Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

Communication compartments in the ectoderm of embryos of Patella vulgata

Summary Patterns of gap junctional communication in the ectoderm of embryos of Patella vulgata have been studied by intracellular injection of the fluorescent dye Lucifer Yellow, and by analysis of its subsequent spread to adjacent cells (dye-coupling). We found that dye-coupling became progressively restricted to different domains of the ectoderm, forming communication compartments. These communication compartments are characterized by their high coupling abilities within the compartment, and reduction of coupling across their boundaries. During development, the pretrochal (anterior) ectoderm becomes subdivided into two communication compartments, the apical organ and the anlage of the head ectoderm. The posttrochal (posterior) ectoderm becomes subdivided into different communication compartments in two successive phases. Firstly, in the 15-h embryo the dorsal and ventral domains of the ectoderm form separate communication compartments. A dorso-ventral communication boundary restricts the passage of dye between the two domains. Secondly, in the 24-h embryo dye-coupling becomes further compartmentalized in both the dorsal and ventral domains. These compartments correspond to the anlagen of different ectodermal structures. In order to study whether any level of coupling persists between the ectodermal compartments we injected currents through a microelectrode inserted into one cell of one compartment and monitored its spread by means of a second microelectrode inserted into one cell of another compartment (electrical coupling). Despite the absence of dye-coupling, electrical coupling between the ectodermal dye-coupling compartments was detected, which suggests that some level of communication is maintained between compartments. Our results demonstrate that within the ectoderm layer of Patella vulgata the transfer of dyes becomes progressively restricted to communication compartments and, concomitantly with the specification of the different ectodermal anlagen, these compartments become subdivided into smaller communication compartments.     

Microinjection of lectins, hyaluronidase, and hyaluronate fragments interferes with cleavage delay and mesoderm induction in embryos of Patella vulgata

In embryos of Patella vulgata at the 32-cell stage, one of the four vegetally located macromeres makes contacts with overlying animal micromeres. As a result, this macromere (designated 3D) divides significantly later than the other macromeres and forms the mesodermal stem cell 4d. Shortly before and during this interaction two types of extracellular matrix are present: a basal lamina-like layer on the tips of the micromeres and a loose fibrillar meshwork in the blastocoel. In this paper we examine the role of the matrix in cleavage delay and mesoderm determination. The microinjection of extracellular matrix-binding lectins, or of hyaluronidase, or of decasaccharide fragments of hyaluronate into the blastocoel results in embryos in which either no or two macromeres are delayed in cleavage and are presumably determined as mesodermal stem cells. We suggest that the fibrillar meshwork is needed for macromere elongation toward the micromeres and that the basal lamina-like layer is involved in the determination process itself.     

Changes in junctional communication associated with cell cycle arrest and differentiation of trochoblasts in embryos of Patella vulgata

In early embryos of molluscs, different clones of successively determined trochoblasts differentiate into prototroch cells and together contribute to the formation of a ciliated ring of cells known as the prototroch. Trochoblasts differentiate after cell cycle arrest, which occurs two cell cycles after the commitment of their stem cell. To study the changes of junctional communication in embryos of Patella vulgata in relation to commitment, cell cycle arrest, and differentiation of the trochoblasts, we have monitored electrical coupling as well as transfer of fluorescent dyes. The appearance of dye coupling in embryos of Patella occurs after the fifth cleavage (at the 32-cell stage), when the cell cycles of all embryonic cells become asynchronous and longer. At the 32- and 64-cell stages all cells are well coupled. However, after the 72-cell stage dye transfer to or from any cell of the four interradial clones of four primary trochoblasts becomes abruptly reduced, whereas electrical coupling between these cells and the rest of the embryo can still be detected. From scanning electron microscopical analysis of the cell pattern we conclude that this change in gap junctional communication coincides with cell cycle arrest and with the development of cilia in all four clones of primary trochoblasts. Similarly, after the 88-cell stage the four radial clones of accessory trochoblasts stop dividing, reduce cell coupling, and become ciliated. By the formation of the prototroch, the embryo becomes subdivided into an anterior (pretrochal) and a posterior (posttrochal) domain which will develop different structures of the adult. At the 88-cell stage, the cells within each of these two domains remain well coupled and form two different communication compartments that are separated from each other by the interposed ring of uncoupled trochoblasts. The relations among control of cell cycle, changes in junctional communication, and differentiation are discussed.     

