Salamander blue-sensitive cones lost during metamorphosis.

The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.

Eel visual pigments revisited: the fate of retinal cones during metamorphosis.

During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P523(2) to P501(1)) and then a total change in gene expression with the down regulation of a "freshwater" opsin and its concomitant replacement by the expression of a typical "deep-sea" opsin (P501(1) to P482(1)). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540-545 nm and 435-440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the "green"-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.

Transient expression of thyroid hormone nuclear receptor TRbeta2 sets S opsin patterning during cone photoreceptor genesis.

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.

Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow trout.

Ultraviolet-sensitive (UVS) cones disappear from the retina of salmonid fishes during a metamorphosis that prepares them for deeper/marine waters. UVS cones subsequently reappear in the retina near sexual maturation and the return migration to natal streams. Cellular mechanisms of this UVS cone ontogeny were investigated using electroretinograms, in situ hybridization, and immunohistochemistry against opsins during and after thyroid hormone (TH) treatments of rainbow trout (Oncorhynchus mykiss). Increasing TH levels led to UVS cone degeneration. Labeling demonstrated that UVS cone degeneration occurs via programmed cell death and caspase inhibitors can inhibit this death. After the cessation of TH treatment, UVS cones regenerated in the retina. Bromodeoxyuridine (BrdU) was applied after the termination of TH treatment and was detected in the nuclei of cells expressing UVS opsin. BrdU was found in UVS cones but not other cone types. The most parsimonious explanation for the data is that UVS cones degenerated and UVS cones were regenerated from intrinsic retinal progenitor cells. Regenerating UVS cones were functionally integrated such that they were able to elicit electrical responses from second-order neurons. This is the first report of cones regenerating during natural development. Both the death and regeneration of cones in retinae represent novel mechanisms for tuning visual systems to new visual tasks or environments.

An urn model of the development of L/M cone ratios in human and macaque retinas.

A model of the development of L/M cone ratios in the Old World primate retina is presented. It is supposed that during gestation, the cone cycles randomly between states in which it transcribes either L or M opsin. The current state determines and increases the probability that it will transcribe the same opsin in future cycles. These assumptions are sufficient to formalize the process as a Markov chain that can be modeled as an urn containing two types of balls, L and M. Drawing one ball results in the increase of its species and the decrease of the other. Over the long run, the urn will become populated with a single type of ball. This state corresponds to the photoreceptor adopting a fixed identity for its lifetime. We investigate the effect of the number of states and the rule that regulates the advantage of transition toward one cone type or another on the relation between fetal and adult L/M cone ratios. In the range of 100 to 1000 states, small variations of the initial L/M ratio or the transition advantage can each generate large changes in the final L/M ratio, in qualitative accord with the variation seen in human adult retinas. The time course to attain stable L/M ratios also varies with these parameters. If it is believed that the cycling follows a circadian rhythm, then final L/M cone ratios would be expected to stabilize shortly after birth in the human being and the macaque.

Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta.

Color vision requires the expression of opsin photopigments with different wavelength sensitivities in retinal cone photoreceptors. The basic color visual system of mammals is dichromatic, involving differential expression in the cone population of two opsins with sensitivity to short (S, blue) or medium (M, green) wavelengths. However, little is known of the factors that directly activate these opsin genes and thereby contribute to the S or M opsin identity of the cone. We report that the orphan nuclear receptor RORbeta (retinoid-related orphan receptor beta) activates the S opsin gene (Opn1sw) through binding sites upstream of the gene. RORbeta lacks a known physiological ligand and activates the Opn1sw promoter modestly alone but strongly in synergy with the retinal cone-rod homeobox factor (CRX), suggesting a cooperative means of enhancing RORbeta activity. Comparison of wild-type and mutant lacZ reporter transgenes showed that the RORbeta-binding sites in Opn1sw are required for expression in mouse retina. RORbeta-deficient mice fail to induce S opsin appropriately during postnatal cone development. Photoreceptors in these mice also lack outer segments, indicating additional functions for RORbeta in photoreceptor morphological maturation. The results identify Opn1sw as a target gene for RORbeta and suggest a key role for RORbeta in regulating opsin expression in the color visual system.

Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina.

Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal-ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone beta2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRbeta2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development.

Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors.

The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl-/- retinas, the GFP+ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP+ photoreceptors from wild-type and Nrl-/- retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.

