonged photoresponse recovery [9]
gcap4	Guanylate cyclase activation protein 4	Cones
gcap5	Guanylate cyclase activation protein 5	Cones
gcap7	Guanylate cyclase activation protein 7	Cones
eml1	CNG-modulin	Cones	Reduced light sensitivity [94]	Eml1

The photoreceptor outer segment is a cylindrical structure comprised of an ordered stack of disc-shaped membranes, allowing a high concentration of transmembrane visual pigments and increasing the probability of photon catch [92].

Rod and cone photoreceptors share a generally similar visual transduction cascade, but adopt rod- or cone-specific protein isoforms for many of the cascade’s components. The evolution of these photoreceptor-specific paralogues is a well-studied paradigm for the fate of duplicated genes in evolution [103]. This is particularly true for teleost genomes that underwent a lineage-specific whole-genome duplication, following two rounds of whole-genome duplications in the early vertebrate lineage [7, 190, 184].

The generation, deletion, and fate of these duplicated genes add a fascinating complexity to the teleost visual transduction cascade that is beyond the scope of this review. However, the multitude of gene variants to be discussed in the following is the direct consequence of whole-genome duplications in the past [63, 103, 104, 106, 99, 100, 60].

The visual transduction cascade is initiated by the absorption of photons by opsins. These G protein–coupled 7-transmembrane receptors are covalently bound to a light-sensitive chromophore via a Schiff base forming the photopigment complex [73]. Upon the absorption of a photon, chromophore (most commonly vitamin A1 11-cis-retinal) in the photopigment complex isomerizes to all-trans-retinal, which activates the opsin (now referred to R*) by inducing a transformational change [51, 52]. Zebrafish cones express a total of 8 cone opsins, namely opn1sw1 (also known as sws1), opn1sw2 (also known as sws2), opn1mw1 (also known as rh2-1), opn1mw2 (also known as rh2-2), opn1mw3 (also known as rh2-3), opn1mw4 (also known as rh2-4), opn1lw1 (also known as lws1), and opn1lw2 (also known as lws2) [2, 145, 182]. Hence, there are four green (short wave length) and two red (long wavelength) opsin variants. These variants have slightly different peak absorption properties potentially allowing a bewildering range of fine-tuning of color perception [30, 23]. The expressions of these multiple rh2 and lws genes follow a spatiotemporal order during development [182]. Rod photoreceptors express only a single-rod opsin gene rho (also known as rh1) [218]. Mutations in human rod opsin may produce night blindness or retinal degeneration, while cone opsin defects may lead to achromatopsia [168, 43, 130, 200, 201]. For years, vitamin A1-based photopigment has been recognized as the sole photopigment existing in zebrafish photoreceptors under standard laboratory conditions [23, 30]. The peak absorption spectra (λmax) of A1-based photopigments differ markedly and cover a wide spectrum from 355 nm (UV) to 558 nm (red) in vivo. However, thyroid hormone (TH) treatment or colder water temperature may result in a transition from A1 to vitamin A2 (11-cis 3,4-didehydroretinal)-based photopigments. This demonstrates a functional A1-A2 photopigment interchange system in zebrafish [159, 3, 48]. The λmax of A2-based photopigments shifts towards longer wavelength relative to A1-based photopigment [115, 66]. This interchange system is frequently observed in freshwater fishes and amphibians, and may be adapted to the red-shifted light environment in fresh water compared with marine and terrestrial environments [149, 197, 222]. Another mechanism to tune photopigments is to change opsin expression levels. TH treatment has been reported to reduce lws2 (548 nm) and rh2-1 (467 nm), while increasing lws1 (558 nm) and rh2-2 (488 nm) in larvae, favoring the opsins with longer λmax [119]. Both the mechanisms red-shift the zebrafish photoreceptor spectral sensitivity. Moreover, in TH receptor-defective fish, retinal progenitors designed to become red cones are transfated into UV cones, providing another mechanism for TH to regulate long-wavelength vision [180, 195, 37].

Besides the visual opsins, the zebrafish genome harbors 32 nonvisual opsin genes, which encode opsins forming functional photopigments with different chromophores [35, 34, 56]. Many, but not all of them, are expressed in the photoreceptor layer. Their functions in photoreceptors are largely unknown, but a role in circadian light entrainment is discussed [56, 174, 26].

