Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.     

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.     

Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina

Transforming growth factor beta (TGF-beta) is an extracellular signaling molecule known to mediate programmed cell death (PCD) in the developing retina. In the present study, we investigated the expression profiles and activity levels of TGF-beta ligand and TGF-beta receptors (TbetaR) during the successive physiological PCD periods of the developing postnatal mouse retina. The peak of TbetaR expression levels--revealed by Western Blots and MLEC assays--coincided with the main periods of postnatal (P) retinal murine PCD at P2, P9, and P15. Immunocytochemical studies showed that the localization of the TbetaRs is restricted to the ganglion cell layer. Application of a neutralizing anti-TGF-beta antibody to E15 and P9 retinal cultures resulted in a significant decrease in the number of TUNEL-positive neurons specifically in the ganglion cell or prospective ganglion cell layer. Treatment of P2 and P15 organotypic murine retinal wholemount cultures with exogenous recombinant TGF-beta significantly increased cell death levels. In the P15 retina, where PCD affects ganglion cells and photoreceptors, TGF-beta induced cell death of large retinal ganglion cells, whereas small ganglion cells and photoreceptor neurons remained unaffected. Our data indicate that TGF-beta mediated apoptosis during all postnatal retinal PCD phases specifically affects the fate of retinal ganglion cells.     

Astrocyte invasion and vasculogenesis in the developing ferret retina

We studied the time course of astrocyte invasion and blood vessel formation in the developing ferret retina using glial fibrillary acidic protein (GFAP)-immunohistochemistry for astrocytes and isolectin B4 histochemistry for blood vessels. As in other mammals, strongly GFAP positive astrocytes invade the ferret retina from the optic nerve. At birth, strongly GFAP positive astrocytes have reached about 22% of the distance between optic disc and outer retinal edge whereas weakly GFAP positive processes already extend to the edge of the retina. At postnatal days P30–P37 about 82% of the distance between optic disc and outer retinal edge and in the adult 88% of this distance is covered with strongly labelled astrocytes. Superficial blood vessels form from the optic disc. They reach up to about 24% of the retinal radius at birth and grow radially across the retina during further development. At P30–P37, the whole retina is covered with superficial blood vessels. The deep vascular layer forms later (around P30) through sprouting from superficial vessels. The radial pattern of astrocyte and vessel growth from the optic disc is not affected by the formation of the area centralis and visual streak.     

Expression profile of heat shock protein 108 during retinal development in the chick

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.     

Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.     

Neuronal and glial cell expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the rat retina

The bio-active peptide, angiotensin II (Ang II), has been suggested to exert a neuromodulatory effect on inner retinal neurons. In this study, we examined the distribution of angiotensin receptors (ATRs) in the developing and mature rat retina and optic nerve using immunofluorescence immunocytochemistry. Double-labeling experiments were performed with established markers to identify different retinal cell populations. In adult retinae, ATRs were observed on neurons involved in “ON” pathways of neurotransmission. Angiotensin II type 1 receptors (AT₁Rs) were expressed by a sub-population of “ON” cone bipolar cells that also labeled for Gα₀ and islet-1. Extra-neuronal expression of AT₁Rs was evident on retinal astrocytes, Müller cells and blood vessels. Immunoreactivity for the angiotensin II type 2 receptor (AT₂R) was observed on conventional and displaced GABAergic amacrine cells. Co-localization studies showed that AT₂R-expressing amacrine cells constituted at least two separate sub-populations. Cell counts revealed that all wide-field amacrine cells expressing protein kinase C-alpha were also AT₂R-positive; a further subset of amacrine cells expressing AT₂Rs and stratifying in sublamina “b” of the inner plexiform layer (IPL) was identified. Developmental expression of AT₁Rs was dynamic, involving multiple inner neuronal classes. At postnatal day 8 (P8), AT₁R immunoreactivity was observed on putative ganglion cells. The characteristic bipolar cell labeling observed in adults was not evident until P13. In contrast, AT₂Rs were detected as early as P2 and localized specifically to amacrine cells from this age onward. These data provide further evidence for the potential role of angiotensin II in the modulation of retinal neurons and glia. The differential pattern of expression of these receptors across these cell types is similar to that observed in the brain and suggests that a similar functional role for Ang II may also exist within the retina.     

