红腹罗非鱼视网膜色素上皮及光感器的胚后分化

硬骨鱼胚后发育中,边缘生长区及胚胎裂缝代表神经形成的活跃位点,同时对新的神经胶质细胞和光感器(视锥和视杆细胞)的形成有积极作用。此项工作首次研究红腹罗非鱼(硬骨鱼类辐鳍亚纲)视网膜中视网膜色素上皮和光感器的胚后生长及分化。采用光学和透射电子显微镜术分析了四个大小不同的鱼群组。正如至目前为止,在已研究过的大多数其它硬骨鱼中所观察到的结果一样,我们研究的所有样本中,成年红腹罗非鱼的视网膜表现为气球样扩张。此外,尚具有一弯曲的、有开口的胚胎裂缝,表明眼的不对称性胚后生长。在胚后分化过程中,视网膜色素上皮表现为自外周向中央的梯度变化,即边缘生长区周围,该上皮仅含有球形的黑色素体,而在中央部位发育为长形的黑色素体及顶端突起。同样,发育中的光感受器亦表现为由侧面向中央逐步发展变化的程序。光感觉器先形成1内节及其附属的突起,随后内节分化为一椭圆体加一肌样结构,这两种成分与其它已研究过的硬骨鱼视网膜中观察到的胚胎发育程序大致相符。因此提供了另一线索：硬骨鱼胚后视网膜的生长极大地刺激(促进)了胚胎视网膜的发育。本研究表明：双体视锥细胞可能是发育中的单体视锥细胞裂开的结果[动物学报49(6)：813～818,2003]。     

The fine structure of the pigment epithelium and photoreceptor cells of the newt,Triturus viridescens dorsalis (Rafinesque)

The morphology of the retinal pigment epithelium and photoreceptor cells has been studied in the common newt Triturus viridescens dorsalis by light, conventional transmission and scanning electron microscopy. The pigment epithelium is formed by a single layer of low rectangular cells, separated by a multilayered membrane (Bruch's membrane) from the vessels of the choriocapillaris. The scleral border of the pigment epithelium is highly infolded and each epithelial cell contains smooth endoplasmic reticulum, myeloid bodies, mitochondria, lysosomes, phagosomes and an oval nucleus. Inner, pigment laden, epithelial processes surround the photoreceptor outer and inner segments. The three retinal photoreceptor types, rods, single cones and double cones, differ in both external and internal appearance. The newt, rod, outer segments appear denser than the cones in both light and electron micrographs, due to a greater number of rod lamellae per unit distance of outer segment and to the presence of electron dense intralamellar bands. The rod outer segments possess deep incisures in the lamellae while the cone lamellae lack incisures. Both rod and cone outer segments are supported by a peripheral array of dendritic processes containing longitudinal filaments which originate in the inner segment. The inner segment mitochondria, forming the rod ellipsoid, arelong and narrow while those in the cone are spherical to oval in shape. The inner segments of all three receptor cell types also contain a glycogen-filled paraboloid and a myoid region, just outside the nucleus, rich in both rough and smooth endoplasmic reticulum. The elongate, cylindrical nuclei differ in density. The rod nuclei are denser than those of the cones, contain clumped chromatin and usually extend further vitreally. Similarly, the cytoplasm of the rod synaptic terminal is denser than its cone counterpart and contains synaptic vesicles almost twice as large as those of the cones. Photoreceptor synapses in rods and cones are established by both superficial and invaginated contacts with bipolar or horizontal cells.     

On the eye of the Goldeye Hiodon alosoides (Teleostei: Hiodontidae)

In the eye of the Goldeye the photoreceptors are arranged in bundles and the pigment epithelium contains a massive reflector or tapetum lucidum. Photoreceptor bundles are arranged in parallel rows, the bundles alternating in position from row to row. Each bundle contains about 60 photoreceptors, of which 30 or so are cones. Rod outer segments lie in the scleral half of the outer retinal region of the light-adapted eye. Processes of the pigment epithelium cells extend vitread almost to the external limiting membrane; they envelop the bundles of rods and cones, and a ring of four processes surrounds each bundle. A process contains two kinds of reflecting crystals (composed of uric acid). A large part of the epithelium cell is packed with small disc-shaped crystals (crystallites) enclosed in thin membranes; the tip of the process, in the region of the photoreceptor bundle, contains orderly arrays of small rod-shaped crystals (rodlets). It is suggested that the crystallites form a diffuse reflector backscattering light into the rods; and that the rodlets reflect light regularly from their surfaces into the photoreceptor bundles. In the light-adapted state, rods are enveloped by pigment and crystallites. The organization is compared with that of other fishes that have photoreceptors in bundles (grouped retinae) and tapeta lucida.     

