Intercellular gap junctions in the developing retina and pigment epithelium of the chick

Summary Gap junctions are found in the pigment epithelium, between retina and pigment epithelium and in the retina of 5–14 day chick embryos, they are identified using block staining and extracellular tracer techniques. In the pigment epithelium gap junctions are found between cell bodies and interdigitating processes and many change their position during development. Gap junctions between retina and pigment epithelium are only made by undiferentiated retinal ventricular cells and may provide intercytoplasmic pathways important for photoreceptor differentiation. Retinal gap junctions are found in an outer zone next to the pigment epithelium and inner zone near the vitreous, they are only seen between ventricular cells but may provide pathways for ganglion cell specification. The role of gap junctions in the generation of retinal neurons is discussed.     

Vimentin intermediate filament protein as differentiation marker of optic vesicle epithelium in the chick embryo

For the study of the differentiation process of optic vesicle epithelium into neural retina, pigment epithelium and pars caeca retinae, vimentin intermediate filament protein in retinal epithelial cells was detected immunohistochemically in chick embryo at stages 11-21. In the late stage of optic vesicle development (stage 14), optic vesicle epithelium was classified into the following 3 different portions on the basis of vimentin staining intensity: latero-central epithelium under the lens placode, medio-central epithelium facing the latero-central epithelium, and peripheral epithelium connecting the latero-central and medio-central epithelia. Latero-central epithelium, the future neural retina, exhibited strongest staining of vimentin of the 3 portions. In contrast, medio-central epithelium, the future pigment epithelium, showed weakest staining. Moderate staining was observed in peripheral epithelium, the future pars caeca retinae. These differences in levels of vimentin expression were observed during optic cup formation. The present results clearly demonstrate that differentiation of retinal epithelium into neural retina, pigment epithelium and pars caeca retinae occurs in the late stage of the optic vesicle, and that retinal differentiation is reflected by the amount of vimentin in epithelial cells.     

Chronotopographical distribution patterns of cell death and of lectin‐positive macrophages/microglial cells during the visual system ontogeny of the small‐spotted catshark Scyliorhinus canicula

The patterns of distribution of TUNEL‐positive bodies and of lectin‐positive phagocytes were investigated in the developing visual system of the small‐spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL‐staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL‐positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL‐positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal‐to‐scleral gradient. Lectin‐positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin‐positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL‐positive bodies and the pattern of distribution of lectin‐labelled macrophages/microglial cells during the shark′s visual system ontogeny.     

Cell death in the ventral region of the neural retina during the early development of the chick embryo eye

The present study deals with morphologic and quantitative changes that take place in the area of cell death in the ventral part of the presumptive retinal wall of the chick embryo. These changes were followed from the optic vesicle stage until the first optic fiber fascicles leave the neural retina. Our results show that both the volume occupied by the area of cell death and the density of its pyknotic fragments undergo considerable variation during the period between Hamburger and Hamilton's (1951) stages 12 to 20. In the optic vesicle stages, cell death in the ventral wall of the vesicle was observed in 50 to 75% of the embryos studied. During stages 14 and 15, this zone was seen in more than 90%. By the time invagination of the optic cup was complete, the ventral retinal zone of cell death had disappeared entirely in a large proportion of embryos; in all others, it shrank significantly both in volume and density of pyknotic fragments. In stage 19, when the first optic fiber fascicles begin to emerge from the retina, a dramatic increase occurs in the number of pyknotic fragments in the posterior pole of the retina. The appearance of dying cells, in a region shortly to be traversed by developing ganglion cell axons, supports the hypothesis that cell death processes are apparently somehow related to the creation of a suitable environment for the emergence of fibers toward the optic stalk. Densities of mitotic and interphasic cells as well as the mitotic index were determined in both the retinal zone of cell death and in areas devoid of dead cells. In all developmental stages analyzed, the mitotic index was notably lower in the former than in non-necrotic zones, suggesting that cell proliferation is partially inhibited in retinal areas of cell death.     

