Enzymatic dissection of embryonic cell adhesive mechanisms

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.     

Intercellular adhesive selectivity. I. An improved assay for the measurement of embryonic chick intercellular adhesion (liver and other tissues)

An improved assay for measuring intercellular adhesive selectivity of embryonic chick liver cells is described. Three major improvements over earlier procedures are noted: (a) enhanced reproducibility of liver cell-liver cell aggregate adhesion (homotypic adhesion) was achieved; (b) 25-70% of the input cells adhered to the collecting aggregates during the course of routine experiments as compared to the 0.25% in earlier assays. This increase in cellular adhesion suggests that the observed cell pick-up is a characteristic of the majority of the dissociated liver cell population; (c) the rate of intercellular adhesion was increased 1,000-fold. The main feature of the assay is that it measures the tissue adhesive selectivities of the dissociated cell population. Studies were undertaken on three embryonic chick tissues (liver, neural retina, and mesencephalon) to determine the tissue selectivity of intercellular adhesion of these dissociated cell types. Some general properties of liver cell homotypic adhesion have been studied and are reported.     

Expression of calcium-dependent cell adhesion during ocular development: a biochemical, histochemical and functional analysis

Previous studies of the adhesive properties of embryonic chick neural retina cells indicate a gradual decrease in the expression of calcium-dependent adhesions during retinal histogenesis, a function which has been attributed in part to gp130/4.8, a retinal calcium-dependent adhesion-associated cell surface membrane glycoprotein with a molecular weight of approximately 130 kDa and an isoelectric point of 4.8 (G. B. Grunwald, R. Pratt, and J. Lilien, 1982, J. Cell Sci. 55, 69-83). The experiments described here were done to define the relationship of gp130/4.8 to N-cadherin, another calcium-dependent adhesion molecule found in chick retina, which has a reported molecular weight of 127 kDa and which is recognized by monoclonal antibody NCD-2 (K. Hatta and M. Takeichi, 1986, Nature (London) 320, 447-449). Using two-dimensional gel electrophoresis followed by Western blotting as well as quantitative solid-phase immunoassays, polyspecific antisera recognizing gp130/4.8 were compared with monoclonal antibody NCD-2 for reactivity with proteins of retina and other tissues. The data lead us to conclude that retinal calcium-dependent adhesion proteins gp130/4.8 and N-cadherin are likely to be the same molecule. In order to obtain evidence for a direct correlation of changes in expression of these adhesion proteins with changes in retinal cell adhesivity and related morphogenetic events, parallel studies were carried out with cells from various ocular tissues to examine the functional, biochemical, and immunohistochemical expression of N-cadherin during ocular development. Immunohistochemical mapping of N-cadherin in the developing chick eye reveals three modes of N-cadherin expression which occur simultaneously in different ocular tissues: (1) down-regulation, (2) up-regulation, and (3) steady-state expression. These patterns of expression correlate with changes in the adhesive behavior of cells as well as with discrete stages in the morphogenesis of several ocular tissues. The results suggest that N-cadherin is a versatile cell adhesion protein with a role in both the development of several ocular tissues and the maintenance of specialized structures in the mature eye.     

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.     

Developmental and biochemical studies of adhesive specificity among embryonic retinal cells.

One pattern of cell-cell adhesion within the chick embryo neural retina follows a dorsoventral gradient in which cells at either end show maximal affinity for each other. Developmental and biochemical approaches have been applied to analyze the basis of this adhesive pattern. When retinal cells were prepared from eyes that had been inverted 180° in situ prior to retinal differentiation, an inverted pattern of adhesive preference resulted. These data suggest that adhesive preference is determined early in embryogenesis. Trypsinization of either dorsal or ventral retinal cells destroyed their adhesive preference. Treatment with neuraminidase resulted in a differential loss of adhesive preference by dorsal retinal cells. This effect could be mimicked by mild oxidation with NaIO₄. Since periodate inactivation of adhesive preference could be reversed by subsequent borohydride treatment, borotritiate was used to label those cell surface molecules crucial to the reactivation of adhesive preference. Fluorographs prepared after polyacrylamide gel electrophoresis revealed that periodate stimulated the appearance of a small number of radioactively labeled bands. These data suggest that adhesive preference is mediated by glycoconjugates, possibly sialoglycoproteins, on the dorsal cell surface.     

A role for adherons in neural retina cell adhesion

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.     

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.     

Development of NADPH-Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 m M calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by N ^G-nitro- l -arginine or l - N ^G-(iminoethyl)ornithine is concentration dependent with IC₅₀ values of ∼1 m M at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC₅₀ values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.     

Promotion of retinal neurite outgrowth by substratum-bound fibronectin

We have examined conditions under which aggregates of embryonic chick neural retina will extend neurities in vitro. Trypsin-dispersed cells from 7-day embryonic chick neural retina were aggregated in rotation culture for 8 hr and maintained in serum-free medium on a variety of standard culture substrate. Aggregates extend few neurites on untreated plastic, glass, or collagen substrata. However, pretreatment of these substrata with human plasma fibronectin enhances their capacity to support retinal neurite outgrowth. Aggregates cultured on fibronectin-treated substrata extend long, radially oriented neurites within 36 hr in vitro. The morphology of these neurites is distinct from that seen when aggregates are cultured on polylysine-treated substrata. In the latter case, neurites are highly branched and grow concentrically around the aggregate perimeter. Addition of fibronectin to polylysine-treated substrata stimulates radial neurite outgrowth. Promotion of neurite outgrowth is dependent on the amount of fibronectin bound to the culture substratum and on the pH at which binding occurs. The requirements for fibronectin-mediated neurite outgrowth are more stringent than those previously reported for fibroblast attachment and spreading.     

