Systematic analysis of E-, N- and P-cadherin expression in mouse eye development

Cadherins are a family of Ca(2+)-dependent cell adhesion molecules. Through their homophilic binding interactions, cadherins play important roles in tissue formation and maintenance during development. Here the authors compare the expression patterns of the three classical cadherins, E-, N- and P-cadherin, during mouse eye development from embryonic day 9.5 (E9.5) to adult. It was found that: (1) The expression patterns of N- and P-cadherin are mutually exclusive in most ocular tissues during development. N-cadherin mRNA is detected specifically in the lens placode during lens induction at E9.5, and is absent in the rest of the surface ectodermal tissues. In contrast, P-cadherin is expressed in the surface ectoderm but not in the lens vesicle. N-cadherin is expressed continuously in the lens pit, lens vesicle, and in the epithelial cells and newly differentiating fiber cells of the mature lens. P-cadherin is expressed in the epithelial cells of the cornea, eyelids and Harderian gland. Reciprocal expression patterns of N-and P-cadherins are also seen during retinal development. N-cadherin is initially expressed in both the inner and outer layers of the optic cup at E9.5. N-cadherin expression persists in the inner layer as it develops into neural retina, but is turned off in the outer layer where the cells differentiate into retinal pigment epithelial (RPE) cells and express P-cadherin. Reciprocal patterns of expression are also seen in the ciliary epithelium. N-cadherin is expressed in the inner layer and P-cadherin in the outer layer of the ciliary epithelium. (2) E- and P-cadherins are epithelial cadherins. Their expression patterns in the eye are not identical. Both cadherins are found in the epithelia of the cornea, eyelid and Harderian gland. In contrast, lens epithelial cells express E- but not P-cadherin, and RPE cells express P- but not E-cadherin. (3) In addition to its high expression in surface ectoderm-derived tissues, E-cadherin mRNA was also detected in some of the retinal ganglion neurons at postnatal day 14 (P14). E-cadherin expression in the neural retina has not been reported before. This study shows that cell fate determination in the eye occurs in conjunction with distinct changes in the patterns of cadherin gene expression.     

Generation and characterization of monoclonal antibodies specific for the retinal pigment epithelium.

Although both the neural retina and the retinal pigment epithelium (RPE) arise as neighboring portions of the embryonic optic cup, these two tissues follow very different developmental pathways. In order to obtain probes for the analysis of RPE development from its earliest divergence from the neural retina to late stages of differentiation, we have developed a panel of monoclonal antibodies which recognize antigens specific to the RPE. These probes have been applied to an immunohistochemical analysis of RPE development. The results indicate that the RPE is antigenically distinct from the neural retina even before the onset of overt differentiation. In addition, the RPE layer of the retina becomes further subdivided antigenically as its distinct anterior and posterior derivatives develop. These antibodies will be useful markers in the analysis of RPE development.     

干细胞诱导分化为视网膜色素上皮细胞的途径探讨

近年来,干细胞在眼科领域的研究及应用受到高度关注,胚胎干细胞(ES)、成体干细胞能够被定向诱导分化成视网膜色素上皮细胞,由此可获得转分化的大量的视网膜色素上皮细胞源,通过体内干细胞及视网膜色素上皮细胞移植有望应用于各种视网膜退行性疾病的细胞替代治疗。本文就各种干细胞诱导分化为视网膜色素上皮细胞的途径及应用进行探讨。     

视网膜色素上皮发育发生学的研究进展

视网膜色素上皮(retinal pigment epithelium,RPE)为一单层排列整齐的六角柱形细胞所组成,位于神经视网膜的光感受器和脉络膜的Bruch's膜之间,在维持光感受器细胞存活和正常功能方面起重要作用。多种先天性视网膜色素上皮疾病的发生与胚胎期的发育发生有着密切的关系。本文对RPE胚胎发育的基本过程、诱导RPE发生发育的信号分子、调控RPE发生发育的基因及维持RPE分化的信号通路等几个方面做一综述。     

视网膜色素上皮细胞直接铺片法与剥离铺片法比较

目的 介绍2种视网膜色素上皮(RPE)细胞铺片实验技术,比较其实验操作的难易程度及对RPE细胞形态和铺片完整性的影响.方法 实验研究.直接铺片法:大鼠眼球取出后用4％多聚甲醛固定30 min,解剖眼球,去除眼前段部分和眼内容物,小心剥去视网膜,将RPE贴附的眼后段部分剪成花瓣形并平整贴附在载玻片上,继续固定过夜.剥离铺片法:大鼠眼球取出后用4％多聚甲醛固定过夜,解剖眼球,去除眼前段部分和眼内容物,小心剥去视网膜,用镊子将RPE-Bruch膜复合物从眼球上分离,保存在PBS液中.结果 剥离铺片法能得到完整RPE细胞铺片,RPE细胞形态完整;直接铺片法中RPE细胞部分丢失,铺片边缘的RPE细胞被部分破坏.结论 直接铺片法操作简便,难度小,但容易破坏边缘的RPE细胞.剥离铺片法操作难度大,但对RPE细胞破坏程度小.     