Activated opsin (R*) interacts with the trimeric G protein transducin [22, 50, 105]. Binding of R* to transducin results in the replacement of GDP by GTP at the active site of the transducin α subunit. This nucleotide exchange dissociates the activated α subunit (Gα*) and the heterodimer of β and γ subunits (Gβγ). Gα* then binds to the cGMP Phosphodiesterase 6 (PDE6) [91, 128].

Zebrafish rod and cone photoreceptors express different variants of three subunits [99, 28]. In rods, gnat1 encodes transducin α subunits, gnb1a and gnb1b encode β subunits, and gngt1 encodes γ subunits (all these variants possibly also in UV cones), while in cones, gnat2 encodes α subunits, gnb3a and gnb3b encode β subunits, and gngt2a and gngt2b encode γ subunits.

Surprisingly, a zebrafish mutant defective in the cone-specific gnat2 gene (no optokinetic response f (nof)) shows a residual photoresponse that needs to be mediated by an unknown transducin-independent mechanism [21].

Interestingly, both Gα and Gβ show massive light-induced translocation from rod outer segment to inner segment in mice, which may contribute to light adaptation in rods [170]. However, Gα translocation has not been observed in zebrafish cones (or mouse cone), indicating light adaptation mechanisms may vary between rods and cones [85, 46, 114].

When Gα* binds to PDE6, two PDE6 inhibitory subunits dissociate from the active sites and allow the activation of PDE6 to hydrolyze cGMP [32]. The rod PDE6 variant is expressed as a heterotetramer consisting of two catalytic α and β subunits encoded by pde6a and pde6b, and two identical inhibitory γ subunits encoded by pde6g. Cone PDE6 comprises two homodimers of two catalytic α′ subunits encoded by pde6c and two inhibitory γ′ subunits encoded by pde6h [106, 62, 32, 72, 122].

Zebrafish retain the same set of catalytic subunit genes as in humans (pde6a, pde6b, and pde6c), while inhibitory subunits are encoded by duplicated paralogues: pde6ga and pde6gb in rods and possibly UV cones and pde6ha and pde6hb in all cones [100, 134]. An additional inhibitory subunit gene pde6i has also been found in zebrafish, and some other lower vertebrates including fish (teleost and non-teleost) and amphibians [100].

Mutations in the cone-specific pde6c gene are associated with cone dysfunction in human patients with achromatopsia [27, 59, 186]. Mutations in cone-catalytic subunit pde6c result in almost diminished cone electroretinogram (ERG) and optokinetic response (OKR), and cone photoreceptor degeneration in zebrafish [134, 172]. The mechanism underlying cone degeneration is unknown and is not linked to increased cytosolic Ca2+ levels [118].

Ultimately, the visual transduction cascade regulates the opening of cyclic nucleotide-gated (CNG) ion channels. These non-selective cation channels are opened by cGMP binding [210]. Falling cGMP concentration due to cGMP hydrolysis by PDE6 leads to the closure of these CNG channels, suppressing the circulating dark current and resulting in photoreceptor hyperpolarization. CNG channels are heteromeric proteins consisting of α and β subunits [81, 125]. Rod channels are assembled from 3 CNGA1 subunits and 1 CNGB1, while cone channels are assembled from 2 CNGA3 subunits and 2 CNGB3 subunits [202, 220, 221, 102].

Mutations in CNGA1 and CNGB1 have been identified in human patients with autosomal-recessive retinitis pigmentosa [14, 5, 93, 42]. In zebrafish, all visual CNG channel genes have retained two paralogues, but no additional information is available.

Regulation of visual transduction

At the biochemical level, visual transduction is mainly regulated by its deactivation kinetics. To deactivate the visual transduction cascade, deactivation of both R* and Gα-PDE* complex and the restoration of cGMP concentrations are required [22, 50].

The lifetime of R* is tightly regulated by arrestin proteins that efficiently inactivate photopigmet by binding to its phosphorylated form. Therefore, the first step of R* inactivation is phosphorylation. R* is phosphorylated by G protein–coupled receptor kinases (GRKs). Mice and rats express only GRK1 in both rods and cones, while humans express GRK1 in rods and GRK1 and GRK7 in cones [219, 117, 165, 199]. In zebrafish, both visual grk genes are present as two paralogues. grk1a is expressed exclusively in rods, grk1b and grk7a in all cones, and grk7b only in UV cones [152, 196] (unpublished data). GRK deficiency in humans leads to Oguchi disease, which is characterized by a delay of rod recovery [209]. A grk7a knockdown model produces largely delayed ERG response recovery and reduced temporal contrast sensitivity in the OKR [152]. Another study demonstrates similar but more modest effects in either grk1b or grk7a mutants [31].

