PURPOSE:The aim of our work was to study apoptosis during the development of the retinal pigment epithelium (RPE) in mice between embryonic day (E) 10.5 and E12.5 and to examine a possible link between apoptosis and pigmentation.METHODS:We collected mouse embryos at E10.5, E11.5, and E12.5 and labeled apoptotic cells in 5-µm paraffin sections, using the terminal deoxynucleotidyl transferase dUTP nick end labeling technique. We counted the total number of cells and the number of apoptotic cells in the early developing RPE and calculated the percentage of apoptosis at each stage.RESULTS:In the C57BL/6J mouse, 17% of the RPE cells were apoptotic at E10.5 compared to 0.9% at E12.5. At E11.5, three-quarters of the RPE cells began to pigment, and apoptotic cells were located mostly in the nonpigmented part. In contrast, in the BALB/c mouse (tyrosinase-deficient) and pJ mouse (carrying mutations in the p gene) hypopigmented strains, the RPE contained significantly fewer apoptotic cells (7.5% and 10.1%, respectively) at E10.5 than controls. Subsequently at E11.5 and E12.5, the two hypopigmented strains displayed different apoptotic patterns; the BALB/c RPE had a similar percentage of apoptotic cells to controls (1.5% and 1.1%, respectively, for BALB/c versus 3.0% and 0.9%, respectively, for C57BL/6J), whereas the pJ RPE contained significantly more apoptosis (7.5% and 3.5%, respectively). Overall we observed differences in the evolution of the relative total number of RPE cells between the three strains.CONCLUSIONS:Apoptosis is a main event during the first stages of normal RPE development, indicating an essential role during RPE differentiation. Moreover, the early apoptotic pattern and possibly the whole early development of the RPE is different between hypopigmented and pigmented strains, as well as between BALB/c and pJ mice. This suggests the existence of regulatory and developmental differences with a more complex origin than just differing pigmentation levels.

La plupart des modèles animaux d’ischémie-reperfusion rétinienne qui ont été mis au point reposent sur l’injection de soluté isotonique dans la chamber antérieure de l’œil ou sur l’occlusion simultanée du nerf optique et des vaisseaux centraux rétiniens situés au cœur du nerf optique (artère et veine centrale de la rétine). Nous avons mis au point un nouveau modèle occlusion vasculaire unilatérale des vaisseaux rétiniens sans atteinte concomitante d’origine mécanique des axones des cellules ganglionnaires de la rétine. Par occlusion unilatérale de l’artère carotide externe et occlusion transitoire de l’artère ptérygopalatine ipsilalatérale, il s’est avéré possible chez la souris adulte (Souris avec amaurose Unilatérale (n = 40) et souris Sham (n = 40) d’éviter la reperfusion d’origine controlatérale et d’obtenir une amaurose unilatérale d’origine purement vasculaire. Il est possible de varier la durée d’ischémie et d’étudier le décours de la reperfusion rétinienne par diverses methods : électrorétinographie et angiographie in vivo puis après injection de fluorescéine intracardiaque et réalisation de rétines montées à plat par des méthods immunohistochimiques, par des méthodes d’étude de l’apoptose et surtout par RTPCR quantitative de certains ARNm, clés des diverses phases de la reperfusion. C’est ainsi qu’ont été étudiés des gènes d’expression précoce tels que Zif268, Arc, Jun, les gènes codant de très nombreux récepteurs au glutamate et des gènes impliqués dans la neurogénèse. Les temps étudiés ont été 4 heures, 24 heures, 72 heures, 7 jours et 1 mois avec 8 animaux groupe. Nous avons pu valider ce modèle comme modèle d’ischémie-reperfusion oculaire tant chez la souris que chez le rat. Des variations d’expression géniques ont pu être rattachées à chacun des stades étudiés. Par exemple celles des gènes COX2, GADD34, HSP70, PAI1. Nous avons mis au point le premier véritable modèle permettant d’étudier l’ischémie-reperfusion rétinienne sans interférences de facteurs mécaniques ou autres. Ce modèle se prête tout particulièrement aux tests précliniques des modulateurs des récepteurs aux glutamates (NMDA, Métabotropiques, AMPA, Kaïnate). Le glutamate joue en effet un rôle fondamenteur de neurotransmetteur dans les synapses des circuits rétiniens d’une part et dans les phénomènes liés à l’excitotoxicité. Ce modèle permettra d’étudier de nouvelles thérapies sur des souris KO, Knock in ou transgéniques atteintes d’hypertension artérielles et/ou d’athérosclérose situation fréquente retrouvée chez les sujets âgés. La thrombolyse peut s’accompagner d’excitotoxicité qui limite ses bénéfices. Ce modèle arrive donc au bon moment pour permettre des avancées thérapeutiques. Les accidents vasculaires oculaires s’accompagnent trop souvent encore d’échecs thérapeutiques du fait du caractère souvent trop tardif de l’examen des patients dû à la méconnaissance ou à l’incompréhension des symptômes ou malaises qu’ils ressentent. Ce modèle devrait permettre de mieux appréhender le potentiel thérapeutique des cellules souches de la rétine stimulées par l’ischémie.