Spatial and temporal coincidence of induction processes and gap-junctional communication in Patella vulgata (Mollusca,Gastropoda)

In the early development of the gastropod mollusc Patella vulgata, two inductive processes take place between the animal and vegetal hemispheres. One is the induction of the vegetally-located stem cell of the mesoderm (the 3D macromere) by the first-quartet micromeres (which are located in the animal hemisphere). The other is the induction of dorsoventral symmetry in the first-quartet micromeres by the 3D macromere. As a consequence of the latter induction process, a dorsoventrally-organised prototroch is formed. In this paper, the moment of 3D induction is determined by deleting the inducing first-quartet micromeres at successive stages of development. Furthermore, the role of gap junctions in the two above-mentioned induction processes is investigated. This was done by studying lucifer yellow dye transfer between the 3D macromere and the first-quartet micromeres, in relation to these two induction processes. Analysis of the pattern of dye-transfer in vivo, in sections and with confocal laser scanning microscopy demonstrated that the moment of dye transfer coincides with the moment of 3D induction and with the moment of induction of the dorsoventral pattern of the prototroch. This indicates that gap-junctional communication may be involved in these two induction processes.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

A useful approach for the screening of active neural-inducing factors

The present study suggests that the membrane-binding molecules of mesodermal cells and/or the modulated extracellular matrix (ECM) with them play an important role in induction of the central nervous system. Artificially mesodermalized ectoderm (mE) or chordamesoderm (cM) was placed on a collagen and flbronectin (CF)-coated dish for 24 h. After mechanical removal of the mesoderm sheet, competent ectoderm of early gastrulae was placed on the same spot. Many melanocytes and neuronal cells were observed after 1 week, along with many cells which reacted specifically with a neuralspecific monoclonal antibody. However, when presumptive ectoderm (pE) instead of mE or cM was used as the control, only epidermal cells with cilia were observed in the competent ectoderm, except for a few melanocytes in rare cases. The proteins synthesized and remaining on the CF substrate during placement of the mE and pE were analysed by two-dimensional polyacrylamide gel electrophoresis (PAGE) fluorography. The fluorography indicated that there were significant differences between the polypeptides spots of mE and pE.Correspondence to: A.S. Suzuki     

Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusk Patella vulgata.

In mollusks with an equal four-cell stage, dorsoventral polarity becomes noticeable in the interval between the formation of the third and fourth quartet of micromeres, i.e., between the fifth and sixth cleavage. One of the two macromeres at the vegetal cross-furrow then partly withdraws from the surface and becomes located more toward the center of the embryonic cell mass than the other three macromeres. Only this specific macromere (3D) contacts the micromeres of the animal pole, divides with a delay, and develops into the stem cell of the mesentoblast (4d). After suppression of the normal contacts between micromeres and macromeres either by dissociation of the embryos or by deletion of first quartet cells, the normal differentiation of the macromeres fails to appear. By deleting a decreasing number of first quartet cells, an increasing percentage of embryos shows the normal differentiation pattern. Deletion of one of the cross-furrow macromeres does not preclude formation of the mesentoblast, which then originates by differentiation of an other macromere. It is concluded that initially the embryo is radially symmetrical and that the four quadrants have identical developmental capacities; mesentoblast differentiation from one macromere is induced through the contacts of the first quartet cells and that single macromere.     

