Thyroid hormone action is required for normal cone opsin expression during mouse retinal development

PURPOSE: The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS: The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS: Hypothyroid newborn mice showed an increase in S-opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TRbeta1 and -beta2 were T3-binding defective, the pattern of opsin expression was similar to TRbeta ablation, showing increased S-opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TRbeta were mutated in the helix 12 AF-2 domain. CONCLUSIONS: The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.     

Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay

Electron-microscope immunocytochemistry and antibody staining of nitrocellulose replicas of SDS gels (Western blots) were used in a developmental study to detect the presence and localization of opsin in the developing photoreceptors of rds (020/A) mutant mice and their BALB/c normal controls. Western blot analysis of isolated retinal membranes first detected opsin at 10 postnatal days in both strains. Opsin levels rose progressively with development in BALB/c normal retinas. In contrast, levels in the rds retina became undetectable by 30 days after peaking at 15 days. Specific binding of anti-opsin antibodies was first observed by immunocytochemistry at postnatal 5 days in the distal plasma membrane of the connecting cilium in both BALB/c and rds retinas. Thereafter, labeling intensity increased progressively with development in the BALB/c retina. Anti-opsin labeling remained localized primarily to the plasma membrane of the distal cilium and to the outer segment with the exception that light labeling of the inner-segment plasma membrane was observed from 5-15 postnatal days. Antibody binding to photoreceptors in the rds mouse retina predominated in the plasma membrane of the connecting cilium at 5 postnatal days, but opsin was present at higher density in the inner segment plasma membrane at 5-, 10-, 15- and 20 postnatal days, when compared with BALB/c photoreceptors. From 10-20 postnatal days opsin-rich vesicles were observed in the ventricular (subretinal) space of the rds retina. Maximum intensity of labeling was observed at 15 postnatal days. By 30 postnatal days, labeling of the ciliary and inner-segment plasma membrane decreased to near background levels.     

斑马鱼视网膜感光细胞的发育及视蛋白的表达

目的研究斑马鱼视网膜感光细胞的发育和视蛋白的表达，明确利用斑马鱼研究视网膜和感光细胞的可行性。方法制备斑马鱼视网膜感光细胞视蛋白（视杆细胞视紫质、视锥细胞紫外线视蛋白和视锥细胞蓝色视蛋白）的RNA探针。收集受精后72，96，120h的斑马鱼眼球组织进行切片、电镜观察和整体原位杂交。结果斑马鱼的神经视网膜分层排列，包括3个细胞层和2个丛状层。随时间发展，视网膜的发育逐渐成熟，3个细胞层的细胞排列更加整齐，感光细胞的外节盘发育更加成熟，3种视蛋白的表达逐渐增强，范围扩大。结论利用斑马鱼进行视网膜和感光细胞的研究是可行的，视蛋白可作为感光细胞的标记。     

Codistribution of S-antigen and opsin in light-adapted retinal rods

The localization of S-antigen in ultra-cryosections of rat retina has been studied and compared with that of opsin by immunoelectron microscopy. Monospecific antisera to both proteins, and Protein A-coated gold particles of two sizes were used for (double) labeling. In the light-adapted retina, S-antigen showed codistribution with opsin in the rod outer segments. S-antigen labeling was diminished in dark-adapted retina. In retina pigment epithelium, multi-layered bodies were found containing S-antigen and rhodopsin representing spent rod outer segment fragments.In addition to theoretical considerations, there is indication that S-antigen is closely associated with the cytoplasmic surface of rod outer segment disc membranes. It seems moreover likely that S-antigen and opsin are closely associated in the illuminated retina. An important reason for this assumption was the observation that double labeling experiments showed interference of the antibodies to both antigens probably due to steric hinderance. The present results are complementary to, and in agreement with biochemical data from literature and point to an important function of S-antigen in the process of phototransduction.     

The development of visual pursuit during the first months of life

· Background: There are few previous investigations of smooth pursuit in infants. The aim of our study was to quantify visual pursuit in infants between 1 day and 16 weeks of age. · Methods: Eye movements of 97 healthy infants between 1 day and 16 weeks of age were recorded one to seven times with infrared photo-oculography. For stimulation of visual pursuit a square of 9.4 deg of visual angle with vertical gratings moved horizontally at a constant velocity of 7.5 deg/s. · Results: In the first 2 weeks of life, segments of smooth pursuit were measured with a maximum velocity of 7.93 deg/s, with a maximum gain of 1.06 and a maximal duration of 3.16 s. In sequential recordings no significant increases of velocity, gain or duration were found. However, the total time the subjects followed the stimulus with smooth plus saccadic pursuit increased significantly with age (from a median of 39.0% to a median of 61.5% of examination time). · Conclusion: This study clearly demonstrates that smooth pursuit is already present in the first week of life. We found no significant increase in velocity, gain and duration of smooth pursuit segments in the first 16 weeks of life with our recording technique. However, the total pursuit time, reflecting attention, increased with age. The ocular machinery to drive pursuit appears to be in place at birth and seems not to be influenced by increased attention in the first months of life. Received: 23 June 1997 Revised version received: 14 August 1997 Accepted: 1 October 1997     