Cell proliferation during the early stages of human eye development

The distribution as well as the ultrastructural and biochemical characteristics of proliferating cells in the human eye were investigated in five conceptuses of 5–9 postovulatory weeks, using morphological techniques and Ki-67 immunostaining. The Ki-67 nuclear protein was used as a proliferation marker because of its expression in all phases of the cell cycle except the resting phase (G0). The labelling indices of Ki-67-positive cells were analysed by means of the Kruskal-Wallis ANOVA test and the Wilcoxon matched-pairs test. In the 5th week, mitotic cells were the most numerous between the two layers of the optic cup, the optic cup and stalk, and between the lens pit and the surface ectoderm. During the 6th week, cells were observed in the lens epithelium covering the whole cavity of the lens vesicle as well as in the neuroblast zone and the pigmented epithelium of the retina. At later stages (7th–9th weeks), Ki-67-positive cells were restricted to the anterior lens epithelium, the outer neuroblast zone, and the pigmented retina. Throughout all stages examined, mitotic figures were found lying exclusively adjacent to the intraretinal space. Early in the lens pit, they were confined to the free epithelial surface, and later were facing the cavity of the lens vesicle. The proliferative activity was the most intensive in the 6th week, whereas it decreased significantly in the later stages. Additionally, when proliferative activities were compared, the peripheral retina appeared to be less mature than the central before the 9th week. In the earliest analysed stage, cell proliferation might be associated with the sculpturing of the optic cup and stalk, the cornea, and the lens. In the 6th week, the most intensive proliferation seems to be involved not only in the further morphogenesis of the optic cup and the lens vesicle but also in the retinal neurogenesis. At later stages, the decreased proliferation might participate in the neurogenesis of the outer neuroblast zone and the secondary lens fibre formation.     

Stereological study on the mode of optic cup expansion and the accumulation of mitoses in the early stages of chick embryo development

The present study was designed to contribute to our understanding of the factors that take part in the developmental transformation of the optic vesicle into the optic cup. The expansion and formation of this structure are dependent upon factors such as cellular proliferation, the space or zone occupied by the growing optic cup, and environmental influences. Our investigation in the chick embryo analyzes the relationship between retinal thickness and ventricular mitotic density. This relationship is shown in the study as PEI (proliferation-expansion index). That index varies in the superior, medial and inferior regions of the retina when the zones of the same stage are compared, as well as in the comparisons of values between the 13-14 stage and the 17-18 stage. These differences indicate a different behavior of the cells constituting the retinal regions. Also discussed is the influence of the retinal fissure on the morphological changes observed during optic cup development.     

扬子鳄眼球的胚胎发生

在２４例扬子鳄胚胎中，观察了眼球的发生及其组织分化过程。孵化后第２ｄ，视泡已从前脑突出形成，第６ｄ形成双层视杯和晶状体泡。晶状体纤维先由晶状体泡后壁上皮生成，然后由赤道部的泡壁上皮生成。虹膜、角膜内皮和基质均由视杯周围的间充质迁入形成。视网膜的色素上皮层最先分化。神经上皮在第１６ｄ出现节细胞的内迁。第１８ｄ可辨认节细胞层和神经纤维层。第２４ｄ可辨认内网层。第３０ｄ开始出现外核层与内核层的分化。第３     

Histology of the ferret retina

The retina of the adult ferret, Mustelo furo, was studied with light and transmission electron microscopy to provide an anatomical basis for use of the ferret as a model for retinal research. The pigment epithelium is a simple cuboidal layer of cells characterized by a zone of basal folds, apical microvilli, and pigment granules at various stages of maturation. The distinction between rod and cone photoreceptor cells is based on their location, morphology, heterochromatin pattern and the electron density of their inner segments. The round, light-staining cone cell nuclei occupy the layer of perikarya along the apical border of the outer nuclear layer. The remainder of the outer nuclear layer consists of oblong, deeply-stained rod cell nuclei. Ribbon type synaptic complexes involving photoreceptor cell axons, horizontal cell processes, and bipolar cell dendrites characterize the outer plexiform layer. The inner nuclear layer is comprised of horizontal, bipolar, and amacrine cell perikarya as well as the perikarya of the Müller cells. The light-staining horizontal cell nuclei are prominent along the apical border of the inner nuclear layer. The light-staining amacrine cell nuclei form a more or less continuous layer along the basal border of the inner nuclear layer. Both conventional and ribbon-type synapses characterize the inner plexiform layer. The ganglion cells form a single cell layer. The optic fiber layer contains bundles of axons surrounded by Müller cell processes. Small blood vessels and capillaries are present in the basal portion of the retina throughout the region extending from the internal limiting membrane to the outer plexiform layer. The adult one-year-old retina is compared with the retina at the time of eye opening.     