Morphometry of cellular protrusions of mesodermal cells and fibrous extracellular matrix in the primitive streak stage chick embryo

Summary Chick mesodermal cells, having become invaginated and beginning to locomote prior to the formation of the mesodermal cell layer at an early primitive streak stage, extend many filopodia and flatten themselves against the basal surface of the epiblast. Morphometry on scanning electron micrographs of chick mesodermal cells revealed two statistically significant tendencies. Each cell took an extended form and protruded filopodia, preferably along its major axis, suggesting that the force extending the cell body was generated by both ends rich in filopodia. The cells also tended to protrude filopodia most frequently in a direction away from Hensen's node. The orientation of the fibrous extracellular matrix (fECM), running on the basal surface of the epiblast, was assessed quantitatively, and it was proved statistically that the orientation of the fECM was radial around the primitive streak: With an immunogold staining technique, fECM, to which the filopodia of the mesodermal cells attached frequently and closely, was confirmed to be rich in fibronectin (FN). These results lead us to conclude that the mesodermal cells in chick gastrula were guided to locomote towards the periphery of the area pellucida by FN-rich fECM laid on the basal surface of the epiblast, and that this movement was due to an in vivo locomotive mechanism using filopodia.Offprint requests to: R. Toyoizumi     

The possible role of mesodermal growth factors in the formation of endoderm inXenopus laevis

We have raised a monoclonal antibody, 4G6, against gut manually isolated from stage 42Xenopus laevis embryos. It is specific for endoderm and recognises an epitope that is first expressed at stage 19 and which persists throughout subsequent development. The antibody maintains gut specificity through metamorphosis and into adulthood. The epitope is conserved in the mouse, where it is also found in the gut. Isolated vegetal poles fromXenopus blastula stage embryos express the epitope autonomously after culturing to the appropriate stage. This shows that certain aspects of endoderm differentiation do not require germ layer interactions. Animal cap cells from stage 9 blastulae cultured in the presence of the mesodermal growth factors FGF, XTC-MIF and PIF form both endodermal and mesodermal tissues, assessed by the binding of tissue-specific monoclonal antibodies. Endoderm is typically found in those caps which form intermediate and ventral forms of mesoderm, that is muscle and lateral plate. Correspondence to: E.A. Jones     

Approaches in extracellular matrix engineering for determination of adhesion molecule mediated single cell function

The native extracellular matrix (ECM) and the cells that comprise human tissues are together engaged in a complex relationship; cells alter the composition and structure of the ECM to regulate the material and biologic properties of the surrounding environment while the composition and structure of the ECM modulates cellular processes that maintain healthy tissue and repair diseased tissue. This reciprocal relationship occurs via cell adhesion molecules (CAMs) such as integrins, selectins, cadherins and IgSF adhesion molecules. To study these cell-ECM interactions, researchers use two-dimensional substrates or three-dimensional matrices composed of native proteins or bioactive peptide sequences to study single cell function. While two-dimensional substrates provide valuable information about cell-ECM interactions, three-dimensional matrices more closely mimic the native ECM; cells cultured in three-dimensional matrices have demonstrated greater cell movement and increased integrin expression when compared to cells cultured on two-dimensional substrates. In this article we review a number of cellular processes (adhesion, motility, phagocytosis, differentiation and survival) and examine the cell adhesion molecules and ECM proteins (or bioactive peptide sequences) that mediate cell functionality.     

Immuno-electron-microscopic study of fibronectin in gastrulating amphibian embryos

Summary The ultrastructural organization of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies directed against amphibian plasmatic FN. Scanning and transmission electron microscopy combined with immunogold labeling of FN revealed that the extracellular matrix that covers the inner surface of the ectodermal layer consists of FN-containing fibrils. During gastrulation, the mesodermal cells appear to be devoid of FN. These cells extend filopodia adhering to the FN-containing fibrils and are spreading along them. These findings suggest that FN may be involved in contact formation between mesodermal cells and the extracellular matrix that serves as a substratum for migration.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Type XIII collagen: a novel cell adhesion component present in a range of cell–matrix adhesions and in the intercalated discs between cardiac muscle cells

Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell–basement membrane interfaces. Some cell–cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell–matrix junctions.     

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

Summary To identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to - and -sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).