Global gene expression analysis of the developing postnatal mouse retina

PURPOSE: Postnatal mouse retinal development involves glial and neuronal differentiation, vascularization, and the onset of vision. In the current study, the gene expression profiles of thousands of genes in the developing postnatal mouse retina were analyzed and compared in a large-scale, unbiased microarray gene expression analysis. METHODS: For each of eight different time points during postnatal mouse retinal development, two separate sets of 30 retinas were pooled for RNA isolation, and gene expression was analyzed by hybridization to gene chips in triplicate (Mu74Av2; Affymetrix, Santa Clara, CA). Genes were sorted into clusters based on their expression profiles and intensities. Validation was accomplished by comparing the microarray expression profiles with real-time RT-PCR analysis of selected genes and by comparing selected expression profiles with predicted profiles based on previous studies. RESULTS: The Mu74Av2 chip contains more than 6000 known genes and 6500 estimated sequence tags (ESTs) from the mouse Unigene database. Of these, 2635 known gene sequences and 2794 ESTs were expressed at least threefold above background levels during retinal development. Expressed genes were clustered based on expression profiles allowing potential functions for specific genes during retinal development to be inferred by comparison to developmental events occurring at each time point. Specific data and potential functions for genes with various profiles are discussed. All data can be viewed online at http://www.scripps.edu/cb/friedlander/gene_expression/. CONCLUSIONS: Expression analysis of thousands of different genes during normal postnatal mouse retinal development as reported in this study demonstrates that such an approach can be used to correlate gene expression with known functional differentiation, presenting the opportunity to infer functional correlates between gene expression and specific postnatal developmental events.     

Differential expression of photoreceptor-specific genes in the retina of a zebrafish cadherin2 mutant glass onion and zebrafish cadherin4 morphants

Cadherins are Ca2+ -dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g. rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.     

Dopaminergic amacrine cells express opioid receptors in the mouse retina

The presence of opioid receptors has been confirmed by a variety of techniques in vertebrate retinas including those of mammals; however, in most reports, the location of these receptors has been limited to retinal regions rather than specific cell types. Concurrently, our knowledge of the physiological functions of opioid signaling in the retina is based on only a handful of studies. To date, the best-documented opioid effect is the modulation of retinal dopamine release, which has been shown in a variety of vertebrate species. Nonetheless, it is not known if opioids can affect dopaminergic amacrine cells (DACs) directly, via opioid receptors expressed by DACs. This study, using immunohistochemical methods, sought to determine whether (1) μ- and δ-opioid receptors (MORs and DORs, respectively) are present in the mouse retina, and if present, (2) are they expressed by DACs. We found that MOR and DOR immunolabeling were associated with multiple cell types in the inner retina, suggesting that opioids might influence visual information processing at multiple sites within the mammalian retinal circuitry. Specifically, colabeling studies with the DAC molecular marker anti-tyrosine hydroxylase antibody showed that both MOR and DOR immunolabeling localize to DACs. These findings predict that opioids can affect DACs in the mouse retina directly, via MOR and DOR signaling, and might modulate dopamine release as reported in other mammalian and nonmammalian retinas.     

Evidence for pigment epithelium-derived factor receptors in the neural retina.

PURPOSE: The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined. METHODS: Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy. RESULTS: Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections. CONCLUSIONS: Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.     

The expression of adhesion molecules in the human retina and choroid

This study examines the expression of some selected adhesion molecules likely to be important in the migration of leucocytes across the blood-retinal and blood-aqueous barriers in samples of normal human retina and choroid by immunoperoxidase staining. ICAM-1, LFA-3 and ELAM-1 were expressed on retinal endothelium and ICAM-1, ELAM-1 and CD44 in the region of the external limiting membrane. VLA-2 was found around the retinal ganglion cells and in the inner and outer fibre layers and is likely to be important in maintaining the structural integrity of the retina. The endothelium on the choriocapillaris showed moderate staining of ICAM-1, and slight staining of LFA-3 and ELAM-1. These molecules may have an important functional role in the pathogenesis of many immunological ocular conditions, including uveoretinitis and cornea! allograft rejection.     