Alteration in protein expression in estrogen receptor alpha-negative human breast cancer tissues indicates a malignant and metastatic phenotype

Ductal carcinoma in situ (DCIS) represents the earliest identifiable breast cancer lesion. Disruption of the myoepithelial cell layer and basement membrane is a prerequisite for DCIS to initiate invasion into the stroma. The majority of epithelial cells overlying a focally-disrupted myoepithelial cell layer are estrogen receptor-alpha negative (ER(−)); however, adjacent cells within the same duct confined by an intact myoepithelial cell layer express high levels of ER. These ER (+) and ER (−) cells were microdissected from the same ducts of breast cancer patients. Differential proteins expressed by ER(+) and ER(−) cells were identified using two-dimensional gel electrophoresis followed by mass spectrometry and Western blot analysis. ER(−) cells express lower levels of superoxide dismutase, RalA binding protein, galectin-1, uridine phosphorylase 2, cellular retinoic acid-binding protein 1, S100 calcium binding protein A11, and nucleoside diphosphate kinase A or non-metastasis protein 23-H1 (nm23-H1). The upregulated protein, Rho GDP-dissociation inhibitor 1 alpha, may induce chemotherapy resistance. The significant findings are that the microdissected ER(−) cells express 12.6 times less cellular retinoic acid-binding protein 1, a protein involved in cellular differentiation, and 4.1 times less nucleoside diphosphate kinase A or nm23-H1, a metastasis suppressor, and express fewer proteins than adjacent ER(+) cells. The collective role of the alterations of protein expression in ER(−) cells may be to promote a more malignant phenotype than adjacent ER(+) cells, including a decreased ability to undergo apoptosis and differentiation, and an increased potential to damage DNA, metastasize, and resist to chemotherapy.     

Characterization of green fluorescent protein-expressing retinal cells in CD 44-transgenic mice

Sensory information in the retina is transferred from rod and cone photoreceptors to higher visual centers via numerous parallel circuits that sample the photoreceptor mosaic independently. Each circuit consists of a unique combination of ganglion cell, bipolar and amacrine cell types. The morphology and physiological responses of many amacrine cells have been characterized. However, the synaptic connections and retinal circuits in which they participate are only rarely understood. A major problem that has prevented fuller characterization of retinal circuitry is the need for specific cellular markers for the more than 50 inner retinal cell types. One potential strategy for labeling cells is to use transgenic expression of a reporter gene in a specific cell type. In a recent study of cluster of differentiation 44 (CD44)-enhanced green fluorescent protein (EGFP) transgenic mice, we observed that the green fluorescent protein (GFP) was expressed in a population of amacrine and ganglion cells in the inner nuclear layer (INL) and the GCL. To characterize the morphology of the GFP-labeled cells, whole mount preparations of the retina were used for targeted iontophoretic injections of Lucifer Yellow and Neurobiotin. Furthermore, immunocytochemistry was used to characterize the antigenic properties of the cells. We found that many GFP-expressing cells were GABAergic and also expressed calretinin. In addition to the somatic staining, there was a strong GFP(+)-band located about 50-60% depth in the inner plexiform layer (IPL). Double labeling with an antibody to choline acetyltransferase (ChAT) revealed that the GFP-band was located at strata 3 inner retina. The best-labeled GFP-expressing cell type in the INL was a wide-field amacrine cell that ramified in stratum 3. The GFP-expressing cells in the GCL resemble the type B1, or possibly A2 ganglion cells. The CD44-EGFP mice should provide a valuable resource for electrophysiological and connectivity studies of amacrine cells in the mouse retina.     

