Chick Embryo Neural Retina Research Articles

Specific factors influencing histotypic aggregation of chick embryo hepatocytes.

Conditions are described for the reproducible assay of substances affecting the in vitro rate of aggregation of isolated chick embryo hepatocytes. Two low molecular weight (less than 1000) fractions--one that promotes hepatocyte aggregation (HAP) and the other that inhibits this stimulation (HAI)--have been isolated and partially purified from adult chicken liver. One major active component of HAP was identified as taurine (2-aminoethanesulfonate). The presence of HAP during the entire time of assay was required for largest aggregate formation. HAP had no effect on aggregation of chick embryo neural retina, kidney, or heart cells. Our results and the fact that puromycin completely inhibits aggregate formation suggest that HAP and HAI influence the specific synthesis and interaction of membrane macromolecules involved in the aggregation process.

Studies on intercellular adhesion. I. Adhesion of neural retina cells to isotypic aggregates in serum free medium.

A quantitative assay for intercellular adhesion, which is a modification of the collecting aggregate assay (Roth et al., '71) is described. The use of mechanically dissociated single cells labeled with 3H-leucine, and of a gyratory shaker increased considerably the efficiency of cell collection. With chick embryo neural retina, it was shown that isotypic cell adhesion occurs in simple synthetic media and even in sodium chloride solution suggesting that divalent cations do not play a major role in adhesion of these cells. Cell collection in media without serum was not affected by metabolic or protein synthesis inhibitors. However, cell adhesion was temperature-dependent since no collection occurred at 4 degrees C but was maximal at 38 degrees C.

Studies on intercellular adhesion. II. Promoting effect of serum and proteins on adhesion of neural retina cells to isotypic aggregates.

The quantitative assay described in the preceding paper (Pessac et al., '77) was used to study the effects of serum and various proteins on isotypic adhesion of chick embryo neural retina cells. Fetal bovine, chicken, horse, rabbit and human sera promoted cell adhesion to the same extent. The same sera also enhanced isotypic adhesion of cells from other organs showing that the cell adhesion promoting activity of sera was not organ specific. Neural retina (NR) cell collection in serum supplemented medium was not modified by protein synthesis or metabolic inhibitors and was temperature dependent with a maximum at 38 degrees C. The higher temperature does not seem to be required for repair of the cell surface after dissociation, but for the process of adhesion itself. Various serum fractions and egg albumin showed a cell adhesion promoting activity similar to that of sera.

Cell interactions and the regulation of cholinergic enzymes during neural differentiation in vitro

Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rembencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates. The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and 10-day-old NR and OL cells show that the interactive capacities of the cells change with development.

Temporal changes in embryonal cell surface recognition.

Plasma membranes have been prepared from optic tectum and neural retina of chick embryos. These membranes specifically inhibit the aggregation of homotypic cells. Plasma membranes prepared from each of these regions obtained from chick embryos at 7,8, and 9 days of development show specificity for homotypic cells of the same age and cross-react weakly with homotypic cells of different ages. Thus cell-surface recognition changes with age of development, and most of the cells in each region have a common cell-surface recognition specificity. Tectal cells react weakly with retinal cell membranes, but no inhibition of retinal cell aggregation by tectal cells has been noted.

The fine structure of the reconstructed neural retina of chick embryos

ABSTRACT The fine structure of reconstructed neural retina formed from dissociated neural retinal cells of -day-old chick embryos on the chorio-allantoic membrane of chick embryos was examined with the electron microscope. Three nuclear layers (ganglion cell layer, inner and outer nuclear layers) and two fibrous layers (inner and outer plexiform layers) are found within the reconstructed retina. Both the outer and the inner limiting membranes of the reconstructed structure are constituted from the processes of differentiated Müller cells. The ganglion cell layer consists of two types of cell, though a typical ganglion cell with axonal process is not observed. Optic nerve fibres are not formed. Amacrine cells are recognized within the inner nuclear layer. Differentiation of the inner segment of the photoreceptor cell occurs, but not of the outer segment. Synaptic structures are recognized in the inner plexiform layer, but not in the outer plexiform layer.

Adhesive recognition and retinotectal specificity.

An assay has been developed to test the hypothesis that neurons from a limited area of the retina will adhere preferentially to that part of the optic tectum near their normal synaptic termini. The method measures the adherence of (32)P-labeled cell bodies from either the dorsal or ventral half of the neural retina of chick embryos to dorsal and ventral tectal halves. When a labeled, single-cell suspension is prepared from dorsal half-retina, more cells bind to the ventral half of the tectum. When the labeled cells are from ventral half-retina, more bind to dorsal half-tecta. This preferential adhesion mimics the retinotectal projection found in vivo. Dorsal retinal cells show this preference shortly after dissociation with crude trypsin, and maintain it for at least 9 hr. Ventral retinal cells, however, require incubation in nutrient medium after trypsinization in order to display this selectivity. Comparable results are obtained when the cell suspension is prepared from pigmented retina. The data support an interpretation of neuronal specificity dependent on cell-surface interactions and demonstrate a clear correlation between selective adhesion and biological function.

