A.A. Moscona

Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular systems

Abstract 1. 1. Evidence is presented for the existence of specific factors associated with the cell surface and intercellular spaces which function as cell ligands and mediate histogenetic attachment and aggregation of sponge cells. 2. 2. Observations on interspecific grafts in sponges determined that within the tissues the cells display the same affinities and sorting-out reactions as they do when aggregating in culture and which also correspond to the specificity of the effects of the aggregation-enhancing ligands isolated from the cells. 3. 3. It was demonstrated that the ligand preparation does not function by repairing “nonspecific damage” due to cell dissociation or by inactivating a hypothetical inhibitor of aggregation released by dissociation. The evidence shows that the ligands are directly involved in the mechanism of cell attachment and aggregation. 4. 4. The ligands bind more rapidly to unwashed than to washed (i.e., depleted of ligands) cells. The functional uptake of the ligands by cell surfaces has been demonstrated by a variety of tests; these included: (a) the use of formalin-killed cells, which respond to the specific aggregation-enhancing effect of the ligand preparations; (b) comparisons between the response of washed and unwashed cells; (c) recovery of ligands from cell aggregates; (d) serological tests. 5. 5. It was demonstrated that the capacity of cells to aggregate is destroyed by heating and so is the characteristic biological activity of the isolated ligands. Heat-inactivated ligands react with normal ligands to form an inactive complex. 6. 6. The activity of the ligands was not destroyed by prolonged treatment with the following enzymes: DNase, RNase, trypsin, collagenase, hyaluronidase, lysozyme; it was destroyed by α-amylase and by pronase. 7. 7. Pronase was found to dissociate sponge tissues into single-cell suspensions, thus providing an experimental situation comparable to trypsin-dissociated embryonic vertebrate cells. The pronase-dissociated cells formed aggregates after a lag period of approximately 4 hours during which they were not receptive to exogenous ligands. Some of the conclusions based on preceding experiments were tested and confirmed on the pronase-dissociated cells. 8. 8. Antiserum prepared against the purified ligand preparation from Microciona cells rapidly agglutinated these cells but not Haliclona cells; the antigenic specificities of the ligands reflected the aggregative affinities of these cells. 9. 9. The results are discussed in the general context of problems of cell contact and “recognition” in the formation of multicellular systems, and in relation to the ECM hypothesis.

Embryonic and neoplastic cell surfaces: availability of receptors for concanavalin A and wheat germ agglutinin.

Embryonic tissue cells dissociated with ethylenediaminetetraacetate are readily agglutinated by the carbohydrate-binding protein concanavalin A. In this property, they resemble transformed, neoplastic cells; and they differ from untransformed adult cells, which are agglutinated by concanavalin A only after their receptors are unmasked by proteolytic treatment. Receptor sites for wheat germ agglutinin are also present on the surface of embryonic cells, but in a masked form, as on untransformed adult culture cells; they can be unmasked by treatment of the cells with trypsin. Concanavalin A binding sites on embryonic cells may function in cell contact and cell organization during embryonic morphogenesis and differentiation and later become masked in adult cells. The unmasking of these sites in neoplastic cells may represent a return, in this respect, to a condition resembling that of embryonic cells and may be related to cell mobility associated with infiltration and metastasis.

Reconstruction of brain tissue from cell suspensions. I. Aggregation patterns of cells dissociated from different regions of the developing brain.

Abstract Cells dissociated from different brain areas of chick and mouse embryos (cerebrum, diencephalon, optic tectum, or corpora quadrigemina, cerebellum, and medulla) reconstructed in vitro aggregates which were diagnostically characteristic in size and shape for their region of origin; in addition, cells from each area exhibited a distinctive age profile of aggregative behavior which reflected their developmental schedule. Mouse cell aggregates from all brain regions tested were consistently larger than aggregates of cells from corresponding chick brain regions; mouse cerebrum cells formed especially massive aggregates in contrast to small aggregates characteristic of chick cerebrum cells. Coaggregation of suspensions of mouse cerebrum cells with chick cells from various brain areas, or from nonnervous tissues resulted in bispecific aggregates which were generally intermediate in size between typical aggregates of each of the component cell populations. The size of these coaggregates and the extent of internal sorting out of cell types was closely related to the degree of homology between the commingled cell populations. The implications of the findings are discussed with reference to fundamental mechanisms governing regional brain differentiation and specificity of neuronal associations.