Generation of retinal cells in the wallaby, Setonix brachyurus (quokka)

We have examined the generation of retinal cells in the wallaby, Setonix brachyurus (quokka). Animals received a single injection of tritiated thymidine between postnatal days 1-85 and retinae were examined at postnatal day 100. Retinae were sectioned, processed for autoradiography and stained with Cresyl Violet. Ganglion cells were labelled by injection of horseradish peroxidase into the optic tracts and primary visual centres. Other cells were classified according to their morphology and location. Retinal cell generation takes place in two phases. During the first phase, which concludes by postnatal day 30, cells destined to lie in all three cellular layers of the retina are produced. In the second phase, which starts by postnatal day 50, cell generation is almost entirely restricted to the inner and outer nuclear layers. Cells produced in the first phase are orthotopic and displaced ganglion cells, displaced and orthotopic amacrine cells, horizontal cells and cones. Glia in the ganglion cell layer, orthotopic amacrine cells, bipolar and horizontal cells. Muller glia, and rods are generated in the second phase. Cells became heavily labelled with tritiated thymidine in the central retina before postnatal day 7, over the entire retina (panretinal) by postnatal day 7 and from postnatal day 18, only in the periphery. The second phase of cell generation is initiated at P50, in a region extending from the optic nerve head to mid-temporal retina. Subsequently, cells are generated in annuli, centred on mid-temporal retina, which are seen at progressively more peripheral locations. Therefore, cell addition to the inner and outer nuclear layers continues for longer in peripheral than in mid-temporal retina. We suggest that such later differential cell addition to the inner and outer nuclear layers contributes to an asymmetric increase in retinal area. This non-uniform growth presumably results in more expansion of the ganglion cell layer peripherally than in mid-temporal retina and may play a role in establishing density gradients of ganglion cells.     

Localization of cellular retinoid-binding proteins in human cervical intraepithelial neoplasia and invasive carcinoma.

Cellular retinoic acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP) were localized in biopsies of normal squamous epithelium, cervical intraepithelial neoplasia (CIN), and invasive squamous cell cancer of the cervix uteri by immunohistochemistry. In both the normal stratified squamous epithelium of the exocervix and low-grade CIN, CRABP I was present predominantly in the basal layer of the epithelium. The more superficial, differentiated cell layers lacked immunoreactive protein. In high-grade CIN (CIN2-3), the distribution of CRABP I was altered. Immunoreactive CRABP I was detected in all layers of high-grade CIN. In squamous cell carcinoma of the cervix, CRABP I was detected in cells throughout the tumor but was minimal in cells demonstrating squamous differentiation. In contrast to CRABP I, CRBP was diffusely present throughout the cervical epithelium irrespective of the state of differentiation or the presence of disease.     

The immunocytochemical localization of neuronal nitric oxide synthase in the developing rat retina

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina.     

The role of nitric oxide in the apoptosis of neurons in the retina of the human fetal eye

The locations of NADPH-diaphorase (NADPH-d), inducible NO synthase (iNOS), and TUNEL-immunoreactive neurons in the retina of human fetuses collected during the first to third trimesters of pregnancy were studied. High levels of NADPH-d activity were seen in the inner segments of light-sensitive cells, amacrine cells, and ganglion cells. The population of NADPH-d-positive amacrine cells included three types of neuron. Type 1 neurons were large and had sparse dendritic fields occupying the inner nuclear and outer retinal layers. Small type 2 neurons were located in the inner retinal layer. Ectopic amacrine cells, type 3, were located in the outer part of the ganglion layer. A high density of NADPH-d-positive neurons was seen in the central part of the retina, surrounding the central fovea and optic disk area. NADPH-d activity increased progressively during ontogenesis and correlated with the appearance of immunoreactive iNOS in neurons. iNOS labeled a subpopulation of amacrine and ganglion cells, which appeared at 20–21 weeks of development and reached a peak of immunoreactivity by the end of the third trimester. TUNEL-immunopositive neuron nuclei with signs of apoptotic destruction were seen at 30–31 weeks of pregnancy. The greatest apoptotic index was seen in the ganglion and amacrine cell populations. These data identify NO as a factor mediating apoptosis of neurons during the critical period of differentiation of interneuronal connections in the human retina. Director: Doctor of Biological Sciences M. A. Vashchenko Director: Doctor of Biological Sciences S. L. Kondrashov __________ Translated from Morfologiya, Vol. 129, No. 1, pp. 42–49, January–February, 2006.     

