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.     

形觉剥夺对豚鼠锥视蛋白表达的影响研究

目的探讨形觉剥夺对豚鼠锥细胞视蛋白表达的影响。方法出生1周的豚鼠28只,分为正常对照组和形觉剥夺组,各14只,按自然亮暗周期饲养。形觉剥夺组随机选取一只眼以半透明橡胶遮盖,正常对照组随机选择一眼作为对照。4周后取眼球行RT-PCR检测神经视网膜M-视蛋白和S-视蛋白的mRNA表达,并进行豚鼠视网膜铺片后免疫组织化学方法观察两种视蛋白的分布和表达密度,一抗为兔抗鼠红／绿锥视蛋白抗体或蓝锥视蛋白抗体,二抗为羊抗兔IgG带488荧光抗体。以Leica光学显微镜和Leica共聚焦显微镜观察豚鼠视网膜腹侧（下方）和背侧（上方）及中央区的锥细胞视蛋白的表达。结果实验前屈光状态为：对照眼（5．63±0．87）D,形觉剥夺处理眼（5．47±1．02）D,4周后屈光状态分别为：对照眼（4．38±1．02）D,形觉剥夺处理眼（-3．03±0．78）D;屈光度的改变分别为：（-1．25±0．53）和（-8．38±1．44）D。正常豚鼠的视网膜短波敏感锥细胞分布为腹侧（下方）多于背侧（上方）;中央密度最高,密度变化为突变。豚鼠的中波敏感锥细胞分布在背部（上方）多于腹侧（下方）,中央密度最高,密度变化为渐变;正常组短波敏感蛋白的表达密度：下方为（805．0±203．3）mm^-2,上方为（100．0±57．7）mm^-2;中央为（1637．2±314．1）mm^-2。形觉剥夺组：下方为（640．9±196．8）mm^-2;中央为：（1016．7±144．6）mm^-2,上方为（70．9±30．8）mm^-2;正常组M-视蛋白的表达密度：上方为（946．2±388．5）mm^-2,中心为（1666．7±137．8）mm^-2,下方为（175．0±100．9）mm^-2;形觉剥夺组：上方为（1436．7±366．0）mm^-2,中心为：（2780．0±180．5）mm^-2,下方为（318．2±172．7）mm^-2。RT-PCR检测示形觉剥夺眼和对照眼的M-视蛋白的相对吸光度值分别为1．06±0．07和0．51±0．10,S-视蛋白的吸光度值分别为0．70±0．07和1．25±0．06。形觉剥夺性近视眼视网膜3个观察区域M-视蛋白的表达均较正常对照增加,S-视蛋白的表达减少,两因素方差分析示差别均有统计学意义（P〈0．05）。结论形觉剥夺性近视眼M-视蛋白的表达增加,S-视蛋白的表达减少,提示感光细胞锥细胞的视蛋白表达有可能在近视的发生发展中起了作用。     

rd11小鼠视锥细胞变性过程中视蛋白的变化特点

背景视网膜变性11（rd11）小鼠是近年来新发现的一种自发突变的视网膜变性小鼠。研究证实,rd11小鼠出生后随着鼠龄的增长出现快速的光感受器变性,且视杆细胞变性早于视锥细胞变性,但对于视网膜不同区域视锥细胞变性的特点还不十分清楚。目的应用视网膜铺片免疫荧光染色技术观察不同鼠龄rd11小鼠M-视蛋白和S-视蛋白在视网膜的表达分布及变化特点,为相关疾病的基因治疗研究提供实验依据。方法取出生后14、28、42d的rd11小鼠各5只,制备视网膜铺片,采用免疫荧光组织化学法分别标记小鼠视网膜后极部颞上、颞下、鼻上和鼻下象限M-视蛋白和S-视蛋白的表达,观察随rd11小鼠鼠龄的变化视网膜各区域M-视蛋白和S-视蛋白的荧光形态和密度,并与相应鼠龄的C57BL／6J小鼠进行比较。结果出生14d的rd11小鼠视网膜M-视蛋白和S-视蛋白的红色荧光形态和密度与C57BL／6J小鼠接近,但出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下4个区域M-视蛋白和S-视蛋白表达密度均明显降低,荧光形态由纺锤形逐渐变为点状,出生42d的rd11小鼠视网膜部分区域M-视蛋白和S-视蛋白表达消失。出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下区域M-视蛋白的表达密度分别为（414±32）、（300±8）、（324±22）和（250±20）个／0．037mm^2,明显低于同龄C57BL／6J小鼠的（484±21）、（442±19）、（459±34）和（436±12）个／0．037mm^2,差异均有统计学意义（t=4．114、15．225、7．505、17．990,均P〈0．05）;上述4个区域S-视蛋白的表达密度下降更明显,分别为（8±4）、（175±16）、（74±13）、（315±20）个／0．037mm^2,明显低于C57BL／6J小鼠的（73±16）、（436±30）、（393±30）和（480±19）个／0．037mm^2,差异均有统计学意义（t=8．555、17．076、21．637、13．498,均P〈0．05）。结论rd11小鼠视锥细胞中的M-视蛋白和S-视蛋白均随着鼠龄的增长而急剧减少,以S-视蛋白更为明显。随着鼠龄的增长,rd11小鼠M-视蛋白变性从视神经周围区向鼻下方,再向颞上方逐渐进展,而S-视蛋白变性由颞上方向鼻下方逐渐进展。     