Characteristics of embryonic retina transplanted to rat and rabbit retina

When aggregates of immature retina are transplanted to lesioned adult retina, the donor cells reorganize themselves into folded sheets and rosettes. With the exception of retinal ganglion cells and an inner limiting membrane, most cell types and all layers develop, corresponding to the normal retina. The transplants can integrate with the adult host retina without the presence of glial barriers. Mouse and human donor retinas have been transplanted to immunosuppressed rat hosts with long-term survival. Transplants of embryonic retina cografted with RPE can contain an apparent ganglion cell layer and an inner limiting membrane, which are not observed in transplants of retina alone. Embryonic rat donor retinas can be transplanted successfully after long-term storage in liquid nitrogen.     

RPE dysplasia with retinal duplication in a mutant mouse strain

An autosomal dominant mutation was produced by quadruple gonadal exposure of a male (C3H x 101)F1 mouse to 500 rad of X-irradiation. This mutation is maintained by the mating of affected heterozygous males to normal (C3H x C57B1)F1 females. Clinically apparent abnormalities were limited to the eyes and, in the affected adults, ranged from apparent anophthalmia to globes that were enlarged and exhibit large uveoscleral colobomas. Sequential evaluations of the embryogenesis of this condition have identified abnormal differentiation of the outer layer of the optic cup (presumptive retinal pigment epithelium-RPE) into a second layer of neural retina. The abnormality is identified as early as day 10 of gestation, during invagination of the optic cup and lens placode. The area of RPE dysplasia may be diffuse or regional with an abrupt transition from normal RPE and often demonstrates excessive and uncontrolled proliferation. The two symmetrical, apposed layers of photoreceptors fail to differentiate and begin to degenerate prenatally. Absence of normal RPE leads to failure of induction of adjacent choroid and sclera, resulting in a posterior segment consisting of a large neuroepithelial-lined cyst. Radiation-induced ocular malformations of this type have not been previously described. In addition, this model presents a unique opportunity to examine the processes leading to differentiation of a single, continuous epithelial layer into tissues as anatomically and functionally distinct as neural retina and RPE.     

Histogenesis of the cat retina

The development of the retina of the cat was studied with light and transmission electron microscopy from prenatal day 36 (E36) through postnatal day 9 (P9; eye opening), and at the adult stage (1 year). Tissue samples were taken from the posterior pole of the retina. At E36, the optic cup was completely formed, the pigment epithelium was a single cell layer, the retina consisted of an inner marginal fiber layer and an outer layer of neuroblastic cells, and the innermost cells of the neuroblastic cell layer, presumptive ganglion cells (which might still migrate through the inner plexiform layer) were displacing away from the neuroblastic cell mass. At E40, a distinct ganglion cell layer was seen. At E46, primitive horizontal cells appeared within the neuroblastic cell layer. Separation of the neuroblastic cell mass into inner and outer nuclear layers was first evident on E48; by E60, the layers were clearly separate. Conventional synapses were seen in the inner plexiform layer in the week prior to birth. Blood vasculature was observed prenatally as deep as the inner plexiform layer. In the newborn a few discs were seen in some photoreceptor cell outer segments. Synaptic ribbons appeared first in the outer plexiform layer in the newborn and then in the inner plexiform layer by P4.     

低氧诱导因子-1α在胚胎及出生后大鼠视网膜中的表达变化

目的 观察大鼠视网膜胚胎期及出生后早期发育过程中低氧诱导因子-1α(HIF -1α)的时空表达,探讨其表达变化与视网膜发育的关系。方法 实验大鼠分为孕12、16、20 d 组,出生后1、5、10 d组及成年组,每组各5只,共35只大鼠。用免疫组织化学方法、半定量逆转录聚合酶联反应（RT-PCR）方法检测视网膜H IF-1α蛋白及HIF-1αmRNA的表达。结果 胚胎期大鼠视网膜神经上皮层及视网膜色素上皮层均有HIF 1α阳性表达,出生后发育早期视网膜神经上皮层及视网膜色素上皮层也可见HIF-1α阳性表达,以神经节细胞、内网层更明显。随着发育进展,其阳性表达主要位于视网膜节细胞层。大鼠视网膜HIF-1α蛋白及mRNA的表达在胚胎期最高、出生后发育期逐渐下降、成年期最低,其差异有统计学意义（P<0.01）。结论 大鼠视网膜胚胎及出生后发育过程中HIF-1α的表达存在时空上的变化,这种变化可能与大鼠视网膜胚胎期及出生后早期的发育密切相关。     