Development and plasticity of the retina in the opossum Monodelphis domestica

We investigated the rate of cell proliferation and death in the retina of the Monodelphis opossum during its postnatal development and the influence of early monocular enucleation on these processes. Our results show that in the opossum, as in other marsupials, the peak of the retinal cells divisions occurs postnatally and that generation of retinal cells continues till the time of eye opening (P34), except of the marginal rim, where it continued till P60. Ganglion and amacrine cells are generated between postnatal days (P) P4 and P9, while bipolar cells and photoreceptors are generated simultaneously between P14 and P25. The peak of ganglion cell death as detected by the TUNEL method occurs around P14-19 in the center of retina. The second peak of apoptosis appears in the inner nuclear layer (INL) at P19-25. Gliogenesis takes place between P25 and P34. We also found that monocular enucleation performed during the early period of retinal development (P0-P7) did not influence proliferation, developmental apoptosis or other developmental processes in the retina of the remaining eye.     

Neuronal and glial localization of homocysteate-like immunoreactivity in the rat retina

Summary To study the distribution ofl-homocysteate in the rat retina, specific polyclonal and monoclonal anti-homocysteate antibodies have been used in combination with a highly sensitive postembedding method for light microscopic immunocytochemistry. In central and peripheral retina, the most strongly immunoreactive cell bodies lay in the inner nuclear layer. They represented about 17% of the total neuronal cell population of the layer and were identified as bipolar cells (19–20% of cells in the outer half of the inner nuclear layer) and amacrine cells (15% of cells in the inner half of the inner nuclear layer). A third cell type showing heavy homocysteate-like immunoreactivity was identified as Müller glial cells. Characteristically, their descending processes formed three immunoreactive bands in the inner plexiform layer. Furthermore, the outer and inner limiting membranes as well as glia around and between ganglion cell axons and in the vicinity of blood vessels were labelled intensely. Photoreceptors and their terminals, and ganglion cells, were not immunostained. These findings indicate the presence of homocysteate in some bipolar and amacrine cells of the inner nuclear layer and support a role for this sulphur-containing excitatory amino acid as a neurotransmitter candidate in the retina.     

Localization of substance P-like immunoreactivity in the adult and developing goldfish retina

Substance P-like immunoreactivity was localized to amacrine cells in both adult and developing goldfish retina using immunohistochemical techniques. These studies utilized a well-characterized monoclonal antiserum directed to substance P. Specificity was established by absorption of the anti-serum with 10 μm synthetic substance P. Specific substance P-like immunoreactivity was localized within a seemingly distinct population of unistratified amacrine cells which were distributed in both central and peripheral retinal regions. The immunoreactive somata were located at the border of the inner nuclear layer and inner plexiform layer and were characterized by a round or ovoid somata which measured about 9μm in diameter. These immunoreactive amacrine cells typically had a single process which descended to and ramified within lamina 3 of the inner plexiform layer.Specific substance P-like immunoreactivity first appeared 60 h after hatching (stage 27) within both somata and processes located in differentiated retinal regions. No substance P-like immunoreactive somata or processes were observed in undifferentiated retinal regions. In retinas from stage 27 to 14 days after hatching, the immunoreactive somata were characterized by an ellipsoidal soma and a large nucleus devoid of immunoreactivity. These immunoreactive cells were also characterized by a single process that descended to and ramified within lamina 3 of the differentiated inner plexiform layer. At 30 days after hatching, the substance P-containing cells were identical in appearance to these same cell types observed within the adult retina.     

Neuronal expression of P2X3 purinoceptors in the rat retina

P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.