Overexpression of grk1a in zebrafish rods shows minor effect on rod photoresponse, suggesting that endogenous GRK1a protein is already at saturation levels. Ectopic expression of cone grk7a in rods resulted in cone-like rod responses [194].

The binding of arrestin completely deactivates the phosphorylated photopigment [98, 203]. In the mouse retina, both rod (ARR1) and cone (ARR3) arrestins are co-expressed in cone photoreceptors [132, 203]. Mutations in ARR1 are a cause of Oguchi disease in human [58]. In zebrafish, arrsa and arrsb (orthologues of Arr1) are expressed in rods while arr3a exists in double cones and arr3b exists in blue and UV cones, indicating subfunctionalization of the two paralogues. arr3a knockdown resulted in a severe delay in ERG response recovery and decreased temporal contrast sensitivity [148].

Regulators of G protein signaling 9 (RGS9) act as GTPase activating protein to deactivate Gα*-PDE complex [17]. Mammals have a single Rgs9 gene, while zebrafish have two rgs9 genes, with rgs9a being expressed in cones and rgs9b in rods [33, 104] (unpublished data). Inactivating mutations in humans lead to bradyopsia, a rare condition characterized by slower photoreceptor deactivation [133]. A landmark study using Rgs9 overexpression in mice demonstrated its crucial role to rate-limit rod visual transduction recovery [96].

To restore the dark current, cGMP needs to be resynthesized by membrane-bound guanylate cyclases (GCs) [88, 167]. Photoreceptor-specific GCs are regulated by the small Ca2+-binding guanylate cyclase activation proteins (CGAPs) [90].

Mammals have two photoreceptor-specific GCs, GC-E (known as GC1) and GC-F (known as GC2), both of which are co-expressed in rods and cones [103, 88, 60]. GC-E is more concentrated in cones, while the expression of GC-F is more prominent in rods. Mutations in GC-E have been shown to cause Leber congenital amaurosis 1 (LCA1), a severe form of pediatric blindness in humans [142]. The zebrafish possess 3 GCs. GC-E (known as GC1), GC-F (known as GC2), and GC-D (known as GC3) are encoded by gucy2e (previous name gucy2f), gucy2f (previous name gc2), and gucy2d (previous name gc3), respectively. Both gucy2e and gucy2f are expressed in rods and UV cones, while gucy2d encodes the only cone-specific GC in all cone subtypes [55, 144].

A zebrafish gucy2d mutant has been identified in behavioral screen by displaying OKR and optomotor response (OMR) impairments [127]. PDE6c protein levels are downregulated in gucy2d knockdown larvae, indicating the interdependence between these two regulators of cGMP metabolism [79]. A knockdown of the gucy2d gene results in the loss and shortening of outer segments and defects in the OMR [176].

In darkness, the open non-selective CNG channels mediate a Ca2+ influx into the photoreceptor outer segment. Ca2+ efflux via Na+/Ca2+, K+ exchanger (NCKX) balances this influx, producing a moderately high intracellular Ca2+ concentration as shown in rods of different species [101, 207]. Under light illumination, CNG channels are closed due to the decrease in cGMP concentration, while Ca2+ efflux continues, resulting in a decrease of intracellular Ca2+ concentration in the outer segment [211]. This light-induced Ca2+ decline can be simultaneously measured with light response in zebrafish UV cones, demonstrating similar kinetics of Ca2+ extrusion via NCKX to that of CNG channel current [109].

NCKX proteins are encoded by SLC24 gene family members. They show a cell-type-specific expression with NCKX1 being expressed in rods and NCKX2 in cones [193, 147, 143, 150]. NCKX2-deficient mice show no or only mild functional defect, suggesting that compensating transporters may mediate ion exchange as well [112, 156]. A recent study proposed that NCKX2 and NCKX4 cooperated to facilitate the rapid and efficient extrusion of Ca2+ from mouse cones. NCKX4 has its well-established function in olfactory sensory neurons and is similarly expressed in all cones in the zebrafish retina [192]. The expression pattern of the other NCKX coding genes is unknown in zebrafish, but studies in the striped bass show expression of nckx1 in rods and four splice variants of nckx2 in cones [137].