Developmental predetermination of the structural and molecular polarization of photoreceptor cells

Through mechanisms still unknown, the apparently homogeneous neuroepithelium of the embryonic optic cup differentiates into such divergent cell types as photoreceptors, glia, and various subsets of neurons. Questions that still remain unanswered in this field include the timing and mechanism of action of the "instructive" events directing each neuroepithelial cell to undergo the sequence of phenotypic changes necessary to develop into a specific retinal cell type. This laboratory is investigating some of these questions using cultures in which dissociated neural retina cells, obtained before the onset of overt photoreceptor differentiation, develop at low density in the absence of glia and pigment epithelium. The cultures initially are a morphologically homogeneous population of process-free, round cells. Some cells retain this morphology throughout the first week in vitro, while others develop either as photoreceptors or as multipolar neurons. Photoreceptors elongate and become very asymmetric as they do in vivo, with characteristic compartments orderly arranged along their longitudinal axis (an outer segment-like process, inner segment, cell body, and a characteristically short, single neurite). Cell polarization can also be observed in the distribution of opsin immunoreactive materials and some cytoskeletal elements. Thus, certain precursor cells present in the embryonic retina seem to be programmed to differentiate into photoreceptors even when developing in the absence of contacts with other retinal cells. However, interactions with other constituents of the retina/pigment epithelium complex are probably necessary to ensure final photoreceptor maturation, including further growth of the opsin-rich outer segment process.     

Müller glia endfeet,a basal lamina and the polarity of retinal layers form properly in vitro only in the presence of marginal pigmented epithelium

Summary Dissociated embryonic chicken retinal cells regenerate in rotary culture into cellular spheres that consist of subareas expressing all three nuclear layers in an inside-out sequence (rosetted vitroretinae). However, when pigmented cells from the eye margin (peripheral retinal pigment epithelium) are added to the system, the sequence of layers is identical with that of an in-situ retina (laminar vitroretinae). In order to elucidate further the lamina-stabilizing effect exerted by the retinal pigment epithelium, we have compared both systems, laying particular emphasis on the ultrastructure of the basal lamina and of Müller glia processes. Ultrastructurally, in both systems, an outer limiting membrane, inner segments of photoreceptors and the segregation of cell bodies into three cell layers develop properly. Synapses are detectable in a premature state, although only in the inner plexiform layer of laminar vitroretinae. Although present in both systems, radial processes of juvenile Müller glia cells are properly fixed at their endfeet only in laminar vitroretinae, since a basal lamina is only expressed here. Large amounts of laminin are detected immunohistochemically within the retinal pigment epithelium and along a basal stalk that reaches inside the laminar vitroretinae. We conclude that the peripheral retinal pigment epithelium is essential for the expression of a basal lamina in vitro. Moreover, the basal lamina may be responsible both for stabilizing the correct polarity of retinal layers and for the final differentiation of the Müller cells.     

Photoreceptor fine structure in the southern fiddler ray (Trygonorhina fasciata).

The fine structure of the retinal photoreceptors has been studied by light and electron microscopy in the southern fiddler ray or guitarfish (Trygonorhina fasciata). The duplex retina of this species contains only rods and single cones in a ratio of about 40:1. No multiple receptors (double cones), no repeating pattern or mosaic of photoreceptors and no retinomotor movements of these photoreceptors were noted. The rods are cylindrical cells with inner and outer segments of the same diameter. Cones are shorter, stouter cells with a conical outer segment and a wider inner segment. Rod outer segment discs display several irregular incisures to give a scalloped outline to the discs while cone outer segment discs have only a single incisure. In all photoreceptors a non-motile cilium joins the inner and outer segments. The inner segment is the synthetic centre of photoreceptors and in this compartment is located an accumulation of mitochondria (the ellipsoid), profiles of both rough and smooth endoplasmic reticulum, prominent Golgi zones and frequent autophagic vacuoles. The nuclei of rods and cones have much the same chromatin pattern but cone nuclei are invariably located against or particularly through the external limiting membrane (ELM). Numerous Landolt's clubs which are ciliated dendrites of bipolar cells as well as Müller cell processes project through the ELM, which is composed of a series of zonulae adherentes between these cells and the photoreceptors. The synaptic region of both rods (spherules) and cones (pedicles) display both invaginated (ribbon) synapses and superficial (conventional) synapses with cones showing more sites than the rods.     