Morphogenesis of the different types of photoreceptors of the chicken (Gallus domesticus) retina and the effect of amblyopia in neonatal chicken.

Despite the great variety in chicken photoreceptors, existing morphogenetic studies only deal with two types: rods and cones. We have therefore examined by scanning electron microscopy the first appearance and maturation of different retinal photoreceptors in 36 chicken embryos (Gallus domesticus), aged 5-19 days prehatching. On day 5 of incubation, chicken retinae were only composed of proliferating ventricular cells devoid of photoreceptors. On day 8, outer mitotic cells were separated from inner differentiating photoreceptors, by the transient layer of Chievitz. Ball-like protrusions appeared at the ventricular surface, representing the first signs of photoreceptor inner segment formation. From day 10 onward, double cones, single cones, and rods could be clearly distinguished, and occasional cilia were detected at their tip. On day 12, inner segments had increased in length and diameter, and frequently carried a cilium representing the beginning of outer segment formation. On day 14, most photoreceptors displayed a distinct outer segment. On day 19, photoreceptors had essentially assumed adult morphology. Based on the shape of their outer segments, two subtypes of cones and three subtypes of double cones could be distinguished. Throughout development, we observed microvilli close to maturing photoreceptors, either originating from their lateral sides, from their tip, or from Müller cells. Microvillus density peaked between day 12 and 14, indicating an important role in photoreceptor morphogenesis. Unilateral occlusion of the eyes of posthatching chicken reduced the proportion of double cones to single cones in the retina, indicating dependence of retinal morphogenesis upon functional activity of visual cells.

Development of the cone ERG in infants.

PURPOSE: To assess cone photoreceptor and cone-mediated postreceptoral retinal function in infants. METHODS: ERG responses to a 1.8-log unit range of long-wavelength flashes on a white, rod-saturating background were recorded in 4-week-old (n = 22) and 10-week-old (n = 28) infants and control adults and children, 8 to 40 years of age (n = 13). A model of the activation of cone phototransduction was fit to the a-waves. Sensitivity (S(CONE)) and saturated-response amplitude (R(CONE)) were calculated. The amplitude and implicit time of the b-wave were examined as a function of stimulus intensity. The cone photoresponse parameters were compared to the rod photoresponse parameters (S(ROD) and R(ROD)) in the same subjects. RESULTS: S(CONE) and R(CONE) in infants were significantly smaller than in the mature control subjects. The mean S(CONE) was 64% and 68%, and the mean R(CONE) was 63% and 72% in 4- and 10-week-olds, respectively. The mean rod photoresponse parameters were considerably less mature, as the mean S(ROD) was 35% and 46%, and the mean R(ROD) was 39% and 43% of mature values at 4 and 10 weeks. The b-wave stimulus-response functions in the 4- and 10-week-old infants did not show the photopic hill that was characteristic of the children's and adults' photopic b-waves. CONCLUSIONS: Peripheral cone function is relatively more mature than rod function in young infants. The lack of a photopic hill is hypothesized to result from immaturity in the relative contributions of ON and OFF bipolar cell responses.

Photopigment coexpression in mammals: comparative and developmental aspects.

In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middle-to-long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently.

The nitric oxide-cGMP signaling pathway differentially regulates presynaptic structural plasticity in cone and rod cells.

Although abundant structural plasticity in the form of axonal retraction, neurite extension, and formation of presynaptic varicosities is displayed by photoreceptors after retinal detachment and during genetic and age-related retinal degeneration, the mechanisms involved are mostly unknown. We demonstrated recently that Ca(2+) influx through cGMP-gated channels in cones and voltage-gated L-type channels in rods is required for neurite extension in vitro (Zhang and Townes-Anderson, 2002). Here, we report that the nitric oxide (NO)-cGMP signaling pathway is active in photoreceptors and that its manipulation differentially regulates the structural plasticity of cone and rod cells. The NO receptor soluble guanylyl cyclase (sGC) was detected immunocytochemically in both cone and rod cells. Stimulation of sGC increased cGMP production in retinal cultures. In cone cells, quantitative analysis showed that NO or cGMP stimulated neuritic sprouting; this stimulatory effect was dependent on both Ca2+ influx through cGMP-gated channels and phosphorylation by protein kinase G (PKG). At the highest levels of cGMP, however, cone outgrowth was no longer increased. In rod photoreceptors, NO or cGMP consistently inhibited neuritic growth in a dose-dependent manner; this inhibitory effect required PKG. When NO-cGMP signaling was inhibited, changes in the neuritic development of cone and rod cells were also observed but in the opposite direction. These results expand the role of cGMP in axonal activity to adult neuritogenesis and suggest an explanation for the neurite sprouting observed in an autosomal recessive form of retinitis pigmentosa that is characterized by high cGMP levels in photoreceptor layers.

