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G B Grunwald

Publications and source records attributed to G B Grunwald.

At least 19 recordsLinked to original sources
PubMed1997

Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development.

N-cadherin, a member of the cadherin family of calcium-dependent cell adhesion molecules, mediates adhesive and signaling interactions between cells during development. N-Cadherin undergoes dynamic spatiotemporal changes in expression which correlate with morphogenetic movements of cells during organogenesis and histogenesis. We have previously shown that N-cadherin expression during development is regulated by several mechanisms, including mRNA expression, cytokine modulation, and proteolytically mediated turnover, yielding the NCAD90 protein. The present study was directed at determining the extent to which N-cadherin in primary embryonic cells is the target of endogenous kinases and phosphatases, as well as the effects of modulation of these enzymes on NCAD90 expression. The results of phosphoamino acid analyses, peptide mapping, and measurements of N-cadherin and NCAD90 expression in embryonic tissues indicate that N-cadherin is indeed the target of endogenous kinase and phosphatase action, and that modulation of different classes of these enzymes can result in either stimulation or inhibition of NCAD90 production. These results provide a mechanistic explanation for observations that cadherin function is downregulated following expression of exogenously introduced viral tyrosine kinases and provide a function for the tyrosine phosphatases recently found in association with cadherins. The results indicate that N-cadherin expression during retinal development is possibly regulated in part by modulation of its phosphorylation state, the balance of which may determine whether N-cadherin remains stably expressed or is targeted for proteolytically mediated turnover to produce NCAD90.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos.

During eye development, optic vesicles evaginate laterally from the neural tube and develop into two bilayered eye cups that are composed of an outer pigment epithelium layer and an inner neural retina layer. Despite their similar embryonic origin, the pigment epithelium and neural retina differentiate into two very distinct tissues. Previous studies have demonstrated that the developmental potential of the pigmented epithelial cells is not completely restricted; until embryonic day 4.5 in chick embryos, the cells are able to switch their phenotype and differentiate into neural retina when treated with fibroblast growth factors (FGF) (Park, C. M., and Hollenberg, M. J. (1989). Dev. Biol. 134, 201-205; Pittack, C., Jones, M., and Reh, T. A. 1991). Development 113, 577-588; Guillemot, F. and Cepko, C. L. (1992). Development 114, 743-754). These studies motivated us to test whether FGF is necessary for neural retina differentiation during the initial stages of eye cup development. Optic vesicles from embryonic day 1.5 chick were cultured for 24 hours as explants in the presence of FGF or neutralizing antibodies to FGF2. The cultured optic vesicles formed eye cups that contained a lens vesicle, neural retina and pigmented epithelium, based on morphology and expression of neural and pigmented epithelium-specific antigens. Addition of FGF to the optic vesicles caused the presumptive pigmented epithelium to undergo neuronal differentiation and, as a consequence, a double retina was formed. By contrast, neutralizing antibodies to FGF2 blocked neural differentiation in the presumptive neural retina, without affecting pigmented epithelial cell differentiation. These data, along with evidence for expression of several FGF family members and their receptors in the developing eye, indicate that members of the FGF family may be required for establishing the distinction between the neural retina and pigmented epithelium in the optic vesicle.

Animals↗
PubMed1996

Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo.

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

Animals↗
PubMed1994

Ca2+ influx and neurite growth in response to purified N-cadherin and laminin.

The signaling mechanisms underlying neurite growth induced by cadherins and integrins are incompletely understood. In our experiments, we have examined these mechanisms using purified N-cadherin and laminin (LN). We find that unlike the neurite growth induced by fibroblastic cells expressing transfected N-cadherin (Doherty, P., and F.S. Walsh. 1992. Curr. Opin. Neurobiol. 2:595-601), growth induced by purified N-cadherin in chick ciliary ganglion (CG), sensory, or forebrain neurons is not sensitive to inhibition by pertussis toxin. Using fura-2 imaging of single cells, we show that soluble N-cadherin induces Ca2+ increases in CG neuron cell bodies, and, importantly, in growth cones. In contrast, N-cadherin can induce Ca2+ decreases in glial cells. N-cadherin-induced neuronal Ca2+ responses are sensitive to Ni2+, but are relatively insensitive to diltiazem and omega-conotoxin. Similarly, neurite growth induced by purified N-cadherin is inhibited by Ni2+, but is unaffected by diltiazem and conotoxin. Soluble LN also induced small Ca2+ responses in CG neurons. LN-induced neurite growth, like that induced by N-cadherin, is insensitive to diltiazem and conotoxin, but is highly sensitive to Ni2+ inhibition. K+ depolarization experiments suggest that voltage-dependent Ca2+ influx pathways in CG neurons (cell bodies and growth cones) are largely blocked by the combination of diltiazem and Ni2+. Our results demonstrate that cadherin signaling involves cell type-specific Ca2+ changes in responding cells, and in particular, that N-cadherin can cause Ca2+ increases in neuronal growth cones. Our findings are consistent with the current idea that distinct neuronal transduction pathways exist for cell adhesion molecules compared with integrins, but suggest that the involvement of Ca2+ signals in both of these pathways is more complex than previously appreciated.