Electron microscopic analysis of aggregation of embryonic cells: the structure and differentiation of aggregates of neural retina cells*

Summary The fine structure of 24-hour aggregates of cells dissociated from neural retinas of 7-, 10-, and 14-day chick embryos was examined. Two basic structural patterns of aggregation of these cells were described: (1) lumicentric rosette , typical of aggregates of 7-day cells; (2) axocentric rosette , typical of aggregates of 14-day cells. Aggregates of 10-day cells contained both kinds of rosettes. The composition and morphogenesis of these cellular configurations are expressions of age-dependent differences in the donor tissue and reflect primarily the types of cells and intercellular junctions that prevail in the retina at the time of dissociation. The formation of rosettes in retina cell aggregates is discussed in the general context of mechanisms of cell specificity, sorting out, and histogenetic association. The results support the hypothesis that qualitative differences in the properties of cell surface consituents are decisive in the sorting out and specific associations of cells. Aggregates of 7-day retina cells undergo in long-term culture advanced differentiation resulting in the development of plexiform layer, synaptic junctions, outer limiting membrane, and photoreceptor cells.

Reconstruction of brain tissue from cell suspensions: II. Specific enhancement of aggregation of embryonic cerebral cells by supernatant from homologous cell cultures☆

Abstract A cell-free supernatant from short-term monolayer cultures of 14-day embryonic mouse cerebrum cells markedly and specifically enhanced the histotypic aggregation of suspensions of dissociated cerebrum cells from mouse and from chick embryos. It did not have this effect on cells from other brain regions, or from nonnervous tissues from either species; its effect was somewhat greater on the aggregation of mouse cerebrum cells than of chick cerebrum cells. Its action was concentration-dependent and critically dependent on the embryonic age of the donor cells and the aggregating cells. The results are discussed in terms of cell surface interactions and their implications for neural histogenesis and differentiation.

Cell Aggregation: Its Enhancement by a Supernatant from Cultures of Homologous Cells

A supernatant medium has been prepared from living embryonic neural retina cells which specifically promotes their histogenetic aggregation. Its function is dependent upon at least two experimentally separable steps: selective uptake and functional utilization.

Induction of Glutamine Synthetase in Embryonic Retina: Its Dependence on Cell Interactions

A relation between enzyme induction in embryonic cells and cellular organization is indicated by the finding that the levels of glutamine synthetase induced by hydrocortisone in the embryonic neural retina in vitro are dependent on the associations between the retina cells. Intact retina tissue, aggregates of dissociated cells, and cells in monolayer culture showed a decreasing response, in this order, to glutamine synthetase induction. With time of culture, the enzyme activity continued to rise in the intact retina and in cell aggregates, but activity declined in monolayer cultures even though the inducer was continuously present. Dispersed cells cultured in monolayer without the inducer showed after 24 hours a loss of inducibility which could not be reversed by reaggregating such modified cells but could be prevented by maintaining the freshly dispersed cells at a low temperature.

Enzyme induction by corticosteroids in embryonic cells: steroid structure and inductive effect.

Glutamine synthetase in the developing retina of the chick embryo can be induced to increase by certain corticosteroids. The inductive effectiveness of various natural corticosteroids has been examined in organ cultures of embryonic retina and correlated with specific groupings on the steroid molecules.

The induction of glutamine synthetase in cell aggregates of embryonic neural retina: Correlations with differentiation and multicellular organization

Abstract A rapid increase in the synthesis and accumulation of the enzyme glutamine synthetase (GS) in the neural retina of the chick embryo characterizes the functional differentiation and maturation of this tissue. A precocious increase of GS can be induced in the embryonic retina by hydrocortisone and related corticosteroids. This paper presents evidence that the responsiveness of neural retina cells to GS induction by the hormonal inducer is dependent on histotypic associations and organization. This was demonstrated, using retina from embryos of different ages, by comparing GS induction in cultures of intact retina tissue with that in aggregates of retina cells and in monolayer cultures of retina cells. At all embryonic ages tested, induced GS activities were always highest in intact retina tissue, intermediate in cell aggregates, and lowest in cell monolayers. While in the intact retina GS inducibility increased with embryonic age (as judged by enzyme levels attained after 24 hours of induction), in cell aggregates it declined with embryonic age; thus, GS activities in aggregates of retina cells from 10-day embryos were higher than aggregates of cells from older embryos. This reduction of GS inducibility in cell aggregates coincided with the age-dependent decline in cell aggregability, i.e., with the fact that suspensions of retina cells from older embryos formed smaller aggregates than younger cells under the same experimental conditions, and (as known from other work) they failed to reestablish in the aggregates patterns or organization similar to those found in aggregates of younger neuroretinal cells. It was demonstrated that the propensity of dissociated neuroretinal cells for histotypic reaggregation reflected inversely the state of differentiation of the cells at the time of their dispersion: the more differentiated were the cells at the time of dissociation, the lesser was their aggregability and the lower was their GS inducibility. The effects of embryonic age and of retina differentiation on the aggregation of dissociated retina cells were experimentally separated: using 12-day embryonic retina it was shown that cells from its least differentiated area formed larger and more inducible aggregates, while cells from the most differentiated formed smaller, much less inducible aggregates. It was established that the size of cell aggregates, as such, is not the decisive factor in GS inducibility: by increasing the speed of gyration of flasks with cell suspensions, the size of aggregates of 10-day retina cells were reduced to that of aggregates of 14-day retina cells, without significantly lowering their inducibility. It was possible to demonstrate that GS induction in older retina was no more sensitive to damage by trypsin than induction in younger retina, and therefore, trypsinization in itself could not account for the lower inducibility of aggregates of older retina cells. The evidence, as a whole, favors the interpretation that GS inducibility of neuroretinal cell aggregates is dependent on the multicellular organization of the cells, i.e., on the ability of the dispersed cells to become reassociated and reorganized in aggregates in a manner conducive to responsiveness to the inducer of GS. In coaggregates of 16-day embryonic retina cells with 10-day retina cells, GS inducibility of the 10-day cells was reduced, presumably due to interference by the older cells with the reorganization of the younger cells. The nature of the cellular associations and organization of retina cells in aggregates specifically required for GS induction are unknown; the general implications of the reported findings to regulatory mechanisms of enzyme induction in embryonic cells are briefly discussed.

Synthesis of experimentally induced glutamine synthetase (glutamotransferase activity) in embryonic chick retina in vitro

Abstract The phenomenon of precocious appearance and rapid enhancement of glutamotransferase activity of embryonic chick neural retina in response to explanation in vitro has been subjected to more detailed scrutiny. On the basis of cofactor requirements, pH optima, response to specific inhibitors, and ratio of transferase to synthetase activity, the enzyme undergoing change was classified as a glutamine synthetase. Tests for the presence of enzyme activators and/or inhibitors undergoing change during increase in enzyme activity were negative, suggesting that increase in activity was due to synthesis of new enzyme. This was substantiated by the finding that low levels of puromycin blocked completely and reversibly increases in enzyme activity under culture conditions; and that actinomycin D similarly blocked such increases irreversibly. Preexisting levels of activity appeared stable in the presence of either inhibitor. These data were interpreted to mean that enhancement of glutamotransferase activity which occurs in culture is due to synthesis of new enzyme; it also suggested a lability of the RNA involved and consequently a rather direct genomic control over the rate of synthesis of the enzyme in early ontogenesis. Pigmented epithelium of embryonic eye, vitreous humor, and embryo extract showed no effect on the increase in glutamine synthetase activity in the explanted retina; this was interpreted as diminishing but not excluding the possibility that a stable, diffusible systemic factor is responsible for control of early ontogenesis of this enzyme in the embryo. Glutamine, glutamate, and γ-aminobutyrate were found to lower synthesis of the enzyme in culture when added to the medium, but none of these amino acids appeared capable of total repression at concentrations approaching physiological values. Glutathione (oxidized and reduced), asparagine, aspartate, and glucose all had no demonstrable affect upon the growth of the enzyme in culture.