Fine structure of the retinal photoreceptors of the ranch mink Mustela vison

The structure of the retinal photoreceptors of the ranch mink (Mustela vison) has been investigated by light and electron microscopy. In this mammalian species, the photoreceptors can be readily differentiated and adequately described by the classical terminology of rods and cones, with the rods being the more numerous. Rods are long slender cells while cones are shorter and stouter in appearance. Both rods and cones are highly differentiated and extremely polarized cells consisting of an outer segment, a non-motile connecting cilium, an inner segment, a nuclear region and a synaptic process extending to an expanded synaptic ending. Morphological differences are noted between rods and cones for most of the various regions of these cells. While rods reach to the cell body of the retinal pigment epithelial (RPE) cells, larger apical processes from the RPE extend to the shorter cone cells, so that both photoreceptor types are in intimate contact with the retinal epithelial cells.     

Functional significance of the taper of vertebrate cone photoreceptors

Vertebrate photoreceptors are commonly distinguished based on the shape of their outer segments: those of cones taper, whereas the ones from rods do not. The functional advantages of cone taper, a common occurrence in vertebrate retinas, remain elusive. In this study, we investigate this topic using theoretical analyses aimed at revealing structure-function relationships in photoreceptors. Geometrical optics combined with spectrophotometric and morphological data are used to support the analyses and to test predictions. Three functions are considered for correlations between taper and functionality. The first function proposes that outer segment taper serves to compensate for self-screening of the visual pigment contained within. The second function links outer segment taper to compensation for a signal-to-noise ratio decline along the longitudinal dimension. Both functions are supported by the data: real cones taper more than required for these compensatory roles. The third function relates outer segment taper to the optical properties of the inner compartment whereby the primary determinant is the inner segment's ability to concentrate light via its ellipsoid. In support of this idea, the rod/cone ratios of primarily diurnal animals are predicted based on a principle of equal light flux gathering between photoreceptors. In addition, ellipsoid concentration factor, a measure of ellipsoid ability to concentrate light onto the outer segment, correlates positively with outer segment taper expressed as a ratio of characteristic lengths, where critical taper is the yardstick. Depending on a light-funneling property and the presence of focusing organelles such as oil droplets, cone outer segments can be reduced in size to various degrees. We conclude that outer segment taper is but one component of a miniaturization process that reduces metabolic costs while improving signal detection. Compromise solutions in the various retinas and retinal regions occur between ellipsoid size and acuity, on the one hand, and faster response time and reduced light sensitivity, on the other.     

Isolation of chick retina cones and study of their diversity based on oil droplet colour and nucleus position

The chick retina has four morphological cone types that differ not only in shape, but also in the visual pigment in the outer segment, in the colour of the oil droplet in the inner segment and in synaptic connectivity. Neither the type of droplet nor the visual pigment has been definitively established for the four cone types. The main aim of the present work has been the isolation of entire live photoreceptors in order to study the oil droplet colour in each cone type and to quantify each type. We have improved an earlier retinal cell isolation method and obtained large numbers of entire cones. Principal cones (27% of the cones) possess a yellow or colourless droplet. Accessory cones (27% of the cones) all contain a small pale green droplet. Straight cones (44% of the cones) have a red, orange, yellow, or colourless droplet. Oblique cones (1.66% of the cones) all have a colourless droplet. We have found that straight cones with a red, orange, or yellow droplet differ in terms of the position of the nucleus and their percentage and conclude that they are distributed in three rows in the outer nuclear layer (ONL) of the central retina. Our study of 4,6-diamidino-2-phenylindole-stained retinal sections has revealed three rows of nuclei instead of the two currently thought to form the ONL. Together, our results show a larger cone diversity than previously known, suggest a larger functional diversity and provide an efficient method for isolating entire chick photoreceptors. This work was supported by grants from the Dirección General de Investigación, Ministerio de Educación y Ciencia of Spain (no. BFU2005-08786-C02-01) and from the Comunidad de Madrid, Spain (no. 920648/2006).     

Electron microscopic studies on the retinal photoreceptors of the spotted snakehead, Channa punctatus

The retinal photoreceptors of Channa punctatus include rods, single cones and the double cones. The double cones have the outer segments having lamellae which are distorted into tubules and vesicles, and the inner segments with hypertrophied mitochondria.     