Inherited retinal degenerations: therapeutic prospects.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerative diseases, characterized by the progressive death of rod and cone photoreceptors. A tremendous genetic heterogeneity is associated with the RP phenotype. Most mutations affect rods selectively and, through an unknown pathway, cause the rod cells to die by apoptosis. Cones, on the other hand, are seldom directly affected by the identified mutations, and yet, in many cases, they degenerate secondarily to rods, which accounts for loss of central vision and complete blindness. Many animal models of RP are available and have led to a better understanding of the disease and to the development of therapeutic strategies aimed at curing the specific genetic disorder (gene therapy), slowing down or even stopping the process of photoreceptor degeneration (growth factors or calcium blockers applications, vitamin supplementation), preserving the cones implicated in the central visual function (identification of endogenous cone viability factors) or even replacing the lost cells (transplantation, use of stem or precursor cells). Still, many obstacles will need to be overcome before most of these strategies can be applied to humans. In this review, we describe the different therapeutic strategies being studied worldwide and report the latest results in this field.

The role of opsin expression and apoptosis in determination of cone types in human retina.

In primates, short wavelength sensitive cones (S cones) and medium- or long-wavelength-sensitive cones (L/M cones) are two separate populations. Each cone type has a different developmental timecourse, contributes to different intra-retinal circuits, and transmits different types of information to the brain. However, in fetal human retina a significant population of cones express both S and L/M opsin (S+L/M cones), raising questions about whether S+L/M cones die or change opsin expression during development. We have utilized fetal, postnatal and adult human retinae to study the immunohistochemical distribution and morphology of S+L/M cones during development. Because S cones appear to be at higher density in fetal compared to adult retinae, we used antibodies to S opsin and alpha-transducin to estimate the proportion of S-cones, and TUNEL labelling to detect apoptotic death in the L/M, S or S+L/M population during development. S cones were present in central retina from fetal week (Fwk)11 and covered the retina by Fwk20. L/M cones appeared in the foveal cone mosaic 3-4 weeks after S-opsin was first detected, and covered the retina by birth. S+L/M cones were detected in all retinae older than Fwk14. They were most numerous at the retinal eccentricity where L/M opsin was just appearing; i.e. at the 'front' of L/M opsin expression. In this region, five morphological types of cones were present. (1) Heavily labelled S cones had thick cell bodies, a thick basal axon and pedicle, and a nucleus at any level of the outer nuclear layer (ONL). (2) Heavily labelled L/M cones were wine goblet shaped with a small round cell body, a large nucleus at the outer ONL edge, and a thin axon with a prominent synaptic pedicle. (3) Goblet-shaped S+L/M cones. (4) Goblet-shaped cones lightly labelled for S-opsin. (5) Cones that were not immunoreactive to either opsin. Only type 1 S cones were present peripheral to the L/M expression front, and their labelling intensity, morphology and distribution indicates that these are the 'true blue' cones of the adult mosaic. Only type 2 L/M cones were present in the foveal cone mosaic. Types 3 and 4 were most numerous within 500-750 microm of the L/M expression front, but type 3 S+L/M cones were also scattered throughout more central regions in fetal, infant and adult retinae. S+L/M cones comprised 5-10% of opsin immunoreactive cones at the L/M front in fetal and early postnatal retinas but 0.01-0.03% throughout P8mo and adult retinae. We found no evidence of significant levels of apoptosis in L/M cones at the expression front, suggesting that this decrease was not due to cell death. The findings suggest that goblet-shaped cones destined to express L or M opsin may initially and transiently express S opsin. Near the optic disc, at Fwk17 S cone density was around 2000 cells mm(-2), which dropped 50% by Fwk20 and stabilized at around 500 cells mm(-2) by birth. Double labelling with alpha-transducin showed that throughout this period 8-10% of all cones expressed S opsin. TUNEL labelling found no significant apoptosis in the S cone population. The decrease in S cone density near the optic disc occurs in the absence of apoptosis, and is likely due to other developmental events acting on the photoreceptor layer, including displacement of cones towards the fovea.