The reduction of cytoplasmic Ca2+ negatively feedbacks to the phototransduction cascade, triggering the rapid photoresponse recovery and facilitating photoreceptor adaptation to background light [120, 129]. During light adaptation, photoreceptor light sensitivity is reduced and response kinetics is accelerated, to avoid saturation and to operate across a wide range of environmental light intensity [50]. This has been achieved by mechanisms that primarily involve the regulation of GRKs by Recoverin, GCs by GCAPs, and CNG channels by CNG-modulin (or Calmodulin) [138, 191].

Recoverin (RCV) is a small neuronal calcium sensor (NCS), which is primarily located in vertebrate photoreceptors. Upon Ca2+ binding, RCV undergoes a pronounced conformational change, the so-called Ca2+-myristoyl switch, which translocates the proteins from a cytosolic form to a membrane tethered conformation, allowing targeting and inhibiting GRK proteins [82, 166, 183, 6, 40, 83, 217]. Light stimulation reduces intracellular Ca2+ concentration, allowing the Ca2+-free RCV releasing GRK. GRK disinhibition accelerates R* phosphorylation, enabling arrestin binding.

While there is only one RCV isoform in mammals (RCV1), four rcv genes are encoded in the zebrafish genome (rcv1a, rcv1b, rcv2a, and rcv2b) [215]. rcv1b, rcv2a, and rcv2b are cone RCV, while rcv1a is expressed in rods and UV cones. Mouse RCV1 experiences a remarkable light-induced translocation from outer and inner segment towards synaptic terminals in rods, which has not been observed in zebrafish photoreceptors by studying all zebrafish RCVs [177] (unpublished observation). Downregulation of cone RCV accelerates photoresponse recovery, but this effect is abolished when cone GRK7a is simultaneously knocked-down. This result not only indicates that RCV regulates opsin deactivation via GRK, but also demonstrates that the cone opsin deactivation kinetics dominates the overall photoresponse shut off kinetics in vivo [215]. Interestingly, different RCVs contribute at distinct light intensities. This implies different Ca2+ sensitivities for these RCVs, since intracellular Ca2+ concentration correlates with light levels [158]. Indeed, a recent biochemical work demonstrated distinct Ca2+ affinities, Ca2+-dependent membrane binding, and Ca2+-induced conformational changes among zebrafish isoforms [45]. Furthermore, salamander cone photoresponse, but not rod response, is also dominated by a Ca2+-sensitive mechanism [121, 216]. If the Ca2+-sensitive dominance is a general feature in cone photoresponse, it may contribute to the more powerful light adaptation of cones compared to rods.

To restore the dark current, cGMP needs to be resynthesized by GC, which is under the regulation of small Ca2+-binding proteins called GCAPs [90, 39]. GCAPs belong to the superfamily of EF-hand Ca2+-binding proteins, harboring four EF-hand Ca2+-binding motifs, three of which are functional [89]. Unlike RCVs, GCAPs do not undergo a classical Ca2+-myristoyl switch, but the myristoyl group does play an important role to regulate GCAP properties, including Ca2+ sensitivity, GC affinity, and the catalytic efficiency of the enzyme. Ca2+-binding GCAPs together with GCs form GC/GCAP complex in darkness. Ca2+ reduction during light exposure triggers a conformational change in GCAPs, which results in a transformational change within the GC/GCAP complex, increases GC catalytic activity and reopens the CNG channels. During light adaptation, the Ca2+-sensitive GCAP activity will also prevent the closure of all CNG channels and keep photoreceptors responsive.

GCAP1 and 2 are expressed in mammalian rods and cones. The human (but not the mouse) genome also processes a cone-specific CGAP3 [75, 140, 160]. Zebrafish photoreceptors express six GCAPs, of which gcap3, 4, 5, and 7 are restricted to cones and gcap1 and 2 are exclusively expressed in rods and UV cones [76, 144, 54]. These isoforms show distinct Ca2+ sensitivities of GC activation, Ca2+/Mg2+-dependent conformational changes, and Ca2+-binding affinities [164, 179]. Light exposure allows intracellular Ca2+ fluctuating to different levels, in which distinct CGAPs may reach their optimal working range.

