Equilibrium-driven mechanism for preferential adhesion between chick embryo cells

The experiments presented here confirm the hypothesis according to which, in our experimental system of differential cell adhesion (where we studied the kinetics of the earliest period of adhesion of a suspension of chick embryo neuroblasts to layers of astroblasts or fibroblasts), the mechanism of adhesion appears to consist of two steps, the first of which is a short-term reversible phase corresponding to a binding equilibrium. In fact, adhesion of neuroblasts to each of the two cell layers occurs according to kinetic constants and attains levels which are characteristic for each of the two adhesion systems. In both systems, neuroblasts that have not adhered at equilibrium are able to adhere if inoculated over a fresh cell layer of the same type, as they do during the first inoculation; conversely, neuroblasts that have adhered to a cell layer can be made to de-adhere by substituting cell-free fresh medium to the inoculation medium containing non-adhering neuroblasts. This shows that, as predicted for a reversible equilibrium system, removal of adhering neuroblasts from the system at equilibrium provokes adhesion, and removal of non-adhered neuroblasts provokes de-adhesion. Furthermore the level of adhesion at equilibrium is, in all cases, the same. The reversibility of adhesion, which is almost quantitative during the onset of the equilibrium, gradually decreases with time, indicating the presence of a process of irreversible attachment between cells after the first reversible step. The developmental implications of the complete sequential mechanisms are briefly discussed.     

Behaviour of neuroblasts in the presence of glial cells, fibroblasts and meningeal cells in culture

Dissociated nerve cells from 7-day-old chick embryo cerebral hemispheres were cultivated on plastic surfaces, astroblast layers, fibroblast layers and meningeal cell layers. The cell suspensions obtained by mechanical dissociation and plated on these layers contained primarily neuroblasts. The neuroblasts cultured on astroblast layers behaved differently from those cultured on fibroblast or on meningeal cell layers. They adhered within 2 h to the preformed astroblast monolayers and remained scattered over it. In contrast, in the two other cases, the neuroblasts formed floating aggregates which adhered to the layers only after 24 h. Neuroblasts behaved on monolayers made of fibroblasts or meningeal cells as on plastic surfaces.The neuronal cells grown on astroblast layers were much more differentiated than those plated on plastic or on the two other layers studied. After 2–3 weeks of culture the neurons were large and the fibres were longer, thicker and more ramified. However, the fibroblast and the meningeal cells enhanced slightly the growth of the neuroblasts relative to plastic surfaces. These results support the possibility of specific interactions between astroblasts and neuroblasts.     

Cell kinetic studies of chick brain cells in culture: an autoradiographic study with [3H]- and [14C]-thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [3H]- and/or [14C]-thymidine. The dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. The labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [3H]- and [14C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. The cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). The results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.     

Cell Kinetic Studies of Chick Brain Cells In Culture: an Autoradiographic Study With [3H]- and [14C]-Thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [³H]- and/or [¹⁴C]-thymidine. the dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. the labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [³H]- and [¹⁴C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. the cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). the results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro

When CNS precursor cells purified from the external germinal layer of the early postnatal mouse cerebellum are cultured in cellular reaggregates, DNA synthesis increased 10-fold above that of cells dispersed in a monolayer or embedded in a collagen matrix. Dividing precursor cells gave rise to neurons immunopositive for the neural antigens N-CAM, L1, and TAG-1, but not to astroglial cells immunopositive for glial filament protein. Moreover, proliferating precursor cells did not generate other types of cerebellar neurons, as judged by the lack of expression of glutamic acid decarboxylase, the synthetic enzyme for gamma-amino-n-butyric acid. By contrast, the addition of astroglial cells, or astroglial cell membranes, to cellular reaggregates of granule cell neuroblasts arrested precursor cell DNA synthesis in a dose-dependent manner. These results suggest that homotypic contact interactions among CNS neural progenitors control precursor cell proliferation and fate in generative zones of developing brain.     

Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface--cell surface interactions

Neurite outgrowth of C 1300 neuroblastoma cells, which were dispersed from adherent cultures or grown in suspension, was studied on different protein-coated surfaces. Of 29 different surface structures studied, including surfaces treated with various fibronectins, lectins, glycosidases, or glycosyltransferases capable of stimulating fibroblast spreading, only the surfaces coated with plasma fibronectin or with a protein mixture secreted by C6 glioma cells displayed an extensive activity in the sprouting assay. Neurite outgrowth was inhibited by brain gangliosides and by colominic acid (a sialic acid polymer). A 50% inhibition of neurite outgrowth of N18 neuroblasts induced by the glioma cell proteins was observed at the following approximate concentration: 100 microM (0.2 mg/ml) GD1A ganglioside, 20 microM (0.04 mg/ml) GT1B ganglioside, and 5 mg/ml colominic acid. Specificity of inhibition was suggested by the finding that a few polyanionic substances tested were not inhibitory in the sprouting assay, and that the type of gangliosides inhibiting sprouting were found to be major sialoglycolipids of the neuroblasts. A hypothesis is discussed, according to which neurite outgrowth of neuroblasts is stimulated by adhesion involving interactions of the adhesion-mediating protein with cell surface carbohydrates characteristic of brain gangliosides.     

Selective adhesion of mast cells to tracheal epithelial cells in vitro

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.     

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.     

RNA biosynthesis during differentiation of various cell types of chicken embryo in cerebral hemispheres

Summary During the development of the chick embryo from the 6th to the 15th day of incubation, the cell types in cerebral hemispheres undergo differentiation. During this period the indifferent cells of the germinal layer migrate away from the neural cavity to form the mantle layer. These cells differentiate into neuroblasts and spongioblasts.RNA biosynthesis is very active in the cells of the germmal layer of the young embryos. From the 10th day on, it decreased becoming very weak in the 15-days old embryos. The RNA is stored in the nucleus and its passage to cytoplasm is very slow.In 6 and 8-days old embryos the RNA biosynthesis in the mantle layer is not very active but increases during embryonic development as the germinal cells differentiate. The biosynthesis is always more intense in the neuroblasts than in the spongioblasts. The RNA is stored in the nucleus and its passage to cytoplasm is slow in the young neuroblasts and the spongioblasts. The formation of Nissl bodies in neuroblasts and the differentiation of neuroblasts into neurons, which corresponds to the development of axons and dendrites, both are accompanied by an activation of the RNA passage from the nucleus into the cytoplasm.With the technical assistance of A. Brossard.     

Rat brain glial cells in culture: Effects of brain extracts on the development of oligodendroglia-like cells

Cells dissociated from brains of newborn rats and grown on plastic surfaces develop into a glial culture, composed of at least three morphologically different cell types. The predominating cell type consists of astroglial cells, which form a monolayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells scattered upon the astroglial layer. After 3 weeks very few of these small cells remain and the glial culture develops into a more homogenous appearance, mainly composed of astroglial cells. The effects of various brain extracts on the development of the small cell type was investigated. The treatment by either rat or chick brain extracts caused an increase in the number of these cells, which were seen to form clusters. Brain extracts from older animals have a stronger effect than brain extracts from younger animals. These data suggest that factors contained in the brain during and after the myelination period influence the development of this cell type in dissociated cultures. The small cells were tentatively identified as oligodendroglial cells by ultrastructural and histochemical criteria. They did not contain acetylcholinesterase (AChE) and did not bind tetanus toxin. Furthermore, they did not contain glial fibrillary acidic (GFA) protein. But carbonic anhydrase II (CAII) was found in them at light and electron-microscopical level. CAII was found to be localized essentially on the plasmic membrane and on the endoplasmic reticulum of these cultured oligodendroglial cells.     