Differential distribution of fibroblast growth factor receptors (FGFRs) on foveal cones: FGFR-4 is an early marker of cone photoreceptors.

PURPOSE: Relatively little is known of the expression and distribution of FGF receptors (FGFR) in the primate retina. We investigated expression of FGFRs in developing and adult Macaca monkey retina, paying particular attention to the cone rich, macular region. METHODS: One fetal human retina was used for diagnostic PCR using primers designed for FGFR1, FGFR2, FGFR3, FGFR4, and FGFR like-protein 1 (FGFrl1) and for probe design to FGFR3, FGFR4, and FGFrl1. Rat cDNA was used to synthesize probes for FGFR1 and FGFR2 with 90% and 93% homology to human, respectively. Paraffin sections of retina from macaque fetuses sacrificed at fetal days (Fd) 64, 73, 85, 105, 115, 120, and 165, and postnatal ages 2.5 and 11 years were used to detect FGF receptors by immunohistochemistry and in situ hybridization. RESULTS: PCR showed each of the FGF receptors are expressed in fetal human retina. In situ hybridization indicated that mRNA for each receptor is expressed in all retinal cell layers during development, but most intensely in the ganglion cell layer (GCL). FGFR2 mRNA is reduced in the adult inner (INL) and outer (ONL) nuclear layers, while FGFrl1 mRNA is virtually absent from the adult ONL. FGFR4 mRNA is particularly intense in fetal and adult cone photoreceptors. Immunoreactivity to FGFR1-FGFR4 was detected in the interphotoreceptor matrix in what appeared to be RPE microvilli associated with developing photoreceptor outer segments, and generally is high in the GCL and low in the INL. Different patterns of FGFR3 and FGFR4 immunoreactivities in the outer plexiform layer (OPL) suggest localization of FGFR3 to horizontal cell processes, with FGFR4 being expressed by both horizontal and bipolar cell processes. FGFR1, FGFR3, and FGFR4 immunoreactivities are present in the inner segments and somata of adult cones. The pedicles of developing and adult cones are FGFR1 and FGFR3 immunoreactive, and the basal, synaptic region is FGFR4 immunoreactive. FGFR4 labels cones almost in their entirety from early in development and is not detected in rods. The fibers of Henle are intensely FGFR4 immunoreactive in adult cones. CONCLUSIONS: The results show high levels of FGF receptor expression in developing and adult retina. Differential distribution of FGF receptors across developing and adult photoreceptors suggests specific roles for FGF signalling in development and maintenance of photoreceptors, particularly the specialized cones of the fovea.

Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina.

Previous studies have analyzed photoreceptor development, some inner retina cell types, and specific neurotransmitters in the zebrafish retina. However, only minor attention has been paid to the morphology of the synaptic connection between photoreceptors and second order neurons even though it represents the transition from the light sensitive receptor to the neuronal network of the visual system. Here, we describe the appearance and differentiation of pre- and postsynaptic elements at cone synapses in the developing zebrafish retina together with the maturation of the directly connecting second order neurons and a dopaminergic third order feedback-neuron from the inner retina. Zebrafish larvae were examined at developmental stages from 2 to 7dpf (days postfertilization) and in the adult. Synaptic maturation at the photoreceptor terminals was examined with antibodies against synapse associated proteins. The appearance of synaptic plasticity at the so-called spinule-type synapses between cones and horizontal cells was assessed by electron microscopy, and the maturation of photoreceptor downstream connection was identified by immunocytochemistry for GluR4 (AMPA-type glutamate receptor subunit), protein kinase beta(1) (mixed rod-cone bipolar cells), and tyrosine hydroxylase (dopaminergic interplexiform cells). We found that developing zebrafish retinas possess first synaptic structures at the cone terminal as early as 3.5dpf. Morphological maturation of these synapses at 3.5-4dpf, together with the presence of synapse associated proteins at 2.5dpf and the maturation of second order neurons by 5dpf, indicate functional synaptic connectivity and plasticity between the cones and their second order neurons already at 5dpf. However, the mere number of spinules and ribbons at 7dpf still remains below the adult values, indicating that synaptic functionality of the zebrafish retina is not entirely completed at this stage of development.

Ontogeny of ultraviolet-sensitive cones in the retina of rainbow trout (Oncorhynchus mykiss).

In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land-locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double-labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength-sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal-temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species.