Detection of interphotoreceptor retinoid binding protein (IRBP) mRNA in human and cone-dominant squirrel retinas by in situ hybridization

Interphotoreceptor retinoid binding protein (IRBP) is a soluble glycolipoprotein located between the neurosensory retina and pigment epithelium, which may serve to transport vitamin A derivatives between these tissues. The specific cell type responsible for IRBP synthesis has not been well established. To address this issue, we have examined the expression of IRBP mRNA in human and cone-dominant ground squirrel retinas by in situ hybridization. Optimal labeling and histological resolution were achieved with 35S- and 3H-labeled anti-sense riboprobes made from a human IRBP cDNA clone, and semi-thin wax-embedded retinal sections. In human retina, label was localized over the inner segments of both rod and cone photoreceptors. Quantitative analysis demonstrated a fourfold higher density of label over rod inner segments. In ground squirrel retina, labeling was found almost exclusively over the inner segments of cones. The results indicate that in human retina both rods and cones express IRBP mRNA, albeit at different levels. In cone-dominant species such as the ground squirrel, cones are the principal cell type responsible for IRBP mRNA synthesis.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: I. Induction of Cone Contraction Is Mediated by D2 Receptors

In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4-dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light-adaptive retinomotor movements in all three cell types. In isolated dark-cultured retinas, dopamine induced light-adaptive cone contraction with a half-maximal effect at 10(-8) M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light-adaptive cone contraction in isolated dark-cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light-induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark-adaptive cone elongation in isolated light-adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light-adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light-adaptive retinomotor movements in isolated distal fragments of dark-adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light-adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.     

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.     

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases.     

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20?%) as the number of retinal ganglion cells (19?%). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.     

Ultrastructural diurnal changes of the retinal photoreceptors in the embryo of a viviparous teleost (Poecilia reticulata P.)

All diurnal changes studied - ellipsosome excepted - start at midgestation, following differentiation of photoreceptors and pigment-epithelium cells. These are: (1) shedding of the tips of the light-sensitive photoreceptor outer segments and subsequent phagocytosis by the pigment-epithelium; (2) retinomotor movements of pigment-epithelium processes, rods and cones; (3) changes of cone square-mosaics into row-mosaics at night. Newly-differentiated photoreceptors in the embryo are, therefore, already vulnerable to disruption of cyclical systems. Several inherited human retinal diseases, such as Retinitis pigmentosa, are thought not to affect differentiation of photoreceptors but their cyclical renewal pathways. The retina of the guppy-embryo is, therefore, a valuable model for such studies.     

The accessory outer segment of rods and cones in the retina of the guppy,Poecilia reticulata P. (teleostei)

Summary The ultrastructure of the accessory outer segment (AOS) — a ciliumlike structure emanating from the inner segment and running alongside the outer segment of photoreceptors — is described. The AOS occurs in both rods and cones of Poecilia reticulata. Its ultrastructure, including the arrangement of microtubules, which originate from the ciliary stalk, is the same in rods and cones. The cone-AOS is connected with the outer segment by a thin plasmabridge, whereas the rod-AOS lies embedded within the outer segment. The outer segment of the cone, in contrast to that of the rod, is separated from the pigment epithelium by a large extracellular space. An intimate contact, however, is secured by the AOS; its membrane is closely appositioned to the pigment epithelium membrane. The functional significance of the AOS and its possible occurrence in other vertebrate classes, are discussed.     

Ontogeny of the photoreceptors in the embryonic retina of the viviparous guppy,Poecilia reticulata P. (Teleostei)

Summary Ultrastructural analyses of retinal development in the guppy embryo show that at midgestation all types of photoreceptors are differntiated in the fundus, and at birth differentiation extends over the whole retina. Formation of discs of outer segments is more rapid in rods than in cones. Double cones differentiate simultaneously with long single cones and are formed by the adhesion of two primordial inner segments; short single cones develop last. Wherever cones are differentiated, they are arranged in an adult-type square mosaic. The rods in the embryo, as opposed to the adult, are likewise regularly arranged within the mosaic unit.These results are at variance with the generally held opinion that adult teleosts which possess duplex retinae have larvae with pure cone retinae, and that rods, double cones and mosaics appear in late larval life or only at metamorphosis.In the double cones of the guppy embryo subsurface cisternae develop along the adjoining primordial inner segments. Additionally, regularly distributed subsurface cisternae are formed in the regions of intimate contact of long single cones with double cones and rods.We suggest that the early development of rods and double cones, and a square-mosaic with regular distribution of rods and subsurface cisternae, provide the newly born with a fully functional optical apparatus, especially suited to perception of movements. This is necessary for its survival against predatory, especially maternal, attacks.This work was in part subsidized by a grant from the Medical Research Council of Ireland to Y.W.K.