The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion

To study the effect of the flavonoid (+)-catechin on cell-matrix interactions two cell types with a different morphology on and adhesion to laminin were used. MO4 virally transformed fetal mouse cells adhere and spread when cultured on top of laminin-coated coverslips or on human amnion basement membrane. M5076 mouse reticulum cell sarcoma cells poorly adhere to these substrates and remain round. Both cell types are invasive in confronting cultures with embryonic chick heart fragments. (+)-Catechin binds to laminin in a pH-dependent way. Pretreatment of laminin-coated coverslips or amnion basement membrane with 0.5 mM (+)-catechin abrogates the effect of laminin on cell morphology and adhesion. MO4 cells do not adhere to the pretreated substrates and remain round, while M5076 cells now adhere and spread. (+)-Catechin inhibits the invasion of MO4 cells but not of M5076 cells into embryonic chick heart in vitro. We speculate that the anti-invasive activity of the flavonoid to MO4 cells is the result of its interference with MO4 cell adhesion to laminin. Invasion of M5076 cells does not imply adhesion to and spreading on laminin.     

Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts

By immunizing mice with cells from embryonic chick motoneuron cultures, an antiserum was produced which recognizes an antigen(s) restricted to cell surfaces of most, or all, neurons. With the use of this antiserum, the appearance of neuron-specific antigenicity in cells of the embryonic spinal cord was examined by indirect immunofluorescence microscopy. The antigen or set of antigens reacting with this antiserum was first detectable in the neural tube of chick embryos at stage 15–16 (V. Hamburger and H. L. Hamilton, 1951, J. Morphol.88, 49–92). In addition to the neuroblasts located in the mantle layer, some mitotic cells as well as some spindle-shaped cells in the germinal layer were antigen positive. Immunofluorescence microscopy combined with autoradiography revealed that none of the antigen-positive cells could be labeled with [³H]thymidine; thus they do not synthesize DNA, and none of the cells in the DNA synthetic phase expressed the antigen(s). As the neuroblasts do not synthesize DNA after they have differentiated from the germinal cells, we believe that the antigen-positive cells are differentiated elements and that the differentiation of membranes specific for neurons begins already before or during the terminal mitosis of cells which will be defined as neuroblasts.     

Effect of basic FGF on the proliferation of rat neuroblasts in culture

Cultures highly enriched in neuronal cells, derived from brain hemispheres of 13-day-old rat embryo, were used. In these cultures, neuroblasts proliferate during the first 3 days in vitro. The addition of the astroglial growth factor (AGF2), also known as basic fibroblast growth factor (bFGF), 4 hrs. after seeding, induces a strong increase of [125I]-deoxyuridine incorporation. We checked by autoradiography combined with specific immunocytochemical staining of the neurones, using an anti-neurofilament antibody, that the number of proliferating neuroblasts is increased after the treatment. These results demonstrate that AGF2, or bFGF, is a mitogenic factor for rat neuroblasts in culture.     

Stimulation of multiplication of chick embryo cultured cells after an increase in density of the cellular population

The secondary culture of chick embryo cells in 2-3 days after seeding was super-inoculated with homologous cells. Suspended cells adhere and spread on the cell layer whereby the culture density increases quickly. After adhesion of exogenous cells to the layer the stimulation of cell proliferation takes place. This activation is not connected with methodical manipulations or with the influence of conditioned medium factors. The results suggest that the increase in cell number itself does not arrest cell multiplication. It is proposed that the known phenomenon of blocking cell proliferation in dense cultures cannot be attributed only to effects of high cell density.     

Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer)

Lactobacillus acidophilus was isolated from washed and homogenized walls of the crop and caecum of an adult fowl. A strain that adhered well in the Fuller adhesion test was subcultured until colonies on Lactobacillus Selective agar changed from rough to smooth. This coincided with a change from aggregate to planktonic growth in liquid medium and a marked loss of ability to adhere. The ultrastructure of cells from both types of culture was studied by electron microscopy. An S-layer formed the outermost part of the cell wall in the strongly-adherent strain, whereas this layer was covered with polymerized material or was absent in strains that lacked the ability to adhere, or those with reduced adherence.     