Development of the cone photoreceptor mosaic in the mouse retina revealed by fluorescent cones in transgenic mice.

PURPOSE: Normal function of the retina relies on the orderly stereotyped organization of different neurons and their synaptic connections. How such neural organization is patterned during development remains poorly understood due to the paucity of adequate developmental markers. This study was to examine the spatial organization and development of cone photoreceptors quantitatively in the mouse retina. METHODS: A transgenic approach was used to generate a living cone cell marker by driving GFP expression in mouse cones with the human red/green opsin gene 5' sequences. The spatial organization and development of the cones in the mouse retinas were examined quantitatively with epifluorescence and scanning laser confocal microscopy. Cone specific GFP expression in the developing retinas was verified with peanut agglutinin (PNA) staining. Developmental expression of mouse cone opsin genes was determined with RT-PCR. RESULTS: The fluorescent retinal cells expressing GFP can be visualized as early as on embryonic day E15. Following up morphological differentiation of these cells revealed features that were consistent with the typical morphology of the mouse cones. Double labeling with cone specific PNA showed that these cells were co-labeled starting from postnatal day P1, and that a subpopulation of PNA positive cones expressed the GFP. The fluorescent cell densities had a similar ventral and dorsal distribution from E15 to P2, increased dramatically in the ventral by P6, and in the dorsal from P7. Nearest neighbor distance analysis demonstrated that this subpopulation of cones was organized into a regular mosaic pattern with a regularity index of 4.82 in the central and 3.55 in the peripheral retina. Quantitative pattern assessment of the developing cones revealed that the fluorescent cells appeared to be distributed in a non-random array before birth. The regularity of the cone array began to rise on P7, in parallel with the onset of mouse green opsin gene expression and the development of cone pedicles. The regular pattern of cone mosaic organization was basically formed by P10, coinciding with the timing of the cone pedicle maturation. CONCLUSIONS: The cones in the mouse retina are organized in a regular mosaic pattern. Patterning the cone mosaic appears to follow a two phase developmental process involving regulated opsin gene expression and cone pedicle maturation: an early phase where a non-random array emerges during cone differentiation, and a late phase where the regular mosaic pattern is mature at the time when cone synaptic contacts are being formed.

Ectopic photoreceptors and cone bipolar cells in the developing and mature retina.

An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.

Double cone dystrophy and RPE degeneration in the retina of the zebrafish gnn mutant.

PURPOSE: To characterize morphologic alterations in the retina of the visual mutant zebrafish gantenbein (gnn) and to examine whether these alterations correlate with those present in human hereditary eye diseases. METHODS: The gnn mutant was isolated by behavioral and macroscopic screening. Retinas of gnn zebrafish larvae were examined at different developmental stages from 2 to 9 days postfertilization (dpf) by standard histologic staining techniques and by immunocytochemistry. Ultrastructural alterations were examined by electron microscopy. The genetic map position of the induced mutation was identified by mapping with two candidate primer pairs on single larvae. RESULTS: The gnn mutant exhibited shortened outer photoreceptor segments and altered RPE morphology. In the photoreceptor layer of the mutant, the total number of lectin-labeled cones was reduced in all developmental stages from 2 to 7 dpf, whereas the amount of rhodopsin-positive cells remained at the wild-type (WT) level. Labeling with zebrafish opsin antibodies revealed dystrophic red cones at 5 dpf, whereas the morphology of all other cone types was largely unaffected. Electron microscopy unveiled electron-dense deposits between the discs of the double cone outer segments. In addition, the onset of progressive RPE degeneration was observed at this stage of development. At later stages, all cone types and the RPE became degenerative. The morphology of distinct second-order neurons remained largely unaffected by the mutation. The gnn mutation was located approximately 4.3 cM from the simple sequence length polymorphism (SSLP) marker Z15453 on linkage group 16. CONCLUSIONS: In gnn mutant zebrafish, cones, and especially red cones, are dystrophic in early retinal development. Subsequent to this cone dystrophy, the RPE becomes dysfunctional and starts to degenerate in later stages of development. Thus, the early developmental morphology of gnn exhibits similarities to cone dystrophies most commonly seen in age-related macular degeneration (AMD) among humans, whereas the later stages of degeneration in gnn resemble RPE alterations in retinitis pigmentosa (RP) in humans. The gnn zebrafish mutant may therefore be a useful model for examining the possible interplay and connection between cone dystrophy and RPE degeneration.