Retrovirus-mediated gene expression during chick visual system development

The chick embryo is an excellent model for studying eye morphogenesis, retinal cell fate determination, and retinotectal projections due to its accessibility and the available molecular tools. Avian replication-competent retroviruses allow efficient infection of proliferating cells and stable integration of the viral genome, including up to 2.3kb of foreign cDNA, into the host chromosome. High-titer retroviruses are produced by transient transfection of avian DF-1 cells followed by centrifugation of the culture medium. Targeted infection of the optic vesicle, the lens vesicle, the retina and pigmented epithelium, the periocular mesenchyme, and the tectum can be performed at different developmental stages in ovo. In addition, retroviruses can be used to transduce genes of interest into various ocular tissue explants or cells in vitro. Virus-mediated gene expression can be detected within 12h of infection. Therefore, avian replication-competent retroviruses serve as powerful tools to misexpress wild-type and mutant gene products and to study molecular mechanisms underlying vertebrate visual system development.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

The development of membrane specializations in the receptor-bipolar-horizontal cell synapse of the chick embryo retina

Retinae of chick embryos and chicks one to six weeks after hatching were examined in ultrathin sections and in freeze-etch specimens. The development of the synaptic contacts between receptor cells and bipolar cells starts at the end of the second week of incubation with the enclosure of the dendritic prolongations, invaginating receptor terminals accompanied by the appearance of electron dense material at the synaptic contact sites. Subsequently receptor terminals become filled with synaptic vesicles which surround the synaptic lamellae that appear on the 16th day of incubation. The application of the freeze-fracture technique demonstrates that the differentiation of the synaptic membranes continues into the first week post hatching. E-fracture faces of the presynaptic membranes are characterized by crater-like structures, called synaptopores. Their number is rather small during incubation and increases after hatching. In the P-fracture faces of the dendrites, which are enclosed by the receptor terminals, small particle aggregations appear on the 16th day of incubation. These small particle clusters increase by the apposition of further particles which become arranged in lines and bring out a lattice-like aspect. This arrangement of particles in the inner part of the cell membrane is the morphological expression of the maturation process. The significance of these aggregations as a postsynaptic receptor for neurotransmitters in excitatory cells is discussed.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer     

GE-25-like immunoreactivity in the rat eye

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Age specific cell sorting within aggregates of chick neural retina cells

Summary Chick retinal cells of different stages of development, and of different areas of the retina, were stained with two vital fluorescent stains, reaggregated, and analysed for sorting-out. Sorting-out of cells within aggregates occurred if one cell sample was derived from the retinae before and the other after day 7 of incubation. No such cell-sorting effects were found in experiments performed on cells of different areas at the same stage. It is suggested that the sorting-out reflects either a change in cell population around day 7 (such as an increase in postmitotic neuronal cells), or the effect of a stage specific signal which changes the surface properties of a substantial part of most or all cell types.     

Spatial and temporal expression of the 5A11/HT7 antigen in the chick embryo

The 5AII/HT7 antigen is the avian homologue of a 45-50X10³ M _r plasma membrane glycoprotein of the immunoglobulin super-gene family also identified in mammalian species (basigin, mouse gp42, MRC OX-47 antigen, M6 antigen). We had previously demonstrated that antibodies to this antigen interfere with heterotypic cell-to-cell interaction-dependent glial cell maturation in vitro. In this report, we sought to gain insight into its developmental role through an analysis of its distribution at various stages of avian embryogenesis. The primary mode of expression progresses from generalized staining of the undifferentiated tissue to intense labelling of specific cell types coincident with biochemical or morphological differentiation. In the retina, expression progresses from the generalized staining of the neural ectoderm, then, during neurogenesis, becomes restricted to Müller cells, photoreceptor cell bodies, the retina pigmented epithelium, the pigmented cells of the ciliary membrane and endothelia of the pecten. Similarly, the uniform staining of undifferentiated skin ectoderm progressively becomes confined to the germinative cells of the epidermis during biochemical differentiation of scutate and reticulate scales and formation of the feather follicle. During mesonephric and metanephric tubule development, labeling appears on those cells induced to form epithelia from the unlabeled mesenchyme and persists on the basal-lateral membranes of the convoluted tubules. Western blot analysis of NP-40 solubilized proteins from hatchling chicken identifies an immunoreactive polypeptide of 45-50X103 M _r in tissues stained immunohistochemically and an additional band of 69X10³ M _r in the neural retina and pineal gland. The localization of the 5A11 antigen at boundaries typically associated with inductive interactions between cells and tissues suggests broad involvement in cell-to-cell interactions associated with cellular maturation.     