Co-expression of murine opsins facilitates identifying the site of cone adaptation

Murine cones contain two opsins in the same cone, one ultraviolet (UV) and the other middle-wavelength sensitive (M). A long-wavelength flash only affecting M-opsin suppresses the cone electroretinogram (ERG) produced by light absorption of UV-cone opsin raising the hypothesis that activation of M-cone opsin suppresses UV-cone opsin responses in the same cone. Here we show that pharmacologic blockade of synaptic transmission in the superfused murine retina, which eliminates interaction from second-order neurons, fails to prevent suppression of the UV-opsin driven pathway by long-wavelength stimuli. This proves that the antagonism must be occurring in the same cone, co-expressing both opsins. Our results show that UV-opsin suppression successively ceases in presence of the M-opsin activating background light, which implies that cone light adaptation is controlled at the opsin stage, before activation of transducin. It also reveals the time course of a transient desensitization of cones due to post-opsin factors in the transduction cascade.     

Photoreceptor distribution in the retina of adult Pacific salmon: corner cones express blue opsin

The retina of salmonid fishes has two types of cone photoreceptors: single and double cones. At the nuclear level, these cones are distributed in a square mosaic such that the double cones form the sides of the square and the single cones occupy positions at the centre and at the corners of the square. Double cones consist of two members, one having visual pigment protein maximally sensitive to green light (RH2 opsin), the other maximally sensitive to red light (LWS opsin). Single cones can have opsins maximally sensitive to ultraviolet (UV) or blue light (SWS1 and SWS2 opsins, respectively). In Pacific salmonids, all single cones express UV opsin at hatching. Around the time of yolk sac absorption, single cones start switching opsin expression from UV to blue, in an event that proceeds from the ventral to the dorsal retina. This transformation is accompanied by a loss of single corner cones such that the large juvenile shows corner cones and UV opsin expression in the dorsal retina only. Previous research has shown that adult Pacific salmon have corner cones over large areas of retina suggesting that these cones may be regenerated and that they may express UV opsin. Here we used in-situ hybridization with cRNA probes and RT-PCR to show that: (1) all single cones in non-growth zone areas of the retina express blue opsin and (2) double cone opsin expression alternates around the square mosaic unit. Our results indicate that single cone driven UV sensitivity in adult salmon must emanate from stimulation of growth zone areas.     