Glycine receptors of A-type ganglion cells of the mouse retina

A-type ganglion cells of the mouse retina represent the visual channel that transfers temporal changes of the outside world very fast and with high fidelity. In this study we combined anatomical and physiological methods in order to study the glycinergic, inhibitory input of A-type ganglion cells. Immunocytochemical studies were performed in a transgenic mouse line whose ganglion cells express green fluorescent protein (GFP). The cells were double labeled for GFP and the four alpha subunits of the glycine receptor (GlyR). It was found that most of the glycinergic input of A-type cells is through fast, alpha1-expressing synapses. Whole-cell currents were recorded from A-type ganglion cells in retinal whole mounts. The response to exogenous application of glycine and spontaneous inhibitory postsynaptic currents (sIPSCs) were measured. By comparing glycinergic currents recorded in wildtype mice and in mice with specific deletions of GlyRalpha subunits (Glra1spd-ot, Glra2-/-, Glra3-/-), the subunit composition of GlyRs of A-type ganglion cells could be further defined. Glycinergic sIPSCs of A-type ganglion cells have fast kinetics (decay time constant tau = 3.9 +/- 2.5 ms, mean +/- SD). Glycinergic sIPSCs recorded in Glra2-/- and Glra3-/- mice did not differ from those of wildtype mice. However, the number of glycinergic sIPSCs was significantly reduced in Glra1spd-ot mice and the remaining sIPSCs had slower kinetics than in wildtype mice. The results show that A-type ganglion cells receive preferentially kinetically fast glycinergic inputs, mediated by GlyRs composed of alpha1 and beta subunits.     

Ionotropic glutamate receptors of amacrine cells of the mouse retina

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at -75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.     

The expression of adhesion molecules in the human retina and choroid.

This study examines the expression of some selected adhesion molecules likely to be important in the migration of leucocytes across the blood-retinal and blood-aqueous barriers in samples of normal human retina and choroid by immunoperoxidase staining. ICAM-1, LFA-3 and ELAM-1 were expressed on retinal endothelium and ICAM-1, ELAM-1 and CD44 in the region of the external limiting membrane. VLA-2 was found around the retinal ganglion cells and in the inner and outer fibre layers and is likely to be important in maintaining the structural integrity of the retina. The endothelium on the choriocapillaris showed moderate staining of ICAM-1, and slight staining of LFA-3 and ELAM-1. These molecules may have an important functional role in the pathogenesis of many immunological ocular conditions, including uveoretinitis and corneal allograft rejection.     

Ontogeny of plasma membrane Ca2+ ATPase isoforms in the neural retina of the postnatal rat

Calcium ion (Ca(2+)) signaling has been widely implicated in developmental events in the retina, but little is known about the specific mechanisms utilized by developing neurons to decrease intracellular Ca(2+). Using immunocytochemistry, we determined the expression profiles of all known isoforms of a key Ca(2+) transporter, the plasma membrane Ca(2+) ATPase (PMCA), in the rat retina. During the first postnatal week, the four PMCA isoforms were expressed in patterns that differed from their expression in the adult retina. At birth, PMCA1 was found in the ventricular zone and nascent cell processes in the distal retina as well as in ganglion and amacrine cells. After the first postnatal week, PMCA1 became restricted to photoreceptors and cone bipolar cells. By P10 (by postnatal day 10), most inner retinal PMCA consisted of PMCA2 and PMCA3. Prominent PMCA4 expression appeared after the first postnatal week and was confined primarily to the ON sublamina of the inner plexiform layer (IPL). The four PMCA isoforms could play distinct functional roles in the development of the mammalian retina even before synaptic circuits are established. Their expression patterns are consistent with the hypothesis that inner and outer retinal neurons have different Ca(2+) handling needs.     

The spatial and temporal expression patterns of netrin receptors, DCC and neogenin, in the developing mouse retina

Recently it has been demonstrated that the guidance of retinal ganglion cell (rgc) axons through the optic disc is dependent on the DCC/netrin-1 axonal guidance system. To gain further insight into the function of the netrin receptors, DCC and Neogenin, in retinal development we have studied the expression patterns of these receptors in the embryonic mouse retina. Neogenin mRNA was restricted to a single neural cell type, the rgc. However, strong Neogenin mRNA expression was observed in the extending fiber cells of the developing lens suggesting a role for Neogenin in the migration events shaping the early lens. Our studies demonstrated that DCC mRNA was expressed at high levels in chains of closely opposed neurons as they migrated towards the emerging mantle layer in the early retina (E12.5-E13.5) suggesting a role for DCC in the migration of neurons out of the ventricular zone. DCC protein expression was high on rgc axons as they actively navigated through the optic disc into the optic nerve. At birth, when the majority of rgc axons had projected through the optic disc, DCC protein was no longer detectable on the distal axonal segments within the optic nerve despite significant DCC protein expression on the proximal axonal membranes in the nerve fiber layer. These observations suggest that a localized down-regulation of DCC protein occurs on projecting axonal membranes once the DCC guidance function is no longer required. We also demonstrated that DCC mRNA and protein were expressed by amacrine cells and Müller glial cells while DCC mRNA was detected in horizontal cells. Taken together, these expression patterns suggest a role for DCC in axon outgrowth and/or pathfinding for a variety of retinal neurons and in the migration of newly born neurons within the developing retina.     