Purpose: Discs large (dlg), scribble (scrib), and lethal giant larvae (lgl) are major suppressor genes in Drosophila melanogaster. They encode proteins that regulate cell polarity and cell proliferation in Drosophila and mammals. However, their basic oncogenic roles have not yet been established in mouse epithelial ocular cancer. We evaluated the potential implication of these proteins in tumorigenesis of adenocarcinomas originating from the retinal pigmented epithelium of the Trp1/Tag transgenic mouse model. We examined the changes in the distribution and levels of these proteins in mouse ocular tissues from the Trp1/Tag mouse model.Methods: The expression patterns of theses genes and their corresponding proteins in normal mouse ocular tissues were studied by in situ hibridization and immunohistofluorescence experiments. In addition, variations in mRNA and proteins levels and protein distributions for Dlg1, Scrib, and Lgl1 were analyzed in the ocular tissues from Trp1/Tag transgenic mouse model by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis, and immunohistofluorescence.Results: We found that mouse Dlg1, Scrib, and Lgl1 are widely distributed in normal ocular tissues, particularly in retinal neurons. We found that the three proteins are mislocalized in retinal layers during ocular carcinogenesis. These mislocalizations were correlated to the early dysplastic stages of ocular tumorigenesis. Additionally, the mislocalization of each protein was associated with its downregulation. Decreased levels of these proteins may be considered as late-stage markers of the disease but also as markers of the invasive stage of this cancerous process. This downregulation may be involved in epithelial-mesenchymal transition in this mouse ocular tumoral model. This would be consistent with the downregulation of E-cadherin and upregulation of N-cadherin expression observed in this model.Conclusions: This is the first study to demonstrate the involvement of Dlg1, Scrib, and Lgl1 in a mouse with ocular adenocarcinoma and the simultaneous involvement of these proteins in the same cancer. Our results indicate that both the mislocalization and downregulation of these proteins may be involved together in ocular carcinogenesis.