Evidence for contact stimulation of growth of homologous cells in superinoculated cultures of chick embryo cells

The secondary cultures of chick embryo cells were suspended and transferred to homologous cell cultures. Cell adhesion and proliferation were studied in these superinoculated cultures. It was shown that added cells soon adhered to the underlying cell layer which results in a prompt increase in culture density followed by the activation of DNA synthesis and cell division. Stimulation of cell proliferation involved both cell subpopulations composing the superinoculated culture: cells seeded on the built-up cell layer and cells of the layer. The contact nature of added cell mitogenic action on overlaid cell proliferation was evidenced. The cell system described can be used to investigate the adhesive properties of the cell layer apical surface, the relationship between cell growth rate and culture density, and the contact stimulation of cell proliferation.     

Neural stem cells and the regulation of adult neurogenesis

Presumably, the 'hard-wired' neuronal circuitry of the adult brain dissuades addition of new neurons, which could potentially disrupt existing circuits. This is borne out by the fact that, in general, new neurons are not produced in the mature brain. However, recent studies have established that the adult brain does maintain discrete regions of neurogenesis from which new neurons migrate and become incorporated into the functional circuitry of the brain. These neurogenic zones appear to be vestiges of the original developmental program that initiates brain formation. The largest of these germinal regions in the adult brain is the subventricular zone (SVZ), which lines the lateral walls of the lateral ventricles. Neural stem cells produce neuroblasts that migrate from the SVZ along a discrete pathway, the rostral migratory stream, into the olfactory bulb where they form mature neurons involved in the sense of smell. The subgranular layer (SGL) of the hippocampal dentate gyrus is another neurogenic region; new SGL neurons migrate only a short distance and differentiate into hippocampal granule cells. Here, we discuss the surprising finding of neural stem cells in the adult brain and the molecular mechanisms that regulate adult neurogenesis.     

Adhesion of lymphoid cells to the carboxyl-terminal heparin-binding domains of fibronectin

Previously, we have shown that some lymphoid cell lines adhere to fibronectin (FN)-coated substratum, whereas others do not. In this study, the adhesion of five adherent lymphoid cell lines to different FN domains was examined. These cell lines ranged in their adherence to substratum coated with FN, the cell-binding domain (CBD) fragment, or the heparin-binding domain (HBD) fragments. None of the cell lines adhered to substratum coated with the gelatin-binding domain fragment. Three of the lymphoid cell lines adhered preferentially to HBD over CBD, whereas two other lymphoid cell lines and BHK fibroblasts adhered preferentially to CBD. These results suggest that two distinct adhesive interactions occur between cells and FN and that the pattern of interaction varies among cell types. Using MOPC 315 (which adheres preferentially to HBD) as a cell model to study the cell-HBD interaction, the HBD-promoted adhesion was found to be independent of the RGD sequence and could be inhibited by anti-FN antibodies. Moreover, the MOPC 315-HBD interaction had the following characteristics: (1) adhesion was temperature dependent, (2) presence of divalent cations was necessary, (3) integrity of cellular microfilaments but not microtubules was required, (4) inhibition of protein synthesis abolished adhesion, (5) pretreatment of cells with trypsin inhibited adhesion, and (6) the adhesion was mediated by the carboxyl-terminal HBD.     

Uropathogenic Escherichia coli adhere to urinary catheters without using fimbriae

Abstract Five well-characterized urinary and fecal isolates of Escherichia coli were found to be hydrophilic irrespective of their serotypes and their ability to express fimbriae. All the strains were able to adhere to silicone latex urinary catheters, although strain 917, which expressed type P fimbriae as its only adhesin, adhered poorly. Although specific adhesins, particularly fimbriae, have been shown to mediate adhesion of E. coli to uroepithelial cells, they do not mediate specific adhesion onto urinary catheter material. The overall surfaces of the strains, tested using microelectrophoresis as a function of pH and X-ray photoelectron spectroscopy, were not significantly different, thus suggesting more non-specific adhesion mechanisms to urinary catheters.