Retinal development in the lamprey (Petromyzon marinus L.): Premetamorphic ammocoete eye

Development of the retina of the ammocoete begins early in embryogenesis, with the formation of the optic vesicle, but development of the rudimentary eye is suspended and remains arrested during larval life. Prior to the onset of metamorphosis, the retina of the ammocoete is completely undifferentiated, with the exception of a small area (Zone II) surrounding the optic nerve head, where all of the adult retinal layers are found. The photoreceptors in this area have developed to include synaptic contacts as well as inner and outer segments. The pigment epithelium in this area, too, has differentiated to include well-formed melanin granules, myeloid bodies and endoplasmic reticulum and is closely associated with the receptor cell outer segments. With the approach of metamorphosis, differentiation of the remainder of the retina (Zone I) begins, taking place in a radial fashion from the optic nerve head. Differentiating pigment epithelial cells adjacent to the differentiated retinal zone begin to accumulate melanin granules. In the neural retina, junctional complexes are established in the form of an external limiting membrane, and connecting cilia project into the optic ventricle. Photoreceptor differentiation begins with the formation of a mitochondria-filled ellipsoid within the inner segment. Development and differentiation of the ammocoete retina is unique to vertebrates in that only a small area of differentiated retina is present during the larval stage. The remainder of the retina differentiates and becomes functional during metamorphosis.     

Histological examination of chick embryos with bilateral microphthalmus with reference to laterality in the visual apparatuses

The histological abnormalities of chick embryos with bilateral microphthalmus were examined in serial paraffin sections with special reference to laterality in the visual apparatuses, including the cornea, lens, neural retina and pigment epithelium. There was marked laterality in the above structures; some eyeballs had individual, if incomplete, sublayers of the cornea and the neural retina, and others not. The sublayers of the neural retina were occasionally observed even in eyeballs at the stage of optic vesicle formation, in contrast to the previous notion that the pigment epithelium induces the maturation of the primordial neural retina after optic vesicle differentiation into the optic cup. There was also a case where developmental differences between the right and left eyeballs were absent except in the lens. These findings suggest that chick embryos with bilateral microphthalmus exhibit a more complex histological profile or diversity than previously considered, possibly as a result of the differential actions of various mutagens and endogenous trophic factors on the developing visual system.     

Retinal histogenesis in an altricial avian species,the zebra finch (Taeniopygia guttata,Vieillot 1817)

Comparative developmental studies have shown that the retina of altricial fish and mammals is incompletely developed at birth, and that, during the first days of life, maturation proceeds rapidly. In contrast, precocial fish and mammals are born with fully differentiated retinas. Concerning birds, knowledge about retinal development is generally restricted to a single order of precocial birds, Galliformes, due to the fact that both the chicken and the Japanese quail are considered model systems. However, comparison of embryonic pre‐hatchling retinal development between altricial and precocial birds has been poorly explored. The purpose of this study was to examine the morphogenesis and histogenesis of the retina in the altricial zebra finch (Taeniopygia guttata, Vieillot 1817) and compare the results with those from previous studies in the precocial chicken. Several maturational features (morphogenesis of the optic vesicle and optic cup, appearance of the first differentiated neurons, the period in which the non‐apical cell divisions are observable, and the emergence of the plexiform layers) were found to occur at later stages in the zebra finch than in the chicken. At hatching, the retina of T. guttata showed the typical cytoarchitecture of the mature tissue, although features of immaturity were still observable, such as a ganglion cell layer containing many thick cells, very thin plexiform layers, and poorly developed photoreceptors. Moreover, abundant mitotic activity was detected in the entire retina, even in the regions where the layering was complete. The circumferential marginal zone was very prominent and showed abundant mitotic activity. The partially undifferentiated stage of maturation at hatching makes the T. guttata retina an appropriate model with which to study avian postnatal retinal neurogenesis.     

Nogo-A在小鼠胚胎新生视网膜节细胞及其轴突的表达

目的 研究小鼠胚胎阶段Nogo-A在视网膜节细胞(RGCs)及其轴突上的表达及时程变化. 方法 取不同发育阶段的小鼠胚胎,采用免疫荧光染色,以激光扫描共焦显微镜观察Nogo-A在视觉传导通路中的表达.并采用免疫双标染色确定视网膜中表达Nogo-A蛋白的细胞类型. 结果 在视网膜发育的早期阶段(E12),Nogo-A密集表达于具有放射状形态的细胞上,Nogo-A免疫阳性产物出现在胞质、胞膜以及轴突上.Nogo-A与Tuj-1双标染色显示,此阶段的视网膜中几乎所有RGCs及其轴突都表达有Nogo-A;在稍晚的发育阶段(E13),视网膜中表达Nogo-A的RGCs数量明显减少,且仅出现在节细胞层以外的室周带和睫状体边缘区.在视网膜的神经纤维层,大部分RGCs轴突不再表达Nogo-A,仅有少量视觉纤维为Nogo-A免疫阳性;RGCs的神经发生基本完成后(E15), 视网膜中几乎检测不到Nogo-A免疫阳性的细胞,但视网膜纤维层仍有少量表达Nogo-A的节细胞轴突.与之类似,视神经盘、视茎、视交叉和视束都观察到少量Nogo-A免疫阳性的轴突.值得注意的是,视束中表达Nogo-A的纤维集中位于表浅部位,而此处恰为新近到达轴突的通过部位. 结论 Nogo-A在视网膜RGCs以及轴突上表达的时程变化和位置特点提示,新生RGCs及其轴突表达Nogo-A,成熟后RGCs内Nogo-A的表达则下调.推测新生RGCs及其轴突中表达的Nogo-A可能与减少轴突分叉等细胞的内在功能有关.     

