Enzymatic dissection of embryonic cell adhesive mechanisms: II. Developmental regulation of an endogenous adhesive system in the chick neural retina

Previous studies have demonstrated the presence of two functionally distinct intercellular adhesive systems operating among embryonic chick neural retina cells. These systems differ in their proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function, and in vitro morphogenetic potential. In this report we demonstrate that functional expression of the calcium-dependent adhesive system of embryonic chick neural retina cells is developmentally regulated between Days 7 and 16 of development, whereas the calcium-independent adhesive system is not. Age-dependent changes are described in terms of the ability to produce adhesive-competent cells bearing the calcium-dependent adhesive system and in terms of the responses of these cells during aggregation to perturbations with various drugs. Enzyme and ion combinations other than calcium and typsin are shown to yield calcium-dependent adhesive-competent cells. We also describe the protective effect of calcium on the histological and ultrastructural organization of trypsinized embryonic neural retina tissue. The possible role of the calcium-dependent adhesive system in retinal development is discussed.     

Calcium-Dependent and Calcium-Independent Adhesive Mechanisms Are Present During Initial Binding Events of Neural Retina Cells

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena, an initial recognition event and a subsequent stabilization, is supported by the use of a new cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4°C in sealed compartments. The compartments are inverted and a centrifugal force is then applied tending to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chick neural retina cells was determined to be on the order of 10^?5 dynes after incubation at 4°C. Brief incubations at 37°C resulted in significant strengthening of the intercellular bond. Using this cell binding assay, neural retina cells were shown to exhibit both a Ca⁺⁺-independent and a Ca⁺⁺-dependent mechanism in their initial binding to one another.     

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.     

Cross Talk between Adhesion Molecules: Control of N-cadherin Activity by Intracellular Signals Elicited by β1 and β3 Integrins in Migrating Neural Crest Cells

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin–mediated intercellular contacts. In the present study we provide evidence that, in these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to β1 and β3 integrins caused rapid N-cadherin–mediated cell clustering. Restoration of stable intercellular contacts resulted essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulated into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. In addition, agents that cause elevation of intracellular Ca²⁺ after entry across the plasma membrane were potent inhibitors of cell aggregation and reduced the N-cadherin– mediated junctions in the cells. Finally, elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompanied the restoration of intercellular contacts. These results indicate that, in migrating neural crest cells, β1 and β3 integrins are at the origin of a cascade of signaling events that involve transmembrane Ca²⁺ fluxes, followed by activation of phosphatases and kinases, and that ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell–cell and cell–substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis.     

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.     

Utilization of -glutamine in intercellular adhesion: Ascites tumor and embryonic cells

-Glutamine is required for the synthesis of complex carbohydrates required for the intercellular adhesion of mouse teratoma cells. It remained to be seen if these pathways were of general importance in the adhesion of other cell types. In this study, using an electronic particle counter assay to measure cell adhesion, Ehrlich ascites, Sarcoma 180 and Taper liver ascites tumor cells require exogenous -glutamine to aggregate. This effect is concentration dependent and the amino sugar, -glucosamine, replaces the glutamine requirement. Structural analogs of the active compounds are substantially less effective and metabolic inhibitors block the activity of the effective compounds. Two specific glutamine antagonists, DON (6-diazo-5-oxo- -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of -glutamine but not of -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require -glutamine to reaggregate, though the rate of aggregation is enhanced after preincubation with glutamine. Dissociation of small clumps of neural retina and inhibition of reaggregation of these cells are facilitated by preincubation with azaserine for 3–5 h. -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that -glutamine is involved in the synthesis of complex carbohydrates necessary for adhesion in a variety of cell types. The defective adhesion of the tumor cells examined may result from inability to produce glutamine synthetase, or effectively store cr transport -glutamine.     

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.     

Membrane alterations during chick neural retina development: A freeze-fracture study

As part of a study of cell surface differentiation during chick retina development, a freeze-fracture study of neural retinas from 5 to 10 day embryonic chicks was undertaken. Three classes of changes have been detected. (1) As cells differentiate and become recognizable by their position within the tissue, they acquire characteristic numbers of intramembrane particles in the surfaces in each layer. (2) Small gap junctions appear between cells at the outer limiting membrane of the 5 day retina. At 6 days, they are larger, more numerous and are also found in deeper layers of the tissue. By the seventh day, the size and number of the junctions is greatly reduced; they are not visible after the tenth day. (3) The characteristic lack of particles in the outer limiting membrane of the mature retina appears at the ninth day of incubation, at the time that presumptive photoreceptors extend through the outer limiting membrane. Tight junctions between cells were not observed during this study.     