GCAP3 is first expressed in a non-myristoylated form in larvae and then becomes myristoylated in the adult retina [54]. Although GCAP3 has been shown to produce the highest Ca2+-dependent activation of GCs in native zebrafish retina, gcap3 knockdown does not induce any visual behavioral abnormalities [55]. In another study, GCAP3 in green cone was inactivated by antibody injections. Whole-cell patch clamp recordings demonstrated that the photoresponse recovery is strongly prolonged, confirming GCAP3 function to activate GC to restore CNG channel current in cones [9].

cGMP affinity of CNG channels is regulated in a Ca2+-dependent manner in all sensory neurons [19]. Ca2+ cannot directly bind to the channels but work via modulator proteins, which have been identified as calmodulin in mammalian rods and CNG-modulin in fish cones [70, 146]. However, the contribution of CNG channel modulation by Ca2+ in regulating light adaptation is very limited in rods [29, 95]. On the other hand, CNG-modulin has been shown to regulate the cGMP dependence of CNG channels in a Ca2+-sensitive manner, and to modulate the light response kinetics in striped bass cone [146]. CNG-modulin is encoded by the eml1 gene in zebrafish. eml1 knockdown reduces the light sensitivity of dark-adapted and light-adapted cones; the sensitivity cannot be restored to wild-type levels [94]. These experiments demonstrate a stronger Ca2+ feedback to CNG channels in cones compared to rods.

Outer segment: a specialized primary cilium

Photoreceptor outer segments are strongly modified specialized primary cilia, sharing many general structural and biochemical features of cilia [77]. Outer segment stacked discs are arranged on the side of a microtubule-based axoneme, anchoring inside the inner segment through a connecting cilium and its basal body. Therefore, the connecting cilium, known as the transition zone in other cell types, connects outer and inner segment, mediating bi-directional protein trafficking [181]. Dysfunctions of primary cilia result in human disorders referred to as ciliopathies, which were reviewed elsewhere [11].

Outer segments are constantly bombarded by photons and their integrity is endangered by radical oxygen species. Since photoreceptors, like most neurons of the central nervous system, cannot be replaced, photoreceptors constantly rejuvenate themselves by renewing their outer segments. New discs are synthesized by ciliary membrane evagination at the base of the outer segment as the ciliary ectosomes, which then is elongated, flattened, and enclosed inside the outer segment [87, 173, 38, 171]. The tips of the outer segments, containing the oldest and potentially damaged membranes, are phagocytosed and digested by RPE cells. Although outer segment renewal/shedding is essential for photoreceptor homeostasis and survival, molecular mechanisms underlying its regulation are still poorly understood.

Recent works on zebrafish have contributed significantly to our understanding of the molecular mechanisms behind photoreceptor outer segment shedding and renewal. The zebrafish lends itself ideally to transgenically label cellular structures or cells, as Willoughby and colleagues have used elegantly for the outer segment [205]. They devised a stable line with heat shock–inducible fluorescent membrane protein that allowed them to follow the renewal and shedding of the rod outer segments as an updated experimental approach to the classic radioactive labeling method [214]. This line was then used in a high-content small-molecule screens that among others identified an involvement of cyclooxygenase in outer segment growth, gamma secretase in outer segment shedding, and mTOR in RPE phagocytosis [25].

Some earlier studies demonstrated that disc shedding in frog and cat was initiated by light [15, 53]. A recent zebrafish study using PDE6 inhibitors to block the visual transduction cascade mimicking constant dark conditions indeed inhibited rod outer segment shedding [24]. Interestingly, mammalian rod outer segment shedding remains in constant darkness, instead showing circadian clock controlling disc shedding mechanism [108, 185, 64, 74].

Given the nature of the outer segment, it comes as no surprise that many genes associated with intracellular and ciliary trafficking are involved in outer segment generation and maintenance.

The most abundant protein that needs to be shipped out to the outer segment is rhodopsin. Every second, around 70 rhodopsin molecules are trafficked from the inner to the outer segment [213, 204, 141]. Detailed studies of rhodopsin transport in frogs showed that RAB8, a small GTPase, coats rhodopsin-carrier vesicles and directs them to a selective barrier at the base of connecting cilium [139, 36]. In live imaging experiments in zebrafish, RAB8-directed rhodopsin trafficking in rods has been directly visualized in vivo [135]. The correct localization of RAB8 at the base of the outer segment is regulated by components of the connecting cilium itself, such as CC2D2A and further interaction partners, such as Ninl and MICAL3 [10, 12].

About 10% of outer segment is renewed every day in mammalian photoreceptors [108]. Therefore, intraflagellar transport (IFT), which contributes primarily to traffic visual transduction proteins into the outer segment, is important for outer segment development and structure [77]. IFT-B complex and kinesin motors mediate anterograde movement towards the distal outer segment, while IFT-A and dynein motors mediate retrograde movement towards the cell body [154].

