Beatrice B. Garber

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.

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.

Selective cell association of catecholamine neurons in brain aggregates in vitro

Brain tissues (aggregates) were reconstructed in vitro from dissociated single cell suspensions derived from 12- to 18-day embryonic mouse midbrain containing the substantia nigra. The application of the Falck-Hillarp histofluorescence method to these cell systems allows the visualization and identification of this specific population of developing catecholamine (CA) neurons during their reassembly, differentiation and histogenetic patterning in vitro. CA neurons are unselectively distributed in the initial dissociated cell suspension and in the reaggregating tissue up to 24 h. By 48 h the CA neurons have selectively associated into small clusters which further coalesce into a thick and elongated band along one margin of the aggregate by 96 h. This structure is similar in organization to the morphology exhibited by substantia nigra neurons in situ during their migratory phase in normal development. In addition, the differentiated neurons observed in the later aggregates appear to produce normal processes. Catecholamine analyses show a significant increase in dopamine and noradrenaline levels during the process of differentiation and histogenetic organization in vitro.

Self-assembly of cortical plate cells in vitro within embronic mouse cerebral aggregates. Golgi and electron microscopic analysis

Mouse isocortical cells were dissociated at 18 days of embryonic development and were reaggregated in vitro by rotation in gyratory incubator shaker. The internal organization of the resulting aggregates was studied by conventional light microscopy, Golgi impregnation, and electron microscopy, establishing the following pattern of reassembly: (1) the predominant cell type in the aggregates was the pyramidal neuron; (2) each of these pyramidal neurons tended to orient its apical dendrite toward the surface of the aggregate; and (3) in larger aggregates (diameter > 600 microns) there was prominent parallel alignment of pyramidal cell apical dendrites. These characteristics resulted in an in vitro reconstruction of the major features of isocortex observed in situ, including formation of a superficial, rather acellular plexiform layer. Reconstruction of isocortical architecture appeared to take place independently of either a germinal epithelium, a radial glial framework, or an outer mesenchymal scaffold. Analysis of the events occurring during aggregate formation suggests that intrinsic cellular information accounts for the expression of basic pyramidal cell morphology. However, dendritic orientation and alignment are most likely determined by cell-cell interactions dependent upon specific cell surface recognition properties, as well as by geometric restraints imposed by the spherical or cylindrical shape of the aggregates.

Inhibition by glucosamine of aggregation of dissociated embryonic cells

Abstract d -Glucosamine free base inhibited the normal adhesion and histogenetic associations of trypsin-dissociated 8- to 10-day chick neural retina and liver cells in vitro. The effects were dependent on concentration and temperature. Cells cultured with N-acetylglucosamine formed normal aggregates. The suggestion is made that d -glucosamine free base acts at the cell surface either by blocking the available sites normally occupied by N-acetylglucosamine or by interfering with glucose metabolism and biosynthesis of cell surface materials necessary for the adhesive mechanisms of cell aggregation. Simple carbohydrate units, such as N-acetylglucosamine, may participate in activities at the cell surface which provide the basis for histotypic aggregation.

Quantitative studies on the dependence of cell morphology and motility upon the fine structure of the medium in tissue culture.

Abstract 1. 1. Standard tissue cultures of chick heart fibrocytes were grown in plasma clots whose physical constitution was systematically altered, either by varying the pH at the time of clotting through a range of pH 8.0–5.6, or by varying the plasma concentration through a range of 10–90 per cent plasma. Other conditions were kept constant. 2. 2. Measurements of 4 759 cells plotted against plasma concentration or pH produced smooth curves, showing transitional responses. 3. 3. With increasing plasma concentration and decreasing pH, there is a corresponding increase in the percentage of spindle-shaped cells, a decrease in the number of pseudopodia per cell, an increase in the ratio of cell length to cell width, and an increase in the ratio of nuclear length to nuclear width. 4. 4. The rate of cell migration increases as the plasma concentration increases or as the pH decreases, but reaches a constant level above 50 per cent plasma concentration. 5. 5. An interpretation of the results in terms of the properties of the fibrin framework and of the reactions of the cells to it has been given on a previous occasion (25).