Malformation of Embryonic Neural Retina Elicited by BrdU

Exposure of neural retina tissue from early chick embryos (5 and 6-days) to 5-bromo-2'-deoxyuridine (BrdU) for 24 h irreversibly prevented normal histogenesis and resulted in the formation of chaotically disorganized tissue. The sensitivity of the retina to this effect decreased with embryonic age and declined sharply after the commencement of cell stratification. Examination by electron microscopy revealed the following progressive morphologic changes resulting from BrdU treatment: complete breakdown of the outer limiting membrane due to disappearance of its constituent tight junctions which normally anchor cells at the outer retinal surface; collapse and endocytosis of cilia, resulting in the absence of photoreceptor processes; increasing disorganization of the cells which commenced at the outer surface of the retina and progressed inward, resulting in chaotic distortion of the histologic architecture of the retina. Ultrastructural differences were noted between cells in the malformed retina, indicating the presence of several cell types. Possible mechanisms of this BrdU-elicited malformation are considered in the Discussion.     

Gicerin,a cell adhesion molecule,participates in the histogenesis of retina

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 769–780, 1997     

Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells.

Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.     

Scanning electron microscopy of cell aggregation: cardiac and mixed retina-cardiac cell suspensions

Reaggregates of cells from 7-day embryonic chick hearts, 10-day neural retinas and respective cell mixtures were examined by scanning electron microscopy (SEM). Cardiac cell aggregates formed during the first 2 hours were composed of rounded cells arranged in linear or branched arrays. By further collection of single cells and accretion of cell clusters, the aggregates increased in size and formed large grape-like masses. By 12 hours, heart aggregates assumed a spherical shape with partial sorting-out of myogenic from non-myogenic cells; the muscle elements occupied the interior of the aggregates, whereas flattened, squamous-like cells, of a non-muscle character, covered the surface in a multilayered epithelium. Single rounded cells were still found on the surface of 12 to 24-hour aggregates; however, they were absent by 48 hours, suggesting that the collection of free cells by the cardiac aggregates ceased during the second day. Early aggregates (2 hours) of retina cells showed initial stages of axonal and dendritic outgrowth characteristic of neural tissue. Examination of heterotypic aggregates of neural retina and myocardial cells after 2 to 6 hours showed small groups of retina cells attached to the surface of the heart cell clusters. The retina cells did not appear to be randomly distributed within the early aggregates but formed small tissue specific clusters even by 2 hours in culture. These results indicate that SEM should be a valuable tool in the further analysis of homo- and heterotypic cellular aggregation.     

A Growth Curve of Cell Numbers In the Neural Retina of Embryonic Chicks

ABSTRACT The growth of cell numbers in a normal embryonic population of ncural retinal cells is described. the numbers were estimated from a time shortly after the neural retina first becomes recognizable to a time when numbers of retinal cells have become steady. Cell numbers were estimated in preparations of an entire neural retina dispersed into a suspension of single nuclei which were then counted in a Coulter counter. the growth curve of the In numbers of cells has three phases of growth: an exponential phase during which there is steady-state exponential growth, a differentiative phase during which cell proliferation ceases and an end phase when no further change in cell numbers can be detected. the variances of the In numbers of cells were highest during the exponential phase. the variances decreased during the differentiative phase and were at their lowest during the end phase. For variances to decrease requires mechanisms which control the final numbers of cells in the neural retina very precisely. the implications of mechanisms which operate by controlling cell lineages are explored.     

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.     

Diffusible rod-promoting signals in the developing rat retina.

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5-6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Vesicle interactions as a model for the retinal cell-cell recognition mediated by R-cognin

The action of R-cognin, a chick neural retina cell recognition glycoprotein, was investigated in vesicles of retina cell membranes. It was found that the aggregation of the vesicles was dependent on membrane proteins, and specifically R-cognin, as vesicle aggregation was inhibited by R-cognin antibody (Fab'). The R-cognin content of the vesicles, and their ability to aggregate, decreased with increasing embryonic age of the tissue. R-cognin mediated aggregation of the vesicles was not dependent on exogenous calcium. Thus, R-cognin was calcium-independent in its membrane linking activity.     

Ionic coupling and mitotic synchrony of siblings in a Drosophila cell line

Following mitosis in many cell lines, siblings remain adjoined in dyads until further cell division. We report here a series of experiments designed to ascertain the nature of this apposition in the embryonic Kc cell line of Drosophila melanogaster. We have found that (1) cell division in siblings is highly synchronized when compared to that in nonsiblings; (2) siblings in dyads are dye coupled with respect to Lucifer Yellow, but intercellular diffusion of larger molecules (FITC-dextran at 6 and 24 kDa) is retarded; (3) siblings are electrically coupled by an ungated, low-resistance intercellular connection which is resistant to treatment with octanol and CO₂, both known to close gap junction channels; and (4) members of a dyad are joined by a cytoplasmic bridge. Structures resembling septate junctions are also found between siblings and between cells in aggregates. The evidence accumulated here suggests that cytokinesis in Kc dyads is incomplete, resulting in an intercellular pathway that may provide for the passage of a molecular or electrical signal that regulates subsequent mitosis.