Animals↗
PubMed1993

Purification and characterization of NCAD90, a soluble endogenous form of N-cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite-promoting function.

The cadherins are calcium-dependent cell adhesion molecules which regulate cell-cell interactions during morphogenesis. During development, cadherin expression is subject to dynamic patterns of regulation. We have previously demonstrated that expression of N-cadherin, the predominant cadherin of neural tissues, is sharply down-regulated during development of the retina and brain during later stages of histogenesis (Lagunowich and Grunwald, Dev Biol 135:158-171, 1989; Lagunowich et al., J Neurosci Res 32:202-208, 1992), and that this down-regulation is due to multiple factors, including decreased mRNA levels and turnover apparently mediated by endogenous metalloproteolytic activity (Roark et al., Development 114:973-984, 1992). In the present study, we describe metabolic studies which provide direct biochemical evidence for turnover of 130-kDa N-cadherin in embryonic retina tissues, yielding a soluble 90-kDa N-terminal fragment. We demonstrate that this form of N-cadherin, which we refer to as NCAD90, accumulates in vivo during development. We further demonstrate that purified NCAD90, obtained from embryonic vitreous humor, retains biological function and promotes cell adhesion and neurite growth in a dose-dependent fashion among chick embryo neural retina cells when present in a substrate-bound form. The morphology of retinal cells and neurites grown on a substrate of NCAD90 differs strikingly from that seen on a laminin substrate, in a manner similar to that described for intact 130-kDa N-cadherin. We conclude that proteolysis of N-cadherin at the cell surface during embryonic retinal histogenesis is an endogenous mechanism for regulating N-cadherin expression which generates a novel and functional form of the protein. The results further indicate that an intact cytoplasmic domain is not essential for all cadherin functions.

Animals↗
PubMed1993

Analysis of protein variations in adult and postnatal day 11 staggerer and lurcher mutant mice.

As an initial attempt at uncovering the molecular basis of cerebellar synaptogenic defects in the lurcher and staggerer mutant mice, the pattern of protein expression was compared between cerebellar and non-cerebellar tissues, between normal and mutant mice and between early postnatal and adult developmental stages. While cerebellar and mutation-specific alterations in the expression of proteins could be easily observed in adult tissues, no such alterations were observed in early postnatal lurcher mice and only one qualitatively unique protein species could reproducibly be identified in early postnatal staggerer mice.

Aging↗
PubMed1993

The structural and functional analysis of cadherin calcium-dependent cell adhesion molecules.

During the past year considerable progress has been made in our understanding of cadherin structure and function. Recent research has concentrated on several aspects of the cell and molecular biology of cadherins, including genomic organization, cytoskeletal interactions, regulation of expression and function by post-translational modifications, differential expression during embryonic development, and the emerging role of cadherin misexpression and malfunction in pathogenesis.

Animals↗
PubMed1993

Structure/function analysis of the integrin beta 1 subunit by epitope mapping.

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.

3T3 Cells↗
PubMed1992

Immunohistochemical and biochemical analysis of N-cadherin expression during CNS development.

The expression of the calcium-dependent adhesion molecule N-cadherin during chick embryo central nervous system (CNS) development was examined by immunohistochemistry and electrophoresis and immunoblotting. During histogenesis, N-cadherin is expressed at high levels in a uniform fashion in many regions of the CNS. However, during later stages of development, expression becomes restricted to the ependymal cells lining the ventricular system and in the choroid plexus. This down-regulation was confirmed by both immunohistochemical and biochemical techniques. The program of expression lags behind in the cerebellum in concert with the delayed development of this region of the brain. A high level of N-cadherin was found to be expressed in the brainstem and spinal cord floorplate, while a low level was detected at the optic nerve head. The results indicate that while, in general, the program of N-cadherin expression is similar in the retina and the brain, certain structures unique to the eye and brain express locally high or low levels of this adhesion protein.

