Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1-Cn protein interactions occurring in vitro and in vivo

Membrane proteins are expressed in a specific manner in developing tissues, and characterization of these proteins is valuable because it allows them to be used as cell surface markers. Furthermore, they are potentially important for the regulation of organogenesis because some may participate in signal transduction. In the present study, we used proteomics to examine the comprehensive protein expression profile of the membrane fraction in the embryonic and adult mouse retina. We purified the retinal membrane fraction by sucrose-density-gradient centrifugation and analysed total proteins using shotgun analysis on a nanoflow LC-MS/MS (liquid chromatography tandem MS) system. Approximately half of the 326 proteins from the adult retina and a quarter of the 310 proteins from the embryonic retina (day 17) appeared to be membrane-associated proteins. Among these, MLP [MARCKS (myristoylated alanine-rich C-kinase substrate)-like protein], which shares approx. 50% amino acid identity with MARCKS, was selected for further characterization. The mRNA and surface protein expression of MLP decreased as retinal development progressed. Overexpression of MLP by retrovirus-mediated gene transfer enhanced the proliferation of retinal progenitor cells without affecting differentiation or cell migration in a retinal explant culture system. In contrast, MLP overexpression did not promote proliferation in fibroblasts (NIH 3T3 cells). Mutation analysis of MLP demonstrated that myristoylation was necessary to promote proliferation and that phosphorylation inhibited proliferation, indicating the functional importance of membrane localization.     

Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure–function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, “pseudo-phosphorylated” form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.     

Ultracytochemical study on in vitro differentiation of neural retinal cells of chick embryos

In order to study cell differentiation and morphogenesis of neural retina, ultracytochemical examination for acetylcholine esterase (AChE) was carried out on neural retinal cells from 6-day-old chick embryos cultured in monolayer for 20 days. AChE is a suitable marker for identifying cell specificity and structure of cultured neural retinal cells, because its specific localization in the intact chick neural retina has been established. After about 2 weeks of culturing a number of cell aggregates formed on the monolayer sheet of glial cells, in which cell bodies were generally located on the periphery regions while their cellular processes were in the center, forming neuropil structures. Among such peripherally located cells presumptive ganglion, amacrine, bipolar, and photoreceptor cells could be distinguished. In the neuropil structures, some cellular processes had typical ribbon synapses indicating that these structures correspond to the plexiform layers of the retina. We could also classify the neuropils into two types of both from the AChE activity and from the structure of the nerve terminals. These findings indicate that our cell culture system can be used for the study of cell differentiation and histogenesis of retinal cells.     

pp60c-src is developmentally regulated in the neural retina

We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.     

Expression of a novel Müller glia specific antigen during development and after optic nerve lesion

To generate monoclonal antibodies, immunogen fractions were purified from embryonic chick retinae by temperature-induced detergent-phase separation employing Triton X-114. Under reducing conditions, the monoclonal antibody (mAb) 2M6 identifies a protein doublet at 40 and 46 x 10(3) Mr, which appears to form disulfide-coupled multimers. The 2M6 antigen is regulated developmentally during retinal histogenesis and its expression correlates with Müller glial cell differentiation. Isolated glial endfeet and retinal glial cells in vitro were found to be 2M6-positive, identified with the aid of the general glia marker mAb R5. mAb 2M6 does not bind to any other glial cell type in the CNS as judged from immunohistochemical data. Cell-type specificity was further substantiated by employing retinal explant and single cell cultures on laminin in conjunction with two novel neuron-specific monoclonal antibodies. MAb 2M6 does not bind either to neurites or to neuronal cell bodies. Incubation of retinal cells in vitro with bromodeoxyuridine (BrdU) and subsequent immunodouble labelling with mAb 2M6 and anti-BrdU reveal that mitotic Müller cells can also express the 2M6 antigen. To investigate whether Müller cell differentiation depends on interactions with earlier differentiating ganglion cells, transections of early embryonic optic nerves in vivo were performed. This operation eliminates ganglion cells. Müller cell development and 2M6 antigen expression were not affected, suggesting a ganglion-cell-independent differentiation process. If, however, the optic nerve of juvenile chicken was crushed to induce a transient degeneration/regeneration process in the retina, a significant increase of 2M6 immunoreactivity became evident. These data are in line with the hypothesis that Müller glial cells, in contrast to other distinct glial cell types, might facilitate neural regeneration.     