A series of zebrafish studies contributed greatly to our understanding of the mechanism underlying IFT. Mutations affecting the IFT-B complex (IFT52, IFT57, IFT88, IFT172) lead to defects in outer segment formation and/or maintenance, finally resulting in both rod and cone degeneration [188, 41, 65]. Biochemical assays indicated that IFT20, a IFT-A member, requires IFT57 to associate with the IFT particle [97]. In another study, TNF receptor-associated factor 3 interacting protein 1 (TRAF3IP1) was shown to bind to IFT20. It can also interact with RAB8 via Rabaptin5, an endocytosis regulator. This demonstrates a connection between the IFT particle and the GTPase pathway, known to facilitate protein complex assembling [136].

Moreover, microtubular motors play an essential role in transporting IFT complexes. KIF17, kinesin-2 family member, is involved in ciliogenesis [206]. It is located all over zebrafish cones but concentrates at the basal body and the distal tip of the axoneme [13]. Knockdown of kif17 disrupts outer segment structure and mislocates visual transduction proteins [78]. Disc shedding is also promoted by KIF17 and eliminated in its absence [110].

Ribbon synapses

Non-spiking photoreceptors respond and adapt to a wide range of light intensities. The light-induced CNG channel closure generates the graded changes in membrane potential, which in turn regulates tonic neurotransmitter glutamate release at the presynaptic terminals [175, 163, 187]. This graded signaling is facilitated by specialized ribbon synapses, which hold a dense array of synaptic vesicles near active zones along their surface and were firstly identified by electron microscopy as electron dense structures in guinea pig rod synapses [169].

Work on zebrafish has helped to identify the key components of ribbon synapses and their function in signal transmission.

Ribeye is the most abundant protein in the synaptic ribbon [163]. In the zebrafish retina, both ribeyea and ribeyeb are present in the photoreceptors while ribeyea also shows expression in bipolar cells. Downregulation of ribeyea diminishes OKR and reduces ribbon length and number [198, 116].

Synaptojanin (Synj1) is a polyphosphoinositide phosphatase regulating clathrin-mediated endocytosis in conventional synapses [155]. A zebrafish synj1 null mutation (nrc) shows unanchored “floating” ribbons and reduced synaptic vesicles in cone but not rod synapses[189, 68], associated with defect in vision [4].

Photoreceptor L-type voltage-dependent calcium channels (Cav1.4) are located in the vicinity of synaptic ribbons and mediate exocytosis [187]. In darkness, they are opened by the depolarized photoreceptor membrane potential, resulting in calcium-dependent glutamate release. Cav1.4 are heteromultimeric protein complexes comprising of a pore-forming α1F subunit, encoded by CACNA1F, and accessory β and α2δ subunits, encoded by CACNB2 and CACNA2D4, respectively. Mutations in CACNA1F gene result in X-linked congenital stationary night blindness type 2 and cone-rod dystrophy in human [16, 178]. Two paralogues, cacna1fa and cacna1fb are identified in zebrafish with cacna1fa being expressed in photoreceptors while cacna1fb only existing in the inner retina [80]. CACNA1Fa protein exclusively accumulates at the outer plexiform layer and its null mutants (wud) present thinner outer plexiform layer, defective ERG, completely absent of synaptic ribbons, and mislocalized Ribeyeb.

Mutations in human CACNA2D4 are related to autosomal recessive cone dystrophy, while rods in different CACNA2D4 knockout mouse lines are even more severely affected, showing missing or largely defective scotopic and photopic ERG response [208, 86, 71]. More recently, another study focused on zebrafish cacna2d4 encoding Cav1.4 α2δ subunit [116]. cacna2d4 is duplicated in zebrafish as cacna2d4a and cacna2d4b. Double KO shows reduced pore-forming CACNA1Fa expression and minor defects in both visual function and ribbon structure. The zebrafish KO model is associated with similar moderate phenotype in human patients, providing a comprehensive tool to study the related human eye disorders.

Zebrafish show a peculiar phenomenon of disassembled ribbon synapses at least in the larval retina during the night. At light onset, the presynaptic structure is rapidly reassembled for function [47]. This unusual mechanism may have evolved to save energy in rapidly growing larvae.

Conclusion

The zebrafish retina serves as an important model of cone photoreceptor and has already contributed significantly to our understanding of photoreceptor maintenance and function. With its ever-increasing toolbox of imaging and genetic techniques, it will continue to crucially help us further in investigating the outer retina and its diseases.