Scanning electron microscopy of the zonular fibers in the rat eye

Summary The three-dimensional arrangement of the zonular fibers of Zinn and their ultrastructure was studied with the aid of scanning and transmission electron microscopy.Most of the thicker zonular fibers are arranged in straight bundles between the ciliary body and the lens, while the thinner fibers form a complex three-dimensional network interconnecting all the zonular fibers. These do originate from the limiting membrane covering the ciliary body. The zonular fibers are subdivided close to lens and form a complicated network on the surface of the lens capsule, i. e. the zonular lamella. The latter consists of a dense network of fibers and is from a structural point of view closely related to the zonular fibers and not to the lens capsule.The zonular fibers are continuous with those in the vitreous body close to the ciliary body but never in the lenticular two thirds of the zonular fibers or in the retrolental area.The ground substance is possible to demonstrate in freeze-dried specimens by scanning electron microscopy. It appeared granular or amorphous and coated the zonular fibers. It does not form membranes or fill all available space in contrast to its properties in the vitreous body. The many structural similarities between the zonular fibers and the vitreous body indicate perhaps a common origin.Supported by grants from Magnus Bergwalls Stiftelse and the Swedish Medical Research Council (B70-12 X -2543-02, B71-12 X -2543-03).     

Changes induces by haloperidol (antidepressant drug) on the developing retina of the chick embryo

Morphological and histological studies were done on the retina of chick embryos of 6th, 10th and 16th days of incubation by a single dose of haloperidol (0.25 mg/egg), injected on day zero and 5 of incubation. To get an idea about the extent of the teratogenic effect of this drug on the development retina. Sign of malformation in the chick embryo after administration of haloperidol were seen as absent of the ear vesicles, eyes or decreased size of them. Retardation of growth of the retina at 6th, 10th and 16th days treated chick embryo were observed as evidenced of reduction of the size of the retina, associated with sign of degeneration of the retina cells.

Conclusion

The injection of haloperidol drug give rise to several side effects as retardation of growth and degeneration of the cells. The decrease of the thickness of the layers and less density of the cells was related to direct effect of the drug on the cells of this organ. Also on the DNA formation and on the retardation of cellular mitotic activates, therefore the retina appeared decrease in thickness and less cell density with degeneration its cells.     

Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling

The identification of neural stem cells with retinal potential in the ciliary epithelium (CE) of the adult mammals is of considerable interest because of their potential for replacing or rescuing degenerating retinal neurons in disease or injury. The evaluation of such a potential requires characterization of these cells with regard to their phenotypic properties, potential, and regulatory mechanisms. Here, we demonstrate that rat CE stem cells/progenitors in neurosphere culture display astrocytic nature in terms of expressing glial intermediate neurofilament protein, GFAP. The GFAP-expressing CE stem cells/progenitors form neurospheres in proliferating conditions and generate neurons when shifted to differentiating conditions. These cells express components of the canonical Wnt pathway and its activation promotes their proliferation. Furthermore, we demonstrate that the activation of the canonical Wnt pathway influences neuronal differentiation of CE stem cells/progenitors in a context dependent manner. Our observations suggest that CE stem cells/progenitors share phenotypic properties and regulatory mechanism(s) with neural stem cells elsewhere in the adult CNS.     

Dominant Expression of Rat Prostanoid DP Receptor mRNA in Leptomeninges, Inner Segments of Photoreceptor Cells, Iris Epithelium, and Ciliary Processes

Abstract: Prostaglandin (PG) D₂ is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD₂ among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD₂.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

An essential role for FGF receptor signaling in lens development

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over 20 years of accumulated evidence using several different vertebrate species has suggested that fibroblast growth factors (FGFs) and/or fibroblast growth factor receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.