Animals↗
PubMed1992

Evidence for endogenous proteases, mRNA level and insulin as multiple mechanisms of N-cadherin down-regulation during retinal development.

Our previous studies of the role of cell adhesion in retinal development have focused on the expression and function of N-cadherin, the predominant calcium-dependent intercellular adhesion protein of neural tissues. During the course of retinal development, N-cadherin expression undergoes significant qualitative and quantitative changes in its pattern of expression, most prominently a sharp down-regulation of expression throughout most of the retina. The present studies were directed at investigating the epigenetic mechanisms that could mediate this loss of N-cadherin from the retina. Using an in vitro intact retinal organ culture system, results were obtained which suggest that insulin enhances the down-regulation of N-cadherin expression in a protein-synthesis-dependent fashion. Furthermore, the metalloprotease inhibitor 1,10-phenanthroline inhibits the loss of N-cadherin from the retina. While N-cadherin is down-regulated in organ culture, other cell adhesion molecules, which are not down-regulated in vivo, are also not down-regulated in organ culture. The defined organ culture medium conditioned by the retina accumulates both a soluble 90 x 10(3) M(r) N-terminal fragment of N-cadherin as well as a number of secreted proteases. Both of these components are also shown to be present in vivo in the vitreous humor. Northern blot analysis indicates a single mRNA encoding N-cadherin in the retina and no evidence for a second message that could encode the 90 x 10(3) M(r) fragment. However, the amount of N-cadherin mRNA detectable on northern blots decreases during development. The results reported here suggest that the down-regulation of N-cadherin that occurs during retinal development is possibly mediated by multiple mechanisms, which include turnover at the cell surface mediated by endogenous proteolysis, reduced levels of N-cadherin mRNA and modulation by growth factors.

Animals↗
PubMed1992

Differential cell adhesion and expression of N-cadherin among retinoblastoma cell lines.

Retinoblastoma (RB) is the most common intraocular childhood cancer, but little is known regarding its cellular biology and the factors that might regulate its progression and metastatic potential. Although alterations in cell-adhesion interactions could contribute to the metastatic behavior of RB, no specific cell-adhesion molecules have been identified in RB cells. The current study examined the adhesive properties of three RB cell lines (Weri-Rb1, Weri-Rb27, and Y79) and determined whether N-cadherin, a specific cell-adhesion protein expressed during normal retinal development, was expressed by these cell lines. This study revealed marked differences in the cell-adhesive properties of the three cell lines with respect to several parameters assayed, including cell morphology, calcium dependence of the cell adhesion, and the presence of N-cadherin. N-cadherin was expressed by human RB cells, but the level of expression, determined both functionally and biochemically, varied among different RB cell lines.

Cadherins↗
PubMed1991

Tissue and age-specificity of post-translational modifications of N-cadherin during chick embryo development.

Our previous studies indicated that regulation of N-cadherin expression differs spatially and temporally among tissues of the eye, possibly reflecting the distinct roles it has in the development and maintenance of eye tissues. To understand this regulation of N-cadherin expression and its function in different tissues during embryonic development, we investigated the post-translational modifications of N-cadherin and its association with the cytoskeleton. We show that N-cadherin is a sulfated and phosphorylated protein. The phosphorylation of N-cadherin occurs in an age- and tissue-specific pattern during development in the neural retina, brain, lens and heart. The extent of sulfation of N-cadherin is also age-dependent, and both sulfated and unsulfated pools of N-cadherin exist in the same tissue as indicated by two-dimensional electrophoresis. The degree of association of N-cadherin with the cytoskeleton differs from one tissue to another, as well as within a single tissue at different stages of development. A positive correlation was found between the extent, developmental timing, and tissue specificity of N-cadherin phosphorylation and the degree of N-cadherin association with the cytoskeleton. Our results suggest the existence of a microheterogeneous population of N-cadherin molecules, within which posttranslational modification of N-cadherin may affect its association with the cytoskeleton and its expression and function during development.

Aging↗
PubMed1991

Identification of the 2A10 antigen of retinal pigment epithelium as a beta 1 subunit of integrin.