Melanopsin is expressed in PACAP-containing retinal ganglion cells of the human retinohypothalamic tract

PURPOSE: The putative circadian photoreceptor melanopsin is found in rodents in a subpopulation of intrinsic light-sensitive retinal ganglion cells (RGCs) constituting the retinohypothalamic tract (RHT). The study was conducted to determine whether melanopsin is expressed in the human retina and costored with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), a marker for the RHT, projecting to the suprachiasmatic nucleus (SCN). Furthermore, whether melanopsin expression is conserved in retinas of blind patients with severe retinal degeneration was investigated. METHODS: In situ hybridization and immunohistochemistry was used to demonstrate melanopsin synthesis in human eyes of 17 donors and two postmortem hypothalami containing the SCN. The coexistence of melanopsin and PACAP in elements of the retinohypothalamic tract was studied by dual-labeling immunocytochemistry. RESULTS: Melanopsin expression was found in a subpopulation of RGCs located in the ganglion cell layer and displaced in the inner nuclear cell layer. Melanopsin-containing cells comprised approximately 0. 8% of all RGCs, with a distinct morphology characterized by two to four dendritic processes constituting a panretinal network. Melanopsin immunoreactivity was primary present at perikaryal boundaries and neuronal processes and to some extent also in the cytoplasm. PACAP and melanopsin were colocalized in the RGCs and PACAP-containing nerve fibers, seemingly innervating the retinorecipient part of the SCN. Melanopsin-expressing RGCs were conserved in retinas of blind patients with severe degeneration of the outer and/or inner layers. CONCLUSIONS: Given the expression of melanopsin in PACAP-containing RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people.     

Cone-mediated retinal function in cats during development

Cone electroretinograms (ERGs) were recorded in kittens from 25 to 150 days of age and in adult cats. B-wave amplitudes in kittens between 25 and 94 days of age were either within the adult range or greater than adult responses. After about 100 days, all kittens had adult-like amplitudes. Cone b-wave implicit time was markedly prolonged at the earliest ages tested and only after 80 days was the timing like that in adult cats. Amplitude and timing of 40-Hz flicker ERGs confirmed the single flash cone ERG data. Critical flicker fusion frequency was mature by 74 days of age. These findings, taken together with previous results, indicate that there are differences in the time course of development of cone- and rodmediated retinal function in the cat.     

Astrocyte proliferation during development of the human retinal vasculature.

Wholemounts of human fetal retinas were labeled with antibodies to Ki67 or proliferating cell nuclear antigen, to map the distribution of proliferating cells in the developing primary vasculature and neural retina. Double labeling was used to determine the relative proportions of endothelial cells (CD34), astrocytes (glial fibrillary acidic protein - GFAP) and microglia (major histocompatability complex class II) associated with the developing vessels. The differentiated region of neural retina (cold spot) was 3.5 mm(2)at 15 weeks gestation (WG), centred on the incipient fovea, and increased in size with age to 80.5 mm(2)by 23-24 WG. Ki67 immunoreactive cells were distributed throughout the developing vasculature at all ages. The mean density of dividing cells in the neural retina increased with gestational age from 146 mm(-2)at 15 WG, to 624 mm(-2)at 23-24 WG. By 20 WG proliferation in the vasculature overlapped the margins of the cold spot, which was almost completely vascularized by 23-24 WG, except for a narrow strip on the horizontal meridian, which included the incipient fovea. Counts of CD34/Ki67 immunoreactive cells indicated that 15-52% of proliferations in the developing vasculature at 18 WG are endothelial cells. In contrast, in the fellow retina 65-85% cells were Ki67/GFAP immunoreactive, indicating proliferation of astrocytes in situ. No dividing microglia were observed. The findings suggest that large numbers of proliferating astrocytes accompany the developing vessels as they migrate across the primate retina.     

Mannose receptor is expressed in normal and dystrophic retinal pigment epithelium.

In normal retinas, the phagocytosis of shed photoreceptor outer segments is mediated in part through a mannose receptor protein located in the apical retinal pigment epithelium membrane. As dystrophic rats of the Royal College of Surgeons have a defect in which the retinal pigment epithelium (RPE) is unable to phagocytize the shed outer segments, it is hypothesized that mannose receptor expression will be lost with the progression of photoreceptor degeneration. Immunohistochemical and molecular techniques have been used to study the developmental expression of the mannose receptor in normal and dystrophic retinal pigment epithelium. By immunofluorescence, the mannose receptor is localized to the retinal pigment epithelium, apical membrane region, beginning around 5 days postnatally in both normal and dystrophic retinas. In immunoblots, bands at 175 kDa are labelled by an anti-mannose receptor antibody in apical membrane samples from both normal and dystrophic RPE at all developmental times sampled. RT-PCR analysis reveals that mannose receptor message is present in normal and dystrophic RPE samples at all developmental time points examined. The present study demonstrates that the expression of the mannose receptor begins prior to outer segment differentiation and the initiation of phagocytosis in both normal and dystrophic RPE. Expression of the mannose receptor continues to be unchanged during the progression of photoreceptor degeneration in the dystrophic retina.     