Utilization of L-glutamine in intercellular adhesion: ascites tumor and embryonic cells.

Abstract l -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 l -glutamine to aggregate. This effect is concentration dependent and the amino sugar, d -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- l -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of l -glutamine but not of d -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require l -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. l -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that l -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 l -glutamine.

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 microg/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.

An assay for intercellular adhesive specificity.

A modification of an assay for intercellular adhesive specificity is described. The method involves the collection of radioactively labeled cells by aggregates of the same (isotypic aggregates) or different (heterotypic aggregates) types of tissue and determination of the number of cells collected by liquid scintillation counting. The use of (32)P to label the tissues permitted a much more rapid estimation of cell collection than was obtained previously. With the use of chick embryo neural retina, liver, forebrain, pectoral muscle, and heart ventricle tissue, it was shown that isotypic was always greater than heterotypic collection. Labeled neural retina cell collection by neural retina aggregates was studied as a function of time, cell suspension density, aggregate diameter, temperature, and aggregate number. Neural retina aggregates were treated with certain enzymes in an attempt to determine whether specific changes on the surface of the aggregates would interfere with labeled neural retina cell collection. Of the various proteases and glycosidases tested, only beta-galactosidase rendered the surface more nonspecific.

Effect of culture media on self-organization and protein synthesis of neural retina cells

1.1. Seven-day-old chick embryo neural retina cells were grown in a totally chemically defined medium for 20 hr in rotation-mediated cultures.2.2. Supplementation of the medium with 5–10 mg/ml of crystalline bovine serum albumin enhanced the degree of aggregation and the morphologic appearance of the preparation.3.3. Small amounts of added protein had a marked stimulatory effect on the incorporation of glucosamine and glycine into the aggregates.4.4. At an albumin concentration of 5 mg/ml in MEM, protein synthesis was only slightly slower and the size of the aggregates were about the same as were obtained in serum containing control media.5.5. Glycine incorporation was more sensitive to the presence of macromolecules than glucosamine incorporation.

Light and electron microscope studies of cell sorting in combinations of chick embryo neural retina and retinal pigment epithelium.

A recent hypothesis, proposed by A. S. G. Curtis, asserts that the formation of homogeneous tissues within heterotypic aggregates occurs during the initial phases of cellular aggregation rather than by a process of cell sorting in mixed aggregates. The present studies of the early stages of aggregation of mixed suspensions of dissociated chick embryo pigmented retinal epithelial and neural retinal cells do not support Curtis' hypothesis. The early aggregates formed from these mixed cell suspensions are disordered mixtures of the two cell types. Establishment of homogeneous neural retinal and pigmented retinal epithelial tissues occurs much later in the aggregates. The hypothesis of Moscona that the process of formation of type-specific tissues in mixed aggregates requires a specificity of cell adhesion was examined by attempting to demonstrate cell contact specificity at the electron microscope level in heterotypic aggregates undergoing cell sorting. Specificity of cell contact was not observed. Instead, fine structural studies of cell contact interactions demonstrate that pigmented retinal epithelial cells have broad areas of cell contact and specialized contact junctions with neural retinal cells within the aggregates.

Protein synthesis and aggregation of embryonic cells

Dissociated 7-day chick embryo neural retina cells exhibited marked biosynthetic activity as measured by 14C-glucosamine and 3H-glycine incorporation. Glucosamine incorporation was more nearly directly related to the degree of aggregation than glycine incorporation. Several different labeled amino acids gave results very similar to glycine. Qualitatively, similar results were obtained using dissociated 7-day embryo liver and neural retina cells. Aggregation and incorporation of labeled glycine and glucosamine were inhibited by actinomycin D, puromycin, phleomycin, cycloheximide, borate, and periodate. Free hexosamines blocked aggregation and the incorporation of the amino acids and glucosamine by neural retina cells, but their acetylated derivatives and hexoses in similar concentrations did not. A number of amines, phenethyl alcohol, and EDTA depressed the incorporation of these precursors into macromolecules. Protein or some other macromolecules were required for maximal activity. Although the biosynthetic activity was dependent on the type of medium, all media showed an optimal protein concentration. Glycine predominantly labeled intracellular structures while glucosamine preferentially labeled the extracellular proteins. The distribution of the amino acid and glucosamine labels was dependent upon the protein concentration of the medium. Chick embryo extract was found unnecessary and occasionally inhibitory. When aggregates were dissociated the material liberated on dissociation had a greater specific activity than the whole aggregates. When the cells were fractionated into membranous and non-membanous components, the membrane material had a greater specific activity than the whole cells.