Spatial and temporal correlation between early nerve fiber growth and neuroepithelial cell death in the chick embryo retina

Summary The distribution of cell death in the ventral pycnotic zone of the chick embryo retina was studied in Hamburger-Hamilton's stages 16 to 25 (2 1/2 to 4 1/2 days of incubation). The number of fragments appearing in the retina increases notably from stage 20 at which stage they are limited almost exclusively to the optic disc region. At the same time optic fibers are seen in this area for the first time. In stage 24 cell death phenomena are numerous in the ventral retina, and become even more extensive in the following stage. Stage 25 meanwhile sees a drop in cell death in the dorsal retina. The overall picture presented by cell remains and young ganglion cells indicates that in stages 19–23 cell death occurs mainly in the zone between the ganglion cells of the posterior pole and the optic stalk. In the stage 25 retina most of the cell fragments of the ventral retina are found on either side of the fissure, while ganglion cells in the process of sending out axons toward the fissure appear laterally (nasally and temporally) to these zones of degeneration. Hence a spatial and temporal correlation is established between fiber growth and neuroepithelial cell degeneration, allowing us to construct a hypothesis with regard to the role that cell death might play in setting up an initial pattern of optic fiber growth.     

Raldh2 expression in optic vesicle generates a retinoic acid signal needed for invagination of retina during optic cup formation.

Three retinaldehyde dehydrogenase genes (Raldh1, Raldh2, and Raldh3) expressed in unique spatiotemporal patterns may control synthesis of retinoic acid (RA) needed for retina development. However, previous studies indicate that retina formation still proceeds normally in Raldh1-/- mouse embryos lacking RA synthesis in the dorsal neural retina at the optic cup stage. Here, we demonstrate that Raldh2-/- embryos lacking RA synthesis in the optic vesicle exhibit a failure in retina invagination needed to develop an optic cup. This was also observed in Raldh1-/-:Raldh2-/- double mutants, which develop similarly. Both mutants retain RA activity in the lens placode associated with Raldh3 expression, but this RA activity is insufficient to induce optic cup formation. Maternal RA administration at the optic vesicle stage rescues optic cup formation in Raldh2-/- and Raldh1-/-:Raldh2-/- embryos, demonstrating that Raldh1 is not required during rescue of optic cup development. The optic cup of rescued Raldh1-/-:Raldh2-/- embryos exhibits normal RA activity and this is associated with Raldh3 expression in the retina and lens. Thus, RA signaling initiates in the optic vesicle in response to Raldh2 but can be maintained during optic cup formation by a gene other than Raldh1, most likely Raldh3. Loss of optic vesicle RA signaling does not effect expression of early determinants of retina at the optic vesicle stage (Pax6, Six3, Rx, Mitf). Our findings suggest that RA functions as one of the signals needed for invagination of the retina to generate an optic cup.     

Retinal cell death occurs in the absence of retinal disc invagination: experimental evidence in papaverine-treated chicken embryos

In an attempt to clarify the relationship between the presence of retinal cell death and the invagination of the optic vesicle, we have tested the occurrence and cytological characteristics of the retinal necrotic areas in the embryonic chicken after the administration in ovo of papaverine. Papaverine, a Ca2+ antagonist, was found to prevent the invagination of the optic vesicle. All embryonic retinae presented two distinct necrotic areas. However, these areas of cell death appeared abnormally located in the experimental, uninvaginated retina. One area was located at the transition between the retinal disc and the ventral wall of the optic vesicle; a second area was located in the dorsal wall of the optic vesicle, close to the optic stalk. We suggest that these necrotic areas represent the normal necrotic areas, should the invagination of the retinal disc have taken place. Retinal cell death appears to be programmed; it occurs whether the retinal disc invaginates or not. Cell death appears, in this experimental model, as a natural marker giving evidence that the embryonic retinal cells move from the optic stalk into the invagination retinal disc during normal eye cup formation. In addition to the uninvaginated optic vesicle the lens placode failed to invaginate in 45% of the cases, forming a lens vesicle in 55% of the remaining cases. This suggests that the two processes of invagination are governed by a different set of factors.     