Competition between retinal and 3-dehydroretinal for opsin in the regeneration of visual pigment

Rhodopsin regenerated faster than porphyropsin in all preparations of bullfrog opsin, bullfrog rod outer segment membrane and cattle opsin. When opsin was incubated with excess amount of an equimolar mixture of 11-cis-retinal and 11-cis-3-dehydroretinal, the composition of the regenerated pigment was simply dependent on the ratio of regeneration rates of rhodopsin and porphyropsin. This result can provide a mechanism to account for the discrepancy in vitamin A1/A2 composition between the retina and the pigment epithelium. The property of opsin preferring retinal to 3-dehydroretinal may be one of the basic factors affecting vitamin A1/A2 visual pigment systems.     

Astrocyte-endothelial cell relationships during human retinal vascular development

PURPOSE: To evaluate evidence for the presence of vascular precursor cells (angioblasts) and astrocyte precursor cells (APCs) in the developing human retina and determine their relationship. METHODS: Pax-2/GFAP/CD-34 triple-label immunohistochemistry was applied to four retinas aged 12, 14, 16, and 20 weeks of gestation (WG) to label APCs, astrocytes, and patent blood vessels. APCs are Pax-2(+)/GFAP(-), whereas astrocytes are Pax-2(+)/GFAP(+). Adenosine diphosphatase (ADPase) enzyme histochemistry, which identifies endothelial cells and vascular precursors, was applied to human retinas aged 12, 16, 17, and 19 WG. Nissl stain, a nonspecific cell soma marker, was applied to 14.5-, 18-, and 21-WG retinas. Established blood vessels were visualized with CD34 and ADPase. RESULTS: Topographical analysis of the distribution of Nissl-stained spindle cells and ADPase(+) vascular cells showed that these two populations have similar distributions at corresponding ages. ADPase(+) vascular precursor cells preceded the leading edge of patent vessels by more than 1 millimeter. In contrast, Pax-2(+)/GFAP(-) APCs preceded the leading edge of CD34(+) blood vessels by a very small margin, and committed astrocytes (Pax-2(+)/GFAP(+)) were associated with formed vessels and nerve fiber bundles. Two populations of ADPase(+) cells were evident, a spindle-shaped population located superficially and a deeper spherical population. The outer limits of these populations remain static with maturation. CONCLUSIONS: A combination of Pax-2/GFAP/CD34 immunohistochemistry, Nissl staining, and ADPase histochemistry showed that the vascular precursor cells (angioblasts), identified using ADPase and Nissl, represent a population distinct from Pax-2(+)/GFAP(-) APCs in the human retina. These results lead to the conclusion that formation of the initial human retinal vasculature takes place through vasculogenesis from the prior invasion of vascular precursor cells.     

An immunohistochemical study of opsin in photoreceptor cells following light-induced retinal degeneration in the rat

Light-induced retinal degeneration has been hypothesized to be rhodopsin-mediated. However, the alterations induced in the opsin moiety of the rhodopsin molecule and its distribution in the rod cell after a photic insult have not been definitively established. We used light and electron immunohistochemistry to study the alterations in retinal opsin immunoreactivity in a rat model of retinal photic injury. In normal unexposed rat retinas, opsin immunoreactivity was restricted to the rod outer segments. At 6 h after a 24-h light exposure, opsin immunoreactivity was present in the rod outer segments in both the superior and inferior retina, but in addition marked immunoreactivity was present in the inner segments in the superior quadrant of the light-damaged retina. At 6 days after exposure, intense immunoreactivity was noted around the severely degenerating rod nuclei and inner segments. However, at 21 days following light exposure, opsin immunoreactivity in areas of recovery was again restricted to the short regenerated rod outer segments. It appears that, despite severe light-mediated retinal degeneration, anti-opsin immunoreactivity persisted in the photoreceptor cells but with an altered pattern in damaged rod outer segments and photoreceptor perikarya. However, opsin immunoreactivity relocated to the regenerated rod outer segments in the recovery phase.Supported in part by grant R01 EY01903 (Pathology of Retinal Dysfunction) and Core Grant EY01792 from the National Eye Institute, Bethesda, Md.; a grant from the Lions of Illinois Foundation, Maywood Ill., and gifts from the Clifford Sawyer Estate and the McGraw Foundation, Arlington Heights, Ill.