Glial and neuronal regulation of the lipid carrier R-FABP

Neuroembryogenesis critically depends on signaling molecules that modulate cell proliferation, differentiation, and the formation of neural networks. In an attempt to identify potential morphogenetic active components that are distributed in a graded fashion in the developing nervous system, we generated substraction libraries of the embryonic nasal and temporal chick retina. Selected clones were analyzed by sequencing, Northern and Western blotting, in situ hybridization, and immunocytochemistry. Retinal fatty acid-binding protein (R-FABP) mRNA displayed the most pronounced topographic gradient. R-FABP was most strongly expressed in nasal retina, though topographic differences were not evident on the protein level. R-FABP expression was subject to a pronounced spatio-temporal regulation. Peak expression was at the period of cell generation/migration and differentiation. To identify the cell types involved in R-FAPB synthesis, ganglion cells as the only retinal projection neurons were enriched by enzymatic delayering. Cell somata, axons, and growth cones were R-FABP immunoreactive. Most interestingly, R-FABP immunoreactivity was critically dependent on the growth substratum. It was abrogated when axons grew on isolated glial endfeet. Radial glia purified by complement-mediated cytolysis also expressed R-FABP at moderate levels. The expression level was significantly increased during mitosis and dropped down again in postmitotic cells. Further on, transient loss of cell-cell and substratum contact induced a subcellular redistribution of R-FABP. In conjunction with the morphogen-binding activity of other FABP family members and their impact on cell migration and tissue differentiation, R-FABP characteristics suggest a regulatory function during retinal histogenesis but not during topographic map formation.     

Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca²⁺ transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca²⁺ fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.     

Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina

To determine whether production of new neurons of a particular type is regulated by the presence of previously differentiated neurons of the same type, we ablated all tyrosine hydroxylase immunoreactive (THIR) cells from larval frog retina with the neurotoxin 6-hydroxydopamine, and examined the retinas in subsequent weeks for newly generated THIR neurons. Three weeks after neurotoxin administration, new THIR cells appeared near the zone of neural proliferation at the ciliary margin at a higher density than that of normal retina, while the densities of other amacrine cell types, serotonin (t-HT) immunoreactive and substance P immunoreactive (SPIR), remained the same as controls. Thus the production of new retinal TRIR cells is selectively up-regulated following ablation of previously differentiated cells of this type.     

Developmental predetermination of the structural and molecular polarization of photoreceptor cells

Through mechanisms still unknown, the apparently homogeneous neuroepithelium of the embryonic optic cup differentiates into such divergent cell types as photoreceptors, glia, and various subsets of neurons. Questions that still remain unanswered in this field include the timing and mechanism of action of the "instructive" events directing each neuroepithelial cell to undergo the sequence of phenotypic changes necessary to develop into a specific retinal cell type. This laboratory is investigating some of these questions using cultures in which dissociated neural retina cells, obtained before the onset of overt photoreceptor differentiation, develop at low density in the absence of glia and pigment epithelium. The cultures initially are a morphologically homogeneous population of process-free, round cells. Some cells retain this morphology throughout the first week in vitro, while others develop either as photoreceptors or as multipolar neurons. Photoreceptors elongate and become very asymmetric as they do in vivo, with characteristic compartments orderly arranged along their longitudinal axis (an outer segment-like process, inner segment, cell body, and a characteristically short, single neurite). Cell polarization can also be observed in the distribution of opsin immunoreactive materials and some cytoskeletal elements. Thus, certain precursor cells present in the embryonic retina seem to be programmed to differentiate into photoreceptors even when developing in the absence of contacts with other retinal cells. However, interactions with other constituents of the retina/pigment epithelium complex are probably necessary to ensure final photoreceptor maturation, including further growth of the opsin-rich outer segment process.     