In previous studies the 2A10 antigen of retinal pigment epithelium (RPE) was described. It is a cell-surface monoclonal antibody-defined antigen whose properties suggest a possible role in cell adhesive interactions. This report identified the 2A10 antigen as a beta 1 subunit of the cell-surface extracellular matrix receptor protein, integrin. Comparative biochemical, immunochemical, and histochemical analyses of the 2A10 antibody and other antibodies directed against the beta 1 subunit of integrin indicate similar tissue distributions, immunoblotting patterns, tryptic digestion profiles, and coimmunoprecipitation of the 2A10 antigen and the beta 1 subunit of integrin. The 2A10 antigen/beta 1 subunit differed in molecular weight in different tissues, with the RPE demonstrating a form intermediate between neural and endoderm-derived tissues. The application of this new antibody to functional studies of the role of integrin in RPE cell-substrate adhesion is described in the accompanying paper.

Animals↗
PubMed1991

Functional inhibition of retinal pigment epithelial cell-substrate adhesion with a monoclonal antibody against the beta 1 subunit of integrin.

In the preceding report, experiments were described which identify the 2A10 antigen of retinal pigment epithelial (RPE) cells as a beta 1 subunit of the cell-surface extracellular matrix receptor protein integrin. In this article, experiments are presented which use the 2A10 and CSAT antibodies, both directed against the beta 1 subunit, to investigate the role of integrin in RPE and fibroblast (FB) cell-substrate adhesion. When added to cultures simultaneously with cells, either the 2A10 or CSAT antibodies inhibit both FB and RPE cell adhesion and spreading on laminin (LM). However, although the 2A10 antibody blocks adhesion and spreading of FB and RPE cells on fibronectin (FN), the CSAT antibody has no effect. The inhibition of the 2A10 antibody is specific for integrin-mediated adhesion; it does not affect FB or RPE cell adhesion and spreading on tissue-culture plastic. When RPE cells are first allowed to attach to and spread on FN and LM and then the 2A10 or CSAT antibody is added to the cultures, both cause detachment and rounding of RPE cells from LM, but neither has any effect on the cells already spread on FN. These results indicate that there are differences in the way FB and RPE cells interact with LM and FN. Furthermore, these results provide the first direct functional demonstration that RPE cell-substrate adhesion is mediated by integrin.

Animals↗
PubMed1990

Identification of mammalian and invertebrate analogues of the avian calcium-dependent cell adhesion protein N-cadherin with synthetic-peptide directed antibodies against a conserved cytoplasmic domain.

N-cadherin, a 130kD transmembrane adhesive glycoprotein, is a mediator of specific cellular interactions during development. Analysis of N-cadherin at the protein level, to date, has been largely dependent upon monoclonal antibody NCD-2 which recognizes only avian N-cadherin. We produced a monospecific polyclonal antiserum, C-NCAD(838-856), to a synthetic peptide corresponding to a portion of the highly conserved c-terminal cytoplasmic domain of chick N-cadherin. Using polyacrylamide gel electrophoresis and immunoblotting to map tissue distribution we show that the antiserum detects chick N-cadherin with a similar tissue distribution as NCD-2. Unlike NCD-2, however, anti-C-NCAD(838-856) recognizes N-cadherin analogues in a wide variety of species, including mouse, human, fish and drosophila. The results of comparative immunoblot studies demonstrate similar tissue-specific patterns and apparent molecular weight variation in the chick, mouse and human. This indicates that N-cadherin structure and expression, and most likely function as well, have been highly conserved in evolution. The antiserum recognizes an epitope unique to N-cadherin which is conserved among N-cadherins from a variety of species but is absent from other members of the cadherin gene family, as no immunoreactivity was detected with tissues bearing these other cadherins. The antiserum is thus a useful tool for the phylogenetic and biochemical investigation of N-cadherin from a variety of tissue sources.

Amino Acid Sequence↗
PubMed1990

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.

Animals↗
PubMed1990

Generation and characterization of monoclonal antibodies specific for the retinal pigment epithelium.

Although both the neural retina and the retinal pigment epithelium (RPE) arise as neighboring portions of the embryonic optic cup, these two tissues follow very different developmental pathways. In order to obtain probes for the analysis of RPE development from its earliest divergence from the neural retina to late stages of differentiation, we have developed a panel of monoclonal antibodies which recognize antigens specific to the RPE. These probes have been applied to an immunohistochemical analysis of RPE development. The results indicate that the RPE is antigenically distinct from the neural retina even before the onset of overt differentiation. In addition, the RPE layer of the retina becomes further subdivided antigenically as its distinct anterior and posterior derivatives develop. These antibodies will be useful markers in the analysis of RPE development.

Animals↗