Sorting out of coaggregated brain cell types mediated by specific cell recognition factors in vitro: I. Experimental histogenesis and quantitative analysis

Abstract Two region-specific cell recognition factors (CRF, cerebrum specific, and OTF, optic tectum specific), which markedly increased the size of aggregates formed by dissociated cells of homologous type, have also been shown to promote selective association of homologous cells within coaggregates formed from commingled cerebrum and tectum cell suspensions. The resulting patterns of cell sorting were examined quantitatively. Nearest-neighbor analysis of cell associations within equatorial histological sections of these coaggregates evaluated the enhanced association of regionally homologous brain cell types in response to each specific brain cell recognition factor. The statistical analysis also examined a characteristic positional segregation of cerebrum and tectum cells in the sorted out coaggregates: tectum cells tended to segregate toward the periphery of the coaggregate while cerebrum cells massed in the core. These data demonstrate that the cells of each population associated preferentially with one another in direct response to their homologous cell recognition factor. The application of this quantitative nearest-neighbor analysis for making assessments of associative affinities between neural cell populations is evaluated and implications of using this analysis for studying regional and functional homology between nerve cells during development are discussed.

Brain histogenesis in vitro: reconstruction of brain tissue from dissociated cells.

SummaryComparative studies of the aggregative behavior of cells dissociated from different areas of embryonic chick and mouse brains show that each of the regionally differentiated lobes (cerebrum, optic tectum, and cerebellum), and the stem areas (diencephalon and medulla), form characteristic aggregates distinctive in size and shape. Bispecific co-aggregates are produced by commingling dissociated mouse cerebrum cells with chick cells from various brain regions, or from non-nervous tissues; the size of these co-aggregates and the extent of internal sorting out of cell types is closely related to the degree of homology between the interacting cell populations, e.g. co-aggregates of the closely homologous mouse and chick cerebral cell types contain homogeneous tissue fabrics of intermingled mouse and chick cells.Cell surface constituents involved in selective recognition and association of nerve cells were sought and cell-free supernatant preparations were obtained from short-term monolayer cultures of embryonic cerebrum cells (of either mouse or chick origin) which caused a striking, specific enhancement of aggregation of homologous cerebrum cells. These materials had no such effect on heterologous tissues tested: optic tectum, cerebellum, medulla, neural retina, liver, kidney or limb bud. These findings are discussed in relation to control mechanisms governing normal brain histogenesis and to the specificity of neural associations.

HISTOGENESIS OF DEVELOPING DOPAMINE NEURONS IN CELL AGGREGATES IN VITRO

ABSTRACT Self-assembly of dissociated embryonic central dopamine neurons in the presence or absence of normal target cells in vitro shows that a) substantia nigra neurons in aggregates formed from mouse midbrain cell suspensions retain the capacity for selective association with one another, segregate into dense bands, and develop increasing levels of dopamine with time in culture; b) neostriatal cells aggregated alone contain no dopamine; c) coaggregates formed by commingled substantia nigra and neostriatal cells exhibit no segregation of dopamine neurons, but contain abundant catecholamine fluorescent varicosities and twice the amount of dopamine formed in aggregates of substantia nigra cells alone.

Immunochemical analysis of fibronectin using monoclonal antibodies

Cloned hybrid cell lines secreting antibodies directed against human plasma fibronectin were prepared according to the methods of Kohler and Milstein (Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 and (1976) Eur. J. Immunol. 6, 511-519). The specificity of each monoclonal antibody for fibronectin was established from autoradiograms of radioimmunoprecipitates following SDS-polyacrylamide gel electrophoresis. The monoclonal antibodies were reactive with both native and SDS-denatured fibronectin. Ascites fluids obtained from infected isogenic mice precipitated 85-95% of the 125I-labelled fibronectin radioactivity in indirect radioimmunoprecipitation tests. Localization of specific epitopes to restricted regions of the fibronectin molecule was carried out by monitoring monoclonal antibody binding to proteolytic fragments. Of the five monoclonal antibodies analyzed in this study, three recognized determinants which resided in the terminal 35 kDa region of the fibronectin monomer. Furthermore, these epitopes were localized to fragments as small as 20 kDa. Competition studies carried out using plasma fibronectins isolated from different species revealed that three monoclonal antibodies recognized sites which were relatively conserved, while two monoclonal antibodies recognized epitopic sequences which were unique to the human protein. The corresponding anti-fibronectin serum also demonstrated discriminatory capabilities. Immunofluorescent analysis of human fibroblasts grown in vitro demonstrated that all the monoclonal antibodies tested were reactive with pericellular fibronectin.