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

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

外源性全反视黄酸对出生后豚鼠锥细胞视蛋白表达和方向性的影响

目的观察一定浓度的外源性全反视黄酸（all-trans retinoic acid,ATRA）对豚鼠锥细胞视蛋白表达和方向性的影响。方法出生1周豚鼠28只，随机分为ATRA组（n=14）和正常对照组（n=14）。ATRA组随机选取一眼于暗红光下球周注射0．4mg／ml浓度ATRA 0．05ml，对照组随机选择一眼球周注射稀释ATRA用的相应浓度二甲基亚砜（0．001ml/L）和注射用水共0．05ml/L。4周后取眼球，行免疫组织化学方法和RT-PCR技术观察M-视蛋白和S-视蛋白的分布和表达密度，并检测两种视蛋白的mRNA表达，方向性以Leica光学显微镜进行观察。结果ATRA处理眼S-视蛋白的表达密度：下方为（499．4±147．6）MM^-2，上方为（87．8±44．9）MM^-2，中央为（968．4±210．2）MM^-2；正常组S-视蛋白的表达密度：下方为（805．0±203．3）mm^-2，上方为（100．0±57．7）mm^-2；中央为（1637．2±314．1）mm^-2。ATRA处理眼M-视蛋白的表达密度：上方为（1326．1±267．0）mm^-2，中央为（2984．0±613．4）mm^-2，下方为（232．9±173．6）mm^-2；正常组M-视蛋白的表达密度：上方为（946．2±388．5）mm^-2，中央为（1666．7±137．8）mm^-2，下方为（175．0±100．9）mm^-2。0．4mg／ml浓度的ATRA0．05ml局部球周注射后使豚鼠视网膜的M-视蛋白的表达密度较正常对照组增加,S-视蛋白的表达密度减少：RT-PCR检测示M-视蛋白的相对光密度值分别为1．25±0．11（ATRA处理眼）和0．51±0．10（对照眼）,S-视蛋白的相对光密度值分别为0．61±0．09（ATRA处理眼）和1．25±0．06（对照眼）。ATRA处理眼与对照眼相比,M-视蛋白的mRNA表达增加，S-视蛋白的mRNA表达下降．两因素方差分析示两组差异均有统计学意义（P〈0．05）。铺片的免疫组化结果经光学显微镜观察提示，正常豚鼠视网膜M-视蛋白有一较小的偏斜角，朝向较垂直,ATRA作用后可见M-视蛋白偏斜角明显,朝向水平     

S-opsin protein is incompletely modified during N-glycan processing in Rpe65 mice

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65^−/− mice lack 11-cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65^−/− mice. Retina-RPE-choroids of Rpe65^−/− mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65^−/− mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at ∼40-kDa as a major band in wild-type mice, whereas ∼42-kDa S-opsin protein was detected in Rpe65^−/− mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65^−/− mice on SDS-PAGE was similar. In addition, ∼25-kDa S-opsin polypeptide was notably detected in Rpe65^−/− mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65^−/− mice, but expression of M-opsin mRNA in Rpe65^−/− mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65^−/− mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65^−/− mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65^−/− mice.     

Guinea pigs reared in a monochromatic environment exhibit changes in cone density and opsin expression

This study aimed to determine if a monochromatic environment will affect the development of cones in a guinea pig model. Thirty 3-day-old guinea pigs were randomized into three groups and exposed to green, violet, and white light (control) for 8 weeks. The animals were sacrificed and the density of middle-wavelength cones (M cones) and short-wavelength sensitive (S cones) and expression of M-opsin and S-opsin were determined. The density of M cones was increased in the green light group as compared to the control group, and decreased in the violet light group as compared to the control group (both, p < 0.05). There was no significant difference in the density of the S cones among the groups (all, p > 0.05). The density of coexpressing cones in the middle retina was significantly increased in the green light group in comparison to the violet light group (p < 0.01). In addition, there was a significant increase in the level of M-opsin as determined by Western blotting and M-opsin mRNA expression as determined by PCR analysis in the green light group as compared to the control group and a significant decrease in violet light group as compared to the control group (all, p < 0.05). No significant difference in S-opsin level or S-opsin mRNA expression was noted among the groups. We concluded that monochromatic lighting affected the density of cones and expression of opsins in a guinea pig model, and this indicates that the retinal color visual system of the guinea pig possess developmental plasticity.     

Immunocytochemical analysis of photoreceptors in the tiger salamander retina

In the tiger salamander retina, visual signals are transmitted to the inner retina via six morphologically distinct types of photoreceptors: large/small rods, large/small single cones, and double cones composed of principal and accessory members. The objective of this study was to determine the morphology of these photoreceptors and their synaptic interconnection with bipolar cells and horizontal cells in the outer plexiform layer (OPL). Here we showed that glutamate antibodies labeled all photoreceptors and recoverin antibodies strongly labeled all cones and weakly labeled all rods. Antibodies against calbindin selectively stained accessory members of double cones. Antibodies against S-cone opsin stained small rods, a subpopulation of small single cones, and the outer segments of accessory double cones and a subtype of unidentified single cones. On average, large rods and small S-cone opsin positive rods accounted for 98.6% and 1.4% of all rods, respectively. Large/small cones, principle/accessory double cones, S-cone opsin positive small single cones, and S-cone opsin positive unidentified single cones accounted for about 66.9%, 23%, 4.5%, and 5.6% of the total cones, respectively. Moreover, the differential connection between rods/cones and bipolar/horizontal cells and the wide distribution of AMPA receptor subunits GluR2/3 and GluR4 at the rod/cone synapses were observed. These results provide anatomical evidence for the physiological findings that bipolar/horizontal cells in the salamander retina are driven by rod/cone inputs of different weights, and that AMPA receptors play an important role in glutamatergic neurotransmission at the first visual synapses. The different photoreceptors selectively contacting bipolar and horizontal cells support the idea that visual signals may be conveyed to the inner retina by different functional pathways in the outer retina.     