The appearance and distribution of microglia in the developing retina of the rat

We have examined the development of microglia in the rat retina, using a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Retinas were studied from animals aged from E(embryonic day)12, just after the invagination of the optic cup and prior to the closure of the optic fissure, to adulthood. The lectin also proved a sensitive label for the endothelial cells of the developing retina. Our results provide some support for the view that microglia are derived from the monocyte-macrophage series of blood cells. At E12, most labeled cells were found at the vitreal surface, suggesting that they had come from the hyaloid circulation, while some had entered the retina and appeared to be migrating towards its ventricular surface. From E14 to early postnatal ages, most labeled cells had processes and resembled the amoeboid microglial cells described in silver carbonate staining studies (Ling, 1982). The number of labeled cells rose from about 700 to E14 to a peak of about 27,000 at P(postnatal day)7, and fell to about 19,600 by P12. As early as E16, a regularity was apparent in the distribution of microglial cells over the surface of the retina, the cells tending to avoid each other. Microglial cells are found throughout the thickness of the very young retina, but as the layers of the retina differentiate, they are increasingly restricted to the inner half of the retina. Our findings indicate that microglia enter the retina well before the period of neuronal death, making it unlikely that they invade the retina solely in response to cell death. Our results confirm however that, once in the retina, microglia become associated with, and appear to phagocytose, the pyknotic debris which appears during the period of neuronal death. They also become closely associated with the retinal vasculature. In the adult, the intensity of the labeling of microglia was much reduced. Those cells which were labeled appeared more differentiated, resembling the "resting microglia" described in earlier studies.     

Expression of the diabetes risk gene wolframin (WFS1) in the human retina

Wolfram syndrome 1 (WFS1, OMIM 222300), a rare genetic disorder characterized by optic nerve atrophy, deafness, diabetes insipidus and diabetes mellitus, is caused by mutations of WFS1, encoding WFS1/wolframin. Non-syndromic WFS1 variants are associated with the risk of diabetes mellitus due to altered function of wolframin in pancreatic islet cells, expanding the importance of wolframin. This study extends a previous report for the monkey retina, using immunohistochemistry to localize wolframin on cryostat and paraffin sections of human retina. In addition, the human retinal pigment epithelial (RPE) cell line termed ARPE-19 and retinas from both pigmented and albino mice were studied to assess wolframin localization. In the human retina, wolframin was expressed in retinal ganglion cells, optic axons and the proximal optic nerve. Wolframin expression in the human retinal pigment epithelium (RPE) was confirmed with intense cytoplasmic labeling in ARPE-19 cells. Strong labeling of the RPE was also found in the albino mouse retina. Cryostat sections of the mouse retina showed a more extended pattern of wolframin labeling, including the inner nuclear layer (INL) and photoreceptor inner segments, confirming the recent report of Kawano et al. [Kawano, J., Tanizawa, Y., Shinoda, K., 2008. Wolfram syndrome 1 (Wfs1) gene expression in the normal mouse visual system. J. Comp. Neurol. 510, 1-23]. Absence of these cells in the human specimens despite the use of human-specific antibodies to wolframin may be related to delayed fixation. Loss of wolframin function in RGCs and the unmyelinated portion of retinal axons could explain optic nerve atrophy in Wolfram Syndrome 1.     

年龄相关性黄斑病变易感因子2蛋白在正常人眼组织中的表达和定位

背景研究表明,年龄相关性黄斑病变易感因子2（ARMS2）基因A69S位点的变异与年龄相关性黄斑变性（AMD）的发病及病变的进展高度相关,但ARMS2蛋白在正常成人眼组织中的定位仍存在争议,相关功能尚未完全明确。目的研究ARMS2蛋白在正常成人视网膜和脉络膜各层的表达定位及其在视网膜色素上皮（RPE）细胞的分布,为进一步从蛋白水平研究ARMS2基因的功能提供实验基础。方法收集死亡后立即摘除的正常供体眼球10只,来自年龄为28～42岁的男性,将其中3只眼球的视网膜和脉络膜组织制作冰冻切片,采用组织免疫荧光法在激光共焦显微镜下检测ARMS2蛋白在视网膜和脉络膜各层的分布。另7只眼球用于分离视网膜组织,并用组织块培养法进行RPE细胞的原代培养,培养的细胞用CK32抗体进行免疫荧光染色鉴定。应用荧光显微镜检测ARMS2蛋白在RPE细胞内的表达及定位情况。结果激光共焦显微镜下可见ARMS2蛋白在正常人视网膜血管、RPE层、Bruch膜、脉络膜血管内呈强阳性表达,而在视网膜神经节细胞层、内核层、外丛状层、外核层、内丛状层ARMS2蛋白表达较弱。组织块法培养的RPE细胞呈多角形,细胞质中可见大量的棕褐色色素颗粒,CK32染色表明原代培养RPE细胞的细胞质中可见大量的绿色荧光颗粒。在RPE细胞内ARMS2阳性蛋白呈簇状红色荧光,主要分布于细胞质中,细胞核为蓝色荧光。结论成年人正常眼视网膜血管中、RPE层、Bruch膜、脉络膜血管内均有ARMS2蛋白的强表达,此外,RPE细胞的细胞质中也可见呈簇状分布的ARMS2蛋白。