NOD小鼠视网膜VEGF表达和视网膜细胞凋亡

目的:研究NOD小鼠早期糖尿病视网膜VEGF表达和视网膜细胞凋亡情况,以及二者间的关系。方法:NOD小鼠分为对照组(非糖尿病小鼠)(2,4,6,8,12wk组,n=30)和糖尿病组(2,4,6,8,12wk组,n=30)。每组小鼠在规定时间处死,提取血液标本,摘除眼球,分离视网膜备用。ELISA法检测视网膜VEGF和血液VEGF。透射电子显微镜检测小鼠视网膜细胞凋亡情况。结果:糖尿病组血液和视网膜VEGF表达与对照组相比明显增高(12wk,血液标本:4.9±0.4μg/gvs0.19±0.1μg/g,P<0.01;视网膜,165.0±9.0μg/gvs18.0±4.0μg/g,P<0.01)。NOD小鼠早期糖尿病视网膜VEGF表达和血液VEGF表达呈正相关(γ=0.9902,P=0.001)。糖尿病组视网膜神经节细胞和血管内皮细胞凋亡明显增加P<0.01。结论:视网膜VEGF表达增加可能与视网膜凋亡增多有关。早期糖尿病NOD小鼠视网膜VEGF表达增加是多因素的。     

Differential effect of the rd mutation on rods and cones in the mouse retina

The retinas of rd/rd C57BL/6J-rd le mice have been examined by light and electron microscopy to determine whether rod cell degeneration precedes cone cell degeneration. In all regions of the eye, a rapid rod degeneration precedes a much slower cone degeneration. Only about 2% of the rods remain in the posterior region at postnatal day 17, and none by the day 36. By contrast, at least 75% of the cone nuclei remain at day 17. Although most of these slowly disappear, about 1.5% of the original population of cone nuclei in the posterior retina is still present at 18 months of age. A central to peripheral temporal gradient of degeneration exists, such that some rod nuclei persist in the far periphery up to day 47, but none is found at day 65. About 5% of the cone nuclei are still present in the far periphery at 18 months of age.     

VEGF expression and cell apoptosis in NOD mouse retina

AIM: To investigate retinal vascular endothelial growth factor (VEGF) level and retinal cells apoptosis in the early stage of diabetic NOD mouse retina. METHODS: Animals were divided into non-diabetes group, (control) (2-, 4-, 6-, 8- and 12-week sub-groups, n=30) and diabetes group (2-, 4-, 6-, 8- and 12-week sub-groups, n=30). Enzyme-linked immunosorbent assay (ELISA) was performed to detect VEGF level in both serum and retina. Transmission electron microscope method was used to examine retinal cell apoptosis. RESULTS: Compared with the control group, VEGF levels in serum and retina were increased significantly in the NOD group (12 weeks: 4.9±0.4μg/g vs 0.19±0.1μg/g in serum sample, P<0.01; 165±9μg/g vs 17±5μg/g in retinal sample, P<0.01). There exists a positive correlation between serum VEGF and retinal VEGF levels in the early diabetic NOD mice (γ=0.9902, P=0.001). The number of the cells apoptosis in the ganglion cells and endothelium can also been found increased significantly in the NOD group (P<0.01). CONCLUSION: The high VEGF expression may be contributed to increased retinal cells apoptosis. Many factors associated with retinal VEGF expression might involve in the early diabetes stage.     

Isolation of the mouse nyctalopin gene nyx and expression studies in mouse and rat retina

PURPOSE: It has been shown recently that mutations in NYX (nyctalopin on chromosome X), encoding a novel protein associated with the leucine-rich repeat (LRR) protein superfamily, are responsible for the complete form of X-linked congenital stationary night blindness (CSNB1). This study describes the isolation and molecular characterization of the mouse orthologue Nyx and its expression pattern in the retina. METHODS: Nyx was isolated by conventional DNA library screening and polymerase chain reaction (PCR)-based approaches. Gene expression in different mouse tissues was studied by RT-PCR. Subsequently, the expression pattern of Nyx and its gene product in mouse and rat retinas was investigated by RNA in situ hybridization and immunohistochemistry with Nyx-specific antibodies. RESULTS: The Nyx gene encodes a protein of 476 amino acids that contain 11 consecutive LRR motifs flanked by amino- and carboxyl-terminal cysteine-rich LRRs. At the amino acid level, Nyx is highly homologous to its human orthologue (86% identity). The gene is expressed in the eye but also, at lower levels, in brain, lung, spleen, and testis. Nyx expression was found during all stages of postnatal retinal development and was confined to cells of the inner nuclear layer and the ganglion cell layer in adult mouse and rat retinas. CONCLUSIONS: These data suggest an important function of the Nyx protein in the inner retina and provide evidence that CSNB1 is based on a defect in the inner retinal circuitry.