Accumulation of CPC-precipitable material at apical cell surfaces during formation of the optic cup

Accumulation of extracellular material at the apical surfaces of cells in the optic vesicle was studied by precipitation with cetylpyridinium chloride (CPC) and scanning electron microscopy. Treatment at low salt concentration to preserve all precipitable material indicated an initial appearance of surface material at the time that the retinal primordium first formed. The amount of precipitate increased as the optic cup formed, particularly at the margins of the cup. Stability of the precipitate during subsequent washing at higher salt concentrations suggested that the apical cell surface material contained highly acidic glycosaminoglycans. The greatest resistance to extraction occurred during the period in which invagination was most pronounced.     

单侧视束损伤后大白鼠视网膜内胆碱能神经元的研究

本实验应用组织化学及免疫细胞化学的方法观察大白鼠视网膜内乙酰胆碱酯酶和胆碱乙酰转移酶的活性和分布,进而对正常大白鼠及左侧视束损伤后的大白鼠视网膜胆碱能神经元进行了研究。结果表明不论是在出生时(即发育早期)还是在成年后损伤大白鼠的左侧视束时,其右侧视网膜内乙酰胆碱酯酶和胆碱乙酰转移酶的活性和分布与正常大白鼠相比都未见改变。提示这两种胆碱酶分布于视网膜的内部的神经元(intrinsic ncuron)不是节细胞,它们包括内核层的无长突细胞及节细胞层的移位无长突细胞等。大白鼠视网膜胆碱能神经元的成熟与分化很可能不直接依赖于视网膜节细胞。     

Large retinal ganglion cells in the rat: their distribution and laterality of projection

Summary The distributions of the ipsilaterally and contralaterally projecting large ganglion cells in the retina of the rat were determined, using the retrograde transport of Horseradish peroxidase (HRP) following injections into one optic tract. Labelled large retinal ganglion cells occur throughout the contralateral retina and throughout the temporal crescent of the ipsilateral retina, but there is a noticeable decrease in their density in the contralateral retina's temporal crescent. This retinal region was identified in these same retinae by injecting a retrogradely transported flourescent tracer into the optic tract opposite that receiving the HRP. The density of large retinal ganglion cells increases in both the contralateral retina and the ipsilateral temporal crescent in the upper temporal periphery such that, together, these two populations of large cells combine to produce a peak density centred on the retinal representation of the visual field's vertical midline. This peak density of large retinal ganglion cells must therefore be further peripheral than the peak density for the total population of retinal ganglion cells, since all evidence indicates that the latter is positioned nasal to the vertical midline's representation. This was verified in one rat, in which the density distribution of the total population of retinal ganglion cells was determined and compared with the distribution of the large cell population. The results suggest that the rat possesses a specialized retinal focus of large ganglion cells for viewing the visual field directly in front of the animal.     

Development of normal retinal organization depends on Sonic hedgehog signaling from ganglion cells

The adult retina is organized into three cellular layers--an outer photoreceptor, a middle interneuron and an inner retinal ganglion cell (RGC) layer. Although the retinal pigment epithelium (RPE) and Müller cells are important in the establishment and maintenance of this organization, the signals involved are unknown. Here we show that Sonic hedgehog signaling from RGCs is required for the normal laminar organization in the vertebrate retina.     

Prenatal development of the eye in the golden hamster

Prenatal development of the eye in golden hamsters is described at the light microscopic level as twelve periods based on salient morphological features. Period one includes shallow and deepened optic sulci in the cephalic neural plate and closing prosencephalon. Period two shows V-shaped prosencephalic evaginations, and Period three, bulbous vesicles. Contact of the retinal disc and lens placode at Period four precedes presumptive retinal invagination and lens pit formation during Period five. During Period six an open lens vesicle, precociously expanded dorsal and lateral optic cup regions and widespread optic fissure margins are evident. A spherical lens lumen, deepened optic cup and narrow optic fissure characterize Period seven. At Period eight posterior lens fibers are elongated, the optic cup is expanded ventrally, and optic fissure margins are juxtaposed or fusing. By Period nine the lens lumen is nearly or completely occluded, the optic fissure is essentially fused and axonal fibers are exiting from the globe. During Period ten corneal components are associated, axonal processes extend to the diencephalon, and lid folds cover more than half of the cornea. By Period eleven eyelids are fused. Anteriorly the pigmented epithelium is thickened and densely pigmented; outer and inner neuroblastic retinal layers are clearly distinguishable. Period twelve is marked by conspicuous anterior folds of pigmented epithelium; a relatively anuclear zone virtually separates outer and inner neuroblastic layers of the retina.