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons¹, which has been difficult with the conventional method of injection and electroporation at E1.5².     

Enhancement of Apoptosis in Developing Chick Neural Retina Cells by Basic Fibroblast Growth Factor

Abstract: To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time- and dose-dependent manner. The effect was inhibited by anti-bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-α, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.     

Tissue-specific cell-surface antigens in embryonic cells

With the use of antisera prepared in rabbits against suspensions of live embryonic chick tissue cells, qualitative differences in cell surface antigens were demonstrated on cells from different embryonic chick tissues by immune agglutination and immunofluorescence. Unabsorbed antisera reacted with both homologous and nonhomologous cells; thorough absorption of the antisera with heterologous tissues removed cross-reacting antibodies, and the antisera acquired a high degree of tissue specificity. Thus, antiretina cell serum absorbed with nonretina cells or tissues, agglutinated only neural retina cells, and was shown by immunofluorescence tests to react specifically with the surface of retina cells, both in cell suspensions and in frozen tissue sections. Comparable results with antisera against cells from embryonic liver and other tissues demonstrated the existence of tissue-specific, phenotypic disparities in the antigenicities of embryonic cell surfaces, in addition to the presence of cell-surface antigens shared by certain classes of cells, and of antigens common to all cells in the embryo. The results are discussed in terms of the possible involvement of such phenotypic determinants in the specification of cell surfaces, in relation to cell recognition and developmental interactions.     

Immunohistochemical characterization of delta crystallin-containing retina/optic nerve "boundary" cells in the chick embryo

The term "transdifferentiation" has been used to describe the apparent phenotypic conversion of chick embryo neural retina Müller glial cells into lens-like cells in vitro. This phenotypic conversion is characterized by expression of such lens-specific proteins as delta crystallin and has been viewed as an example of cells transforming from the phenotype of a given tissue to that of another. We have identified a population of neuroglia-like cells in the embryonic chick retina which express high levels of delta crystallin as a function of normal development. The position and morphology of these cells is quite distinctive in that they form a loose meshwork which defines the boundary between the neural retina and the optic nerve head. These "boundary" cells are detectable as early as Day 5 of development through hatching. However, the meshwork structure formed by the cells is only readily observed between Days 8 and 9 of development. Double-immunolabeling procedures comparing delta crystallin staining to that of glial and neuronal markers suggest that these cells are a form of retinal Müller glial cell. The results show that under appropriate microenvironmental conditions, expression of delta crystallin falls into the normal repertoire of retinoblast cells. The results also demonstrate the presence of a cellular boundary defining the junction between the neural retina and the optic nerve, tissues that are ontogenetically and structurally continuous but functionally distinct.     

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.     

Immunologic detection of retina cognin on the surface of embryonic cells

The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.     

Possible Roles for Cognin in Chick Embryo Neural Retina

In this review, we discuss current information about a cell-cellrecognition protein present in chick embryo neural retina. Thisprotein, retina cognin, has cell adhesion or aggregation promotingpropertiesin vitro. We discuss five questions. First, what isretina cognin (R-cognin)? Second, what do we know about cogninin chick retina? We discuss its histological distribution inretina and how that distribution changes during embryonic andearly post-hatching development. Third, where is cognin withincells? We review light microscopy evidence for its localizationin plasma membranes of somas and neurites of selected retinalneurons as an intrinsic membrane protein. Fourth, how is cognindistributed in membranes? We summarize evidence that cogninmight not be uniformly distributed over cellsurfaces and thatit might bind to specific proteins on the surfaces of otherretina cells. From the available information, we ask what wecan deduce about cognin's biological role in the neural retina.     

CELL SORTING IN THE PRESENCE OF CYTOCHALASIN B

The ability of cytochalasin B to inhibit ruffled membrane activity and cellular locomotion of vertebrate cells in monolayer culture prompted its use to study the necessity for this kind of active cellular locomotion in cell sorting in heterotypic cell aggregates. Cell sorting was inhibited in chick embryo heart-pigmented retina aggregates but a remarkable degree of sorting did occur in neural retina-pigmented retina aggregates. In these experiments, the levels of cytochalasin B employed (5 or 10 µg/ml) are sufficient to inhibit completely locomotion of these cell types in monolayer culture. It is proposed that the degree of cell movement achieved during sorting in neural retina-pigmented retina aggregates in the presence of cytochalasin B is the result of changes in cell contact resulting from adhesive interaction of cells. The effect of cytochalasin B on the initial aggregation of dissociated cells was also tested. With the cell types used in this study (chick embryo neural retina and limb bud), aggregation was not affected for a period of several hours.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.