PURPOSE:Asymmetric cell division (ACD) is the fundamental mechanism underlying the generation of cellular diversity in invertebrates and vertebrates. During Drosophila neuroblast division, this process involves stabilization of the apical complex and interaction between the Inscuteable (Insc) and Partner of inscuteable (Pins) proteins. Both cell-intrinsic factors and cell-cell interactions seem to contribute to cell fate decisions in the retina. The Pins protein is known to play a major role in the asymmetric segregation of cell fate determinants during development of the central nervous system in general, but its role in asymmetric cell divisions and retinoblast cell fate has never been explored. The primary aim of this study was to determine the spatial distribution and time course of mouse homolog of Drosophila Partner of Inscuteable (mPins) expression in the developing and adult mouse eye.METHODS:The expression pattern of mPins was studied in the mouse eye from embryonic (E) stage E11.5 until adulthood, by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry. In addition, variations in mRNA and protein levels for mPins were analyzed in the developing postnatal and adult lens, by semiquantitative RT-PCR, western blot analysis, in situ hybridization, and immunohistochemistry.RESULTS:We detected mPins mRNA at early stages of mouse embryonic eye development, particularly in the neuroblastic layer. In early postnatal development, mPins mRNA was still detected in the neuroblastic layer, but also began to be detectable in the ganglion cell layer. Thereafter, mPins mRNA was found throughout the retina. This pattern was maintained in differentiated adult retina. Immunohistochemical studies showed that mPins protein was present in the neuroblastic layer and the ganglion cell layer during the early stages of postnatal retinal development. At these stages, mPins protein was colocalized with Numb protein, a marker of the ACD. At later postnatal stages, mPins protein was present in all retinal nuclear layers and in the inner plexiform layer. It continued to be detected in these layers in the differentiated retina; the outer plexiform layer and the photoreceptor inner segments also began to display positive immunostaining for mPins. In the adult retina, mPins was also detected in the retinal pigment epithelium and choroidal melanocytes. Throughout development, mPins protein was detected in nonretinal tissues, including the cornea, ciliary body, and lens. We focused our attention on lens development and showed that mPins protein was first detected at E14.5. The most striking results obtained concerned the lens, in which mPins protein distribution switched from the anterior to the posterior region of the lens during embryonic development. Interestingly, in the postnatal and adult lens, mPins protein was detected in all lens cells and fibers.CONCLUSIONS:We provide the first demonstration that mPins protein is expressed from embryonic stages until adulthood in the mouse eye. These results suggest that mPins plays important roles in eye development. This work provides preliminary evidence strongly supporting a role for mPins in the asymmetric division of retinoblasts, and in the structure and functions of adult mouse retina. However, the link between the presence of mPins in different ocular compartments and the possible occurrence of asymmetric cell divisions in these compartments remains to be clarified. Further studies are required to elucidate the in vitro and in vivo functions of mPins in the developing and adult human eye.

PURPOSE:Choroidal melanoma is the most common primary malignant ocular tumor in human adults. Relevant mouse models of human uveal melanoma still remain to be developed. We have studied the transgenic mouse strain, Tyrp-1-TAg, to try to gain insight into possible molecular mechanisms common to pigmented ocular neoplasms occurring spontaneously in the eyes of these mice and human choroidal melanoma. The role of two members of the ETS (E26 avian leukemia oncogene) family of transcription factors, ETS-1 and ETS-2, has been investigated in many cancers but has not yet been studied in ocular tumors.METHODS:This is the first study describing the production and distribution of ETS-1 and ETS-2 mRNAs and proteins using in situ hybridization and immunohistochemistry in murine ocular tissue sections of normal control eyes and tumoral eyes from mice of the same age. Using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blots experiments, we compared changes in ETS-1 and ETS-2 expression, their protein levels, and the regulation of some of their target gene expressions at different stages of the ocular tumoral progression in the transgenic mouse model, Tyrp-1-TAg, with those in normal eyes from control mice of the same age.RESULTS:In normal control adult mouse eyes, ETS-1 was mostly present in the nuclei of all neuroretinal layers whereas ETS-2 was mostly localized in the cytosol of the cell bodies of these layers with a smaller amount present in the nuclei. Both were found in the retinal pigmentary epithelium (RPE). ETS-1 and ETS-2 mRNA and protein levels were much higher in the ocular tissues of Tyrp-1-TAg mice than in control ocular tissues from wild-type mice. This upregulation was correlated with tumor progression. We also demonstrated upregulation of ETS-1 and ETS-2 target expressions in Tyrp-1-TAg mice when comparing with the same target expressions in control mice.CONCLUSIONS:Our findings suggest that ETS-1 and ETS-2 are upregulated in ocular tumors derived from the retinal epithelium and may be involved in one or several signaling pathways that activate the expression of a set of genes involved in ocular tumor progression such as those encoding ICAM-1 (intercellular adhesion molecule-1), PAI-1 (Plasminogen activator inhibitor-1), MCP-1 (monocyte chemoattractant protein-1) and p16 (Cyclin dependent kinase inhibitor 2A).

PURPOSE:Musashi-1 (Msi1) is an RNA-binding protein produced in various types of stem cells including neural stem/progenitor cells and astroglial progenitor cells in the vertebrate central nervous system. Other RNA-binding proteins such as Pumilio-1, Pumilio-2, Staufen-1, and Staufen-2 have been characterized as potential markers of several types of stem or progenitor cells. We investigated the involvement of Msi1 in mouse eye development and adult mouse eye functions by analyzing the profile of Msi1 production in all ocular structures during development and adulthood.METHODS:We studied Msi1 production by in situ hybridization and immunohistochemistry of ocular tissue sections and by semi-quantitative RT-PCR and western blot analysis from the embryonic stage of 12.5 days post coitum (E12.5 dpc) when the first retinal ganglion cells (RGCs) begin to appear to the adult stage when all retinal cell types are present.RESULTS:Msi1 mRNA was present at all studied stages of eye development. Msi1 protein was detected in the primitive neuroblastic layer (NbL), the ganglion cell layer (GCL), and in all major differentiated neurons of postnatal developing and adult retinae. During postnatal developing stages, faint diffuse Msi1 protein staining is converted to a more specific distribution once mouse retina is fully differentiated. The most striking result of our study concerns the large amounts of Msi1 protein and mRNA in several unexpected sites of adult mouse eyes including the corneal epithelium and endothelium, stromal keratocytes, progenitor cells of the limbus, equatorial lens stem cells, differentiated lens epithelial cells, and differentiating lens fibers. Msi1 was also found in the pigmented and nonpigmented cells of the ciliary processes, the melanocytes of the ciliary body, the retinal pigment epithelium, differentiated retinal neurons, and most probably in the retinal glial cells such as Müller glial cells, astrocytes, and the oligodendocytes surrounding the axons of the optic nerve. Msi1 expression was detected in the outer plexiform layer, the inner plexiform layer, and the nerve fiber layer of fully differentiated adult retina.CONCLUSIONS:We provide here the first demonstration that the RNA-binding protein, Msi1, is produced in mouse eyes from embryonic stages until adulthood. The relationship between the presence of Msi1 in developing ocular compartments and the possible stem/progenitor cell characteristics of these compartments remains unclear. Finally, the expression of Msi1 in several different cell types in the adult eye is extremely intriguing and should lead to further attempts to unravel the role of Msi1 in cellular and subcellular RNA metabolism and in the control of translational processes in adult eye cells particularly in adult neuronal dendrites, axons, and synapses.

Purpose: The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca2+-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl− channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. Methods: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl− current was examined using whole-cell patch clamp analysis. Results: Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibited the function of wild-type bestrophin-1 channels in a dominant negative manner. Conclusions: This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.

The effect of high glucose concentration (3 g/l) on bovine corneal total protein and collagen biosynthesis was studied, using 3H-proline incorporation in explant cultures with protein and collagen determinations. The high glucose concentration increased the incorporation of 3H-proline in total corneal proteins as well as in collagens. The specific radioactivity of stromal collagens was strongly increased in these conditions. Mannitol was used to control the osmotic effect of the high glucose concentration, both at 1 and 3 g/l concentrations. Mannitol did not increase the incorporation of 3H-proline in total proteins or collagens, but on the contrary decreased it. The high glucose concentration decreased the excretion of neosynthesized proteins and collagens in the culture medium, but did not affect the total protein or collagen content of the corneas. The strong increase in the specific radioactivity of corneal collagens in the presence of 3 g/l glucose suggests an increased turnover of collagens in diabetic corneas. The increased biosynthesis of collagens together with their decreased elimination in the extracellular compartment can create the conditions for the formation and accumulation of advanced glycation endproducts by the Maillard reaction. This can induce and stimulate the liquefaction of the vitreous body leading to sight-threatening disorders such as diabetic retinopathy, retinal detachment, glaucoma, cataract formation and age-related macular degeneration.

PURPOSE. It is important to understand the development of the normal retinal vascular system, because it may provide clues for understanding the mechanisms underlying the neovascularization associated with several retinopathies of infancy and adulthood. However, little is known about normal human ocular vascularization. VEGF is a key growth factor during vascular development and one of its receptors, KDR, plays a pivotal role in endothelial cell proliferation and differentiation. The purpose of this study was to analyze VEGF and KDR gene expression patterns during the development of the human eye during the embryonic and fetal stages.METHODS. The gene expression of VEGF and KDR was analyzed by in situ hybridization in 7-week-old embryos and in 10- and 18-week-old fetuses. In addition, we performed VEGF and KDR immunohistochemistry experiments on 18-week-old fetus tissue sections.RESULTS. These results clearly demonstrated that the levels of VEGF and KDR transcripts are correlated during the normal development of the ocular vasculature in humans. The complementarity between the patterns of VEGF and KDR during the early stages of development suggests that VEGF-KDR interactions play a major role in the formation and regression of the hyaloid vascular system (HVS) and in the development of the choriocapillaris. In later stages (i.e., 18-weeks-old fetuses), the expression of KDR seems to be linked to the development of the retinal vascular system. VEGF and KDR transcripts were unexpectedly detected in some nonvascular tissues-that is, in the cornea and in the retina before the development of the retinal vascular system.CONCLUSIONS. The expression of VEGF and KDR correlates highly with the normal ocular vascularization in humans, but VEGF may also be necessary for nonvascular retinal developmental functions, especially for the coordination of neural retinal development and the preliminary steps of the establishment of the definitive stable retinal vasculature.

PURPOSE. To identify proteins secreted by the retinal pigment epithelium (RPE) and to analyze their cellular distribution in normal and pathologic rat retinas at various stages of eye development.METHODS. A cDNA library was constructed with RNA isolated from porcine RPE sheets and screened by using the yeast signal sequence trap system. In situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analysis were performed on rat retinas.RESULTS. The cDNA encoding prosaposin was isolated. This is the first time this gene has been shown to be expressed in the retina. Prosaposin mRNA was detected in the rat RPE cell monolayer and in ganglion cells 14, 2 1, and 45 days after birth. The amount of prosaposin mRNA increased between days 14 and 45 after birth in normal retinas (rdy(+)), but not in the pathologic retinas (rdy(-)) of RCS rats.CONCLUSIONS. Several techniques were used to determine the localization of prosaposin in rat retinas. The increase in the amount of prosaposin mRNA in normal retinas coincided with the maturation of photoreceptor cells and the beginning of the phagocytosis process. In addition, the RCS rdy- RPE cells, characterized by the abrogation of the ingestion phase of the photoreceptor outer segments, are deficient in prosaposin expression.

Integrins are a family of cell surface molecules that mediate the attachment of cells to the extracellular matrix (ECM). These alphabeta heterodimers are involved in many biological processes. We used northern blotting and in situ hybridization to study the pattern of beta3 integrin gene expression during mouse embryogenesis. Northern blotting detected two species of beta3 mRNA from 7 to 17 days post coitum (dpc). These transcripts were abundant in the adult testis, kidney, liver, spleen, and heart. In situ hybridization experiments detected high levels of beta3 in the major haematopoietic and lymphoid organs: yolk sac, liver, and thymus. Moreover, beta3 transcripts were also detected in the vascular system, where beta3 integrin probably plays a key role in angiogenesis and vasculogenesis. We also detected a hybridization signal in the gut, the bronchioles of the lungs, and the bladder wall. beta3 transcripts were also present in the medullary regions of the adrenal glands and in the developing skeleton. Our study shows that beta3 gene expression is not restricted to the liver and gut during mouse development. We also detected beta3 integrin mRNA in the yolk sac, vessels, lung, bladder, and developing bones. Our data suggest that beta3 integrin plays a key role in many important physiological processes like haematopoiesis, angiogenesis, phagocytosis, and bone resorption.

Shed photoreceptor outer segments (POS) are phagocytosed by RPE cells in a circadian manner. The homozygous deletion of the c-mer gene abolishes the ingestion phase of this phagocytosis in the Royal College of Surgeons (RCS) rat strain, which in turn leads to the death of photoreceptor cells. We identified RPE transcripts for which the expression is modulated by the abrogation of POS phagocytosis. A microarray approach and the differential display (DDRT-PCR) technique revealed 116 modulated known genes, 4 modulated unknown genes, and 15 expressed sequenced tags (ESTs) corresponding to unknown genes. The microarray and DDRT-PCR analyses detected alterations in signaling pathways such as the phosphatidylinositol 3-kinase-Akt-mTOR pathway and the DLK/JNK/SAPK pathway. The abrogation of POS phagocytosis caused a decrease in endomembrane biogenesis and altered endocytosis, exocytosis, transcytosis, and several metabolic and signaling pathways in RCS RPE cells. We also found differential levels of transcripts encoding proteins involved in phagocytosis, vesicle trafficking, the cytoskeleton, retinoic acid, and general metabolism.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.