Step-wise specification of retinal stem cells during normal embryogenesis

The specification of embryonic cells to produce the retina begins at early embryonic stages as a multi-step process that gradually restricts fate potentials. First, a subset of embryonic cells becomes competent to form retina by their lack of expression of endo-mesoderm-specifying genes. From these cells, a more restricted subset is biased to form retina by virtue of their close proximity to sources of bone morphogenetic protein antagonists during neural induction. During gastrulation, the definitive RSCs (retinal stem cells) are specified as the eye field by interactions with underlying mesoderm and the expression of a network of retina-specifying genes. As the eye field is transformed into the optic vesicle and optic cup, a heterogeneous population of RPCs (retinal progenitor cells) forms to give rise to the different domains of the retina: the optic stalk, retinal pigmented epithelium and neural retina. Further diversity of RPCs appears to occur under the influences of cell-cell interactions, cytokines and combinations of regulatory genes, leading to the differentiation of a multitude of different retinal cell types. This review examines what is known about each sequential step in retinal specification during normal vertebrate development, and how that knowledge will be important to understand how RSCs might be manipulated for regenerative therapies to treat retinal diseases.     

Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.     

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Differentiation Markers of Retinal Cell Types in Studies on Vertebrate Eye Development and Regeneration

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labeled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.     

Markers of retinal-type cell differentiation in studies of eye development and regeneration in vertebrates]

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labelled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.     

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.     

Isolation of an adhesion-mediating protein from chick neural retina adherons

Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells. They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina. The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M. LaCorbiere, 1985, J. Cell Biol., 100:56-63). This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor. The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture. The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans. Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions. The RP protein is found in neural retina, but not in other tissues such as brain and muscle. These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells.     

Differential enhancement of neural and photoreceptor cell differentiation of cultured pineal cells by FGF-1, IGF-1, and EGF

There are several common features between the pineal organ and the lateral eye in their developmental and evolutionary aspects. The avian pineal is a photoendocrine organ that originates from the diencephalon roof and represents a transitional type between the photosensory organ of lower vertebrates and the endocrine gland of mammals. Previous cell culture studies have shown that embryonic avian pineal cells retain a wide spectrum of differentiative capacities, although little is known about the mechanisms involved in their fate determination. In the present study, we investigated the effects of various cell growth factors on the differentiation of photoreceptor and neural cell types using pineal cell cultures from quail embryos. The results show that IGF-1 promotes differentiation of rhodopsin-immunoreactive cells, but had no effect on neural cell differentiation. Simultaneous administration of EGF and IGF-1 further enhanced differentiation of rhodopsin-immunoreactive cells, although the mechanism of the synergistic effect is unknown. FGF-1 did not stimulate proliferation of neural progenitor cells, but intensively promoted and maintained expression of a neural cell phenotype. FGF-1 appeared to lead to the conversion from an epithelial (endocrinal) to a neuronal type. It also enhanced phenotypic expression of retinal ganglion cell markers but rather suppressed expression of an amacrine cell marker. These results indicate that growth factors are important regulatory cues for pineal cell differentiation and suggest that they play roles in determining the fate of the pineal organ and the eye. It can be speculated that the differences in environmental cues between the retina and pineal may result in the transition of the pineal primordium from a potentially ocular (retinal) organ to a photoendocrine organ.     

Identification of neural progenitors in the adult mammalian eye

We have shown that the embryonic mammalian retina contains neural progenitors which display stem cell properties in vitro. Here we report the characterization of neural progenitors isolated from the adult mammalian eye. These quiescent cells, located in the pigmented ciliary bodies, proliferate in the presence of FGF2 and express the neuroectodermal marker nestin. The proliferating cells give rise to neural spheres and are multipotential; they express cell type-specific markers corresponding to neurons and glia. In addition, neural progenitors can generate secondary neural spheres, thus displaying potential to self-renew. The ciliary body-derived neural progenitors display retina-specific properties; the undifferentiated cells express Chx10, a retinal progenitor marker, and upon differentiation express markers corresponding to specific retinal cell types. Therefore, the pigmented ciliary body in the adult mammalian eye harbors neural progenitors that display stem cell properties and have the capacity to give rise to retinal neurons in vitro.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.