Synapse formation and modification between distal retinal neurons in larval and juvenile Xenopus

A serial section analysis of photoreceptor synaptic bases was undertaken in the clawed frog Xenopus laevis. The developmental period from tadpole stage 48 through metamorphosis was studied. Horizontal cells contacted rod and cone photoreceptors at ribbon synapses; the number of such contacts per receptor base was constant for rods, but increased for cones as a function of developmental stage. In pre-metamorphic animals bipolar cells contacted receptors only through basal junctions; their number in cone bases increased dramatically during development but was unchanged in rod bases. A densitometric estimation of the cleft width of basal junctions showed that it ranged from 10 to 18 nm, but the junctions could not be divided reliably into the 'wide' and 'narrow' categories reported for other vertebrate species. Near metamorphic climax a new type of ribbon-related bipolar cell junction appeared. Gap junctions between horizontal cells and conventional synapses of horizontal cell onto bipolar cell processes were first seen in mid-larval developmental stages.     

Retinal development in mandarinfish Siniperca chuatsi and morphological analysis of the photoreceptor layer

We describe the process of retinal development in mandarinfish Siniperca chuatsi from larvae to young fish. The developmental characteristics of the retinal structure and related cells were identified. Siniperca chuatsi were found to exhibit an altricial mode of retinal development that required considerable time to be completed after hatching. The retina was classed as a pure cone type during the early developmental stage. In the subsequent developmental stages, however, double cones gradually occupied the majority of the cone cells, while rod cells represented the majority of the photoreceptor cells. The outer segment (OS) of the rod cells were significantly longer compared with other morphological features, the OS of the two kinds of cone cells were significantly elongated and the diameters of the inner segment (IS) and OS of the double cone cells were significantly narrower in the later developmental stages. Combined with the scattered arrangement of cone cells at the different stages, the retina was found to have sacrificed a considerable part of visual acuity in the developmental process. The distribution of cone cells was observed to have gradually become regionalised during development. The findings of the present study also indicated that S. chuatsi have a high photosensitivity under dim light conditions as a result of specialised structures of the OS of photoreceptor cells and an increased number of rod cells. The loose arrangement of the cone mosaic presumably resulted in a poor imaging quality and, to some extent, the regionalisation of the cone-cell distribution compensated for the above shortcomings, which would enhance the ability of S. chuatsi to perceive targets in important directions for effective predation behaviour.     

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.     

Retinal rod photoreceptor-specific gene mutation perturbs cone pathway development.

Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.     

Phototransduction in primate cones and blowfly photoreceptors: different mechanisms, different algorithms, similar response

Phototransduction in primate cones is compared with phototransduction in blowfly photoreceptor cells. Phototransduction in the two cell types utilizes not only different molecular mechanisms, but also different signal processing steps, producing range compression, contrast constancy, and an intensity-dependent integration time. The dominant processing step in the primate cone is a strongly compressive nonlinearity due to cGMP hydrolysis by phosphodiesterase. In the blowfly photoreceptor a considerable part of the range compression is performed by the nonlinear membrane of the cell. Despite these differences, both photoreceptor cell types are similarly effective in compressing the wide range of naturally occurring intensities, and in converting intensity variations into contrast variations. A direct comparison of the responses to a natural time series of intensities, simulated in the cone and measured in the blowfly photoreceptor, shows that the responses are quite similar.     

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells

Transgenic mice were derived containing the cytotoxic dt-α gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.     

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.     

Thioredoxin-like 6 protects retinal cell line from photooxidative damage by upregulating NF-kappaB activity

Apoptosis is the common pathway to photoreceptor cell death in many eye diseases including age-related macular degeneration which affects more than 8 million individuals in the United States alone. RdCVF, a truncated mouse thioredoxin is specifically expressed by rod photoreceptor cells and prevents the apoptosis of cone cells. However the protective mechanism of RdCVF and the implications of its human homologue, thioredoxin-like 6 (TXNL6), on the apoptosis of retinal cells remain unknown. In this study, we examined the function of TXNL6 and investigated its mechanism of protection using a cone photoreceptor cell line, 661W. We found that the photooxidative stress-induced degradation of NF-kappaB proteins is rescued by overexpression of TXNL6, which enabled the NF-kappaB transactivation activity. Furthermore, the overexpression of TXNL6 rescued the photooxidative stress-induced apoptosis of 661W cells. Interestingly, this protective effect was significantly blocked by NF-kappaB specific inhibitors demonstrating that TXNL6 exerts its protective effect against apoptosis via NF-kappaB. Taken together, our study shows that the TXNL6 probably protects retinal cells from photooxidative damage-induced apoptosis via upregulation of NF-kappaB activity. The identification of TXNL6 and the demonstration of its protective mechanism offer new insights into treatment possibilities for photoreceptor cell degradation.     

Notch 1 inhibits photoreceptor production in the developing mammalian retina

The transmembrane receptor Notch1 plays a role in development and homeostasis in vertebrates and invertebrates. The mammalian retina is an excellent tissue in which to dissect the precise role of Notch signaling in regulating cell fate and proliferation. However, a systematic analysis has been limited by the early embryonic lethality of Notch1-null mice. Here, Notch1 was conditionally removed from the murine retina either early or late in development. Removal of Notch1 early led to a reduction in the size of the retina as well as aberrant morphology. A decrease in the number of progenitor cells and premature neurogenesis accounted for the reduction in size. Unexpectedly, ablation of Notch1 in early progenitor cells led to enhanced cone photoreceptor production, and ablation of Notch1 at later points led to an almost exclusive production of rod photoreceptor cells. These data suggest that Notch1 not only maintains the progenitor state, but is required to inhibit the photoreceptor fate. These cone enriched mutant mice should prove to be a valuable resource for the study of this relatively rare mammalian photoreceptor cell type.     

Sequential genesis and determination of cone and rod photoreceptors in Xenopus

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 227–244, 1998     

NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD^EGFP fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.     

p53 Selectively Regulates Developmental Apoptosis of Rod Photoreceptors

Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.     

Function for Hedgehog genes in zebrafish retinal development

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.     

Two cellular inductions involved in photoreceptor determination in the Xenopus retina.

Cellular determination in the Xenopus retina is not a strict consequence of cell lineage or cell birthdate. This suggests that a retinal cell gets its fate by either local cellular interactions, diffusible factors, or an indeterminate stochastic mechanism. We have performed an in vitro experiment in which cellular contact is controlled to test the first possibility directly. We use these experiments to demonstrate that two cellular inductions are involved in photoreceptor determination in vitro and that these inductions also occur during development in the retina in vivo. The first interaction is responsible for biasing cells toward either a generic photoreceptor or a cone fate, while the second directs cells toward a rod cell fate.     

Cellular processing of cone photoreceptor cyclic GMP-gated ion channels: a role for the S4 structural motif

We examined cellular protein processing and functional expression of photoreceptor cyclic nucleotide-gated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel alpha subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG beta subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane.