Formation of junctions and cell sorting in aggregates of chick and mouse cells

The frequency of desmosome formation was examined in aggregates of old cells, which form many junctions, combined with young cells, which form few. Cells of chick corneal epithelium and mouse epidermis, which can be distinguished morphologically, were combined. Desmosomes between these cell types are stable. Further, young cells make more desmosomes than they otherwise would on those surfaces adjoining old cells. Desmosomes increase in number in aggregates while cell sorting is occurring. Cells consistently sort, with those which form most desmosomes lying internally. Gap junctions and intermediate junctions are also present, but are uncommon. A carbohydrate cell-surface coat has regenerated by the time desmosome formation starts. The possible relation of desmosome formation to cell sorting is discussed.     

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.     

Control of desmosome formation in aggregating embryonic chick cells

Corneal epithelial cells have been used to study cell surface changes during cell aggregation. Tissue was taken from developmental stages in which desmosomes were forming rapidly. When corneal cells are dispersed, adjacent desmosome plaques are separated and single plaques are left on the cell surface. As cells aggregate, changes in the frequency of single plaques or of full desmosomes (double plaques) per micrometer of cell surface cross section can be followed. Single plaques are lost from the surface by endocytosis. Quantitative studies show a loss of single plaques beginning in the first hour of culture and formation of double plaques at 2 to 3 hr. In cells treated with cytochalasin B or D, single plaques are not lost during the first 2 hr and double plaques form with a higher frequency. Formation of double plaques is suppressed by actinomycin D, cycloheximide, and dinitrophenol. Thus desmosome formation requires de novo protein synthesis. In addition, inhibition of cell surface turnover by drugs which modify the cytoskeleton will enhance the rate at which desmosomes form.     

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.     

Brain histogenesis in vitro: Reconstruction of brain tissue from dissociated cells

Summary Comparative 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. This work was supported by United States Public Health Service research grant HD-01253 to Aron Moscona and by the Louis Block Fund of the University of Chicago.     

DO MORPHOGENETIC TISSUE REARRANGEMENTS REQUIRE ACTIVE CELL MOVEMENTS? : The Reversible Inhibition of Cell Sorting and Tissue Spreading by Cytochalasin B

Previous studies have indicated that cell sorting and tissue spreading are caused by cell combination-specific differences in intercellular adhesive energies, acting in a system of motile cells. We wished to determine whether these adhesive energies could drive cell rearrangements as well as guide them. Accordingly, aggregates of intermixed embryonic cells were cultured in solutions of the drug cytochalasin B (CCB) at a concentration shown to inhibit the locomotion of cells on a solid surface. In addition, spherical aggregates of several kinds were cultured in mutual contact under similar conditions. Both cell sorting and tissue spreading were found to be inhibited. The prompt release of this inhibition upon removal of the CCB showed that the inhibited cells were not merely injured. Moreover, aggregation experiments showed that CCB did not prevent cells of several kinds from initiating mutual adhesions. In fact, heart cell aggregation was enhanced by CCB. We conclude that interfacial forces, originating outside the cell, act together with forces originating inside it in bringing about the morphogenetic movements of cell sorting and tissue spreading. We propose the term "cooperative cell locomotion" to describe translational movements of cells arising from such a combination of intrinsic and extrinsic forces.     

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Light-induced Polysome Formation in Etiolated Leaves: Kinetics of Inhibition by Antibiotics

Red light-induced, far red light-reversible increase in etiolated bean (Phaseolus vulgaris, var. Asgrow Valentine) leaf polyribosomes was shown to be sensitive to actinomycin D, cycloheximide, and rifampicin inhibition. Actinomycin prevented response to red light if administered simultaneously with a 10-minute illumination, had no immediate effect if given 2 hours after illumination, but was again rapidly inhibitory at 4 and 6 hours. The effects of actinomycin and far red light were more than additive.     

LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.     

Two candidate G1 polypeptides in chick embryo fibroblasts

Resting cultures of primary chick embryo cells have been labeled with ³H-leucine for the first 2 or 3 hours after feeding basal medium or basal medium supplemented with 10% dialyzed serum. The labeling patterns of the ³H-polypeptides of the soluble cell fraction have been compared by fluorography of two-dimensional gels. Large and consistent differences are seen in only three of the more than 900 spots that can visualized. This report concerns two of the three spots. The ³H contents of the two polypeptides (41,000 daltons, pI 7.1, designated 41–7.1, and 34,000 daltons, pI 6.2, designated 34–6.2) are increased by serum by about ten-fold. The highly selective effect of serum on the labeling of the two spots does not appear to be an artifact related to the extractability, solubility, or state of aggregation of the polypeptides. The radio-intensities of both polypeptides decrease markedly when the cells are labeled later than 3 hours after “shift-up”. Drugs that inhibit RNA synthesis and are known to stop the progression of the chick cells through the G1 period, camptothecin, cordycepin and 5,6-dichloro-β-D-ribofuranosylbenzimidazole, depress, with great specificity, the enhanced labeling of polypeptide 41–7.1 in the stimulated cells, and all but camptothecin have a similar action on polypeptide 34–6.2. A high level of actinomycin D (10 μg/ml), but neither a low level of the drug (0.02 μg/ml) nor 5-fluorouridine prevents the increased labeling of the two polypeptides in serum-fed cells. That 5-fluorouridine enters the chick cells and is converted to its active form is shown by the inhibition of the processing of pre-ribosomal RNA. The observations with the RNA inhibitors are at least consistent with the conclusions that the enhanced labeling of the two sports results from increased rates of synthesis of the polypeptïdes that depend upon mRNA production but not on the formation of ribosomal RNAs, and that the polypeptides play a role in the regulation of DNA replication in the chick cells.     

Induction of differentiation in cultured mouse neuroblastoma cells by N-methyl-N'-nitro-N-nitrosoguanidine.

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent mutagen and carcinogen, induces differentiation uniquely in N-18 mouse neuroblastoma cells, when compared with that of dibutyryl cyclic adenosine monophosphate. After treatment with 10 μM MNNG for only 2 h, RNA and protein synthesis are stimulated together with neurite formation, while DNA synthesis and growth of the cells are inhibited indefinitely. Induction of neurite formation by MNNG is irreversible, being inhibited by actinomycin D or cycloheximide but recovering after withdrawal of these inhibitors.     

Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule

Nr-CAM is a membrane glycoprotein that is expressed on neurons. It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region. We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule. To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence. After transfection, L cells expressed Nr-CAM on their surface and aggregated. Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments. To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Escherichia coli. Addition of FGTNr to the transfected cells blocked their aggregation. Further analysis using a combination of cell aggregation assays, binding of cells to FGTNr-coated substrates, aggregation of FGTNr-coated Covaspheres and binding of FGTNr-coated Covaspheres to FGTNr-coated substrates revealed that Nr-CAM mediates two types of cell interactions: a homophilic, divalent cation-independent binding, and a heterophilic, divalent cation-dependent binding. Homophilic binding was demonstrated between transfected L cells, between chick embryo brain cells and FGTNr, and between Covaspheres to which FGTNr was covalently attached. Heterophilic binding was shown to occur between transfected and untransfected L cells, and between FGTNr and primary chick embryo fibroblasts; in all cases, it was dependent on the presence of either calcium or magnesium. Primary chick embryo glia or a human glial cell line did not bind to FGTNr-coated substrates. The results indicate that Nr-CAM is a cell adhesion molecule of the nervous system that can bind by two distinct mechanisms, a homophilic mechanism that can mediate interactions between neurons and a heterophilic mechanism that can mediate binding between neurons and other cells such as fibroblasts.     

Protein content and enzyme levels of cultured chick embryo cells treated with camptothecin and actinomycin D.

The alkaloid camptothecin uncouples the growth and adivision of chick embryo cells. At a moderate dose (0.5 microgram/ml) it inhibits the incorporation of thymidine but not of uridine and leucine and the cell protein content increases and reaches twice that of control after 4 days of treatment. Twelve hours after addition of the drug, the activities per cell of the mitochondrial enzymes poly A hydrolase (EC 3.1. 4.21), cytochrome c oxidase (EC 1.9.3.1), and succinate dehydrogenase (EC 1.3.99.1) are greater than that of the control and keep increasing for at least 96 H. The increase in the activities of the mitochondrial enzymes precede that of NADPH-cytochrome c reductase (EC 1.6.2.4) and cytidine triphosphatase (EC 3.6.1.15), which are microsomal and plasma membranes enzymes respectively. Actinomycin D (0.01 microgram/ml) also inhibits the multiplication of the chick cells and the synthesis of DNA. The protein content of the actinomycin D treated cells decreases to 70% of the control by day 2. Nevertheless, the activities of the mitochondrial enzymes increase over that of the control but to a smaller extent that with camptothecin. The activities of the enzymes of the other organelles are not stimulated. Camptothecin at a higher dose (5.0 microgram/ml) induces effects similar to those of actinomycin D.     

Cell sorting and chondrogenic aggregate formation in micromass culture

A fundamental feature of cartilage differentiation in the developing limb is the formation of a prechondrogenic cell condensation. An apparently similar process of prechondrogenic cell aggregation occurs in micromass cultures of limb bud mesenchyme with the formation of cellular aggregates which often differentiate into cartilage nodules. We have investigated the process of aggregate formation in micromass culture using chimaeric mixtures of potentially chondrogenic and nonchondrogenic cell types. Two systems were studied: mixtures of distal and proximal limb mesenchyme cells and mixtures of distal limb cells with avian tendon fibroblasts. In both cases cultures of varying proportions of each cell type have been prepared. The results demonstrate that aggregate formation in vitro is the consequence of a cell sorting process which can involve prechondrogenic cells of widely different spatial origins within the developing limb. This contrasts with in vivo prechondrogenic condensation in which there is no evidence of cell sorting (Searls, R.L. (1967), J. Exp. Zool. 166, 39-50). However, our findings do indicate that cell surface differences occur in apparently undifferentiated limb mesenchyme. The results also suggest that mesenchymal cell aggregates must achieve a threshold size before chondrogenesis can proceed. In addition, the results show that under some culture conditions nonchondrogenic cells will form aggregates.     

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.     

An ultrastructural study of adhesive junctions in reaggregates of unincubated chick embryos.

Cell suspensions obtained by the dissociation of unincubated chick embryo blastoderms were allowed to reaggregate on a gyratory shaker for 24-48 hours. The reaggregates which form during this period consist of an inner phase of tightly packed cohesive cells surrounded by an external phase of loosely packed cells. This sorted out arrangement achieves its definitive form between 24 and 48 hours of rotation culture. It was determined that the external phase consists of primitive ectoderm and that the internal phase consists of primitive endoderm. Both 24- and 48-hour reaggregates were examined in the electron microscope and observations were directed to areas of close membrane apposition between cells. In 48-hour reaggregates, primitive endoderm cells were joined by many specialized junctions (desmosomes). The formation of desmosomes in reaggregates of dissociated unincubated chick embryo cells was correlated with the sorting out process.     

Can retinal adhesion mechanisms determine cell-sorting patterns: a test of the differential adhesion hypothesis

Embryonic chick neural retina cells possess two classes of adhesion mechanism, one Ca2+-independent, one Ca2+-dependent, responsible for short-term cell aggregation. This study investigates the role of these mechanisms in the long-term cell sorting potentially relevant to in vivo histogenesis. Retina cells are prepared either with both (E cells) or with only one mechanism (TC cells, CD; LTE cells, CI), respectively. The two types of cell preparations are differentially labelled using fluorescein or rhodamine isothiocyanate, mixed and allowed to aggregate in the presence or absence of cycloheximide at 0.5 microgram ml-1 to retard metabolic recovery of the removed adhesive mechanism. When observed by fluorescence and phase-contrast microscopy, the aggregates formed in cycloheximide show cell sorting, the cells with both mechanisms assuming a more interior position relative to those with a single adhesion mechanism. In parallel hanging-drop experiments, preformed aggregates of cells with a single adhesion mechanism are seen to spread upon aggregates of cells with both mechanisms. No sorting occurs amongst cells from a given stage prepared using any single dissociation protocol. The observed cell sorting would thus seem to derive exclusively from differential cell adhesiveness dependent upon the different dissociation conditions and maintained in the presence of cycloheximide. The experiments support the hypothesis that the dual CI and CD adhesion mechanisms in question can play a central role in governing cell-sorting behaviour during normal histogenesis.