Erratum: Temporal and spatial patterns of opsin gene expression in the zebrafish (Danio rerio): corrections with additions.

We report here a reexamination of the developmental expression of cone opsins in the zebrafish retina. The red- and blue-sensitive opsins appear at 51 h postfertilization (hpf) whereas ultraviolet (UV) opsin is not seen until after 55 hpf. More cells show red cone opsin expression than blue at 51 and 55 hpf, indicating the sequence of cone opsin expression in zebrafish is first red, then blue, and finally UV. Curiously, morphological development of the cones is in reverse order; UV cones appear quite mature by day 6-7 postfertilization (pf), but morphologically, red cones do not appear adult-like until 15-20 days pf.     

Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors

Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11-14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.     

Distribution and density of medium- and short-wavelength selective cones in the domestic pig retina

The topography of medium (M)- and short (S)-wavelength sensitive cone photoreceptors was studied in the domestic pig retina. Antisera specific for M or S opsin as well as cone photoreceptor proteins arrestin and alpha-transducin were used to label cone types. Retinal wholemounts and their blood vessel patterns were drawn and specific regions removed. The wholemounts were immunocytochemically labelled to detect both M and S cones, and the specific regions labelled to detect S cones. Cones were counted in a 1 mm grid pattern, using the drawings as a guide. Pig retina has a high cone density retinal streak extending across the retina covering the optic disc (OD) and horizontal meridian. Densities in the streak are 20,000-35,000 mm(-2). Two higher peaks occur in the streak, one in temporal retina near the OD (39,000 mm(-2)) and the other in nasal retina 5-7 mm from the OD (40,500 mm(-2)). The lowest cone density is in far peripheral inferior retina (7000 mm(-2)). The total number of cones in pig retina is 17-20 million. Both types of cones are found throughout the retina, with S cone percentages ranging from 7.4 to 17.5% in no consistent topographical pattern. S cones have an irregular local distribution which can vary from a regular hexagonal pattern to small clusters of adjacent S cones to small areas lacking S cones. Double-label immunocytochemistry found that virtually all S cone outer segments (OS) contain some M opsin. M cone OS do not label at detectible levels for S opsin. Domestic pig retina is widely available, large, has a high cone density and has two types of cones. This tissue should be an excellent source for biochemical analysis of cone proteins, and for in vitro approaches to understanding cone survival factors.     

The spatial and temporal expression of outer segment proteins during development of Macaca monkey cones

PURPOSE: To characterize the spatial and temporal expression of key structural and phototransduction cascade proteins in the monkey cone outer segment (OS). METHODS: Retinas from Macaca monkeys from ages fetal day (Fd) 89 through adulthood were double labeled using immunofluorescence for short (S) or long/medium (L/M) wavelength-sensitive cone opsin and either a structural protein (peripherin) or a phototransduction cascade protein (alpha-transducin [alpha-T], phosphodiesterase [PDE], or rhodopsin kinase [RK]). The spatial and temporal patterns of expression for each protein at each age were determined and graphed as a percentage of retinal coverage. RESULTS: In both cone types, opsins and phototransduction proteins appear first in the fovea and last at the retinal edge. Peripherin appears concomitantly with opsin in both S and L/M cones, but S cones express peripherin and opsin 1 to 3 weeks before neighboring L/M cones. Alpha-T, PDE, and RK are expressed together in the L/M cone OS shortly after L/M opsin appears. Phototransduction proteins are not expressed in S cones until 1 to 3 weeks after the appearance of S opsin and at the same time that neighboring cones are expressing both L/M opsin and phototransduction proteins. CONCLUSIONS: The concomitant appearance of opsin and peripherin strongly suggests roles in promoting the structural integrity of the developing OS. Phototransduction cascade proteins appear in the developing OS at the same time as one another, but after opsin. The significant lag between their expression and that of S cone opsin indicates that phototransduction proteins are not essential for OS formation, nor does opsin expression trigger their expression. The different temporal but similar spatial expression patterns of phototransduction proteins within S and L/M cones suggests that some local signal(s) coordinates their appearance.