Abstract. Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4°C were on the order of 10 -5 dynes/cell and did not involve the cytoskeleton. Adhesions to fibronectin after 15 min at 37°C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37°C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37°C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37°C, glioma cells increased their surface area within close contact to the substrate by ",,125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex. C ELL-SUBSTRATUM adhesion to fibronectin (FN) ~ orto glass often has been associated with several morphological and cytological changes including cell spreading and the formation of focal contacts, close contacts, and stress fibers (1, 9, 12, 19, 32-34, 40, 42, 45, 49, 51). While a spread morphology and focal contacts provide physical evidence that an adhesion has occurred, the actual adhesion and its relationship to the cell's behavioral changes that follow must be defined sequentially and on functional terms. As in cell-cell adhesion (39, 48), cell-substrate adhesion has been suggested to involve at least two measurable steps: the first involves initial contact between cell and substrate and the second step strengthens the adhesion through processes requiring metabolic energy (10,27,35,44,48).A number of extracellular matrix proteins including FN and laminin have been found to serve as adhesive substrates for cells. A more recently discovered extracellular matrix Dr. Lotz's present address is New England

During gastrulation, an embryo acquires the three primordial germ layers that will give rise to all of the tissues in the body. In amniote embryos, this process occurs via an epithelial to mesenchymal transition (EMT) of epiblast cells at the primitive streak. Although the primitive streak is vital to development, many aspects of how it forms and functions remain poorly understood. Using live imaging and immunohistochemistry, we have shown that the murine primitive streak arises in situ by progressive initiation of EMT beginning in the posterior epiblast, without large-scale movement or convergence and extension of epiblast cells. Loss of basal lamina (BL) is the first step of this EMT, and is strictly correlated with ingression of nascent mesoderm. This is the first description of dynamic cell behavior during primitive streak formation in the mouse embryo, and reveals mechanisms that are quite distinct from those observed in other amniote model systems.

Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. Here we show, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior. The initial events in the onset of CE are mediolateral elongation, alignment and orientation of mesoderm cells as they exit the primitive streak. This cell shape change occurs prior to, and is required for, the subsequent onset of mediolaterally polarized protrusive activity. In embryos mutant for PTK7, a novel cell polarity protein, the normal cell elongation and alignment upon leaving the primitive streak, the subsequent polarized protrusive activity, and CE and axial elongation all failed. The mesoderm normally thickens and extends, but on failure of convergence movements in Ptk7 mutants, the mesoderm underwent radial intercalation and excessive thinning, which suggests that a cryptic radial cell intercalation behavior resists excessive convergence-driven mesodermal thickening in normal embryos. When unimpeded by convergence forces in Ptk7 mutants, this unopposed radial intercalation resulted in excessive thinning of the mesoderm. These results show for the first time the polarized cell behaviors underlying CE in the mouse, demonstrate unique aspects of these behaviors compared with those of other vertebrates, and clearly define specific roles for planar polarity and for the novel planar cell polarity gene, Ptk7, as essential regulators of mediolateral cell intercalation during mammalian CE.

We review what is known about amphibian limb regeneration from the prospective of developing strategies for the induction of regeneration in adult mammals. Prominent in urodele amphibian limb regeneration is the formation of a blastema of undifferentiated cells that goes on to reform the limb. The blastema shares many properties with the developing limb bud; thus, the outgrowth phase of regeneration can be thought of as cells going through development again, i.e., redevelopment. Getting to a redevelopment phase in mammals would be a major breakthrough given our extensive understanding of limb development. The formation of the blastema itself represents a transition phase in which limb cells respond to injury by dedifferentiating to become embryonic limb progenitor cells that can undergo redevelopment. During this phase, rapid wound closure is followed by the dedifferentiation of limb cells to form the blastema. Thus, the regeneration process can be divided into a wound-healing/dedifferentiation phase and a redevelopment phase, and we propose that the interface between the wound-healing response and gaining access to developmentally regulated programs (dedifferentiation) lies at the heart of the regeneration problem in mammals. In urodele amphibians, dedifferentiation can occur in all of the tissues of the limb; however, numerous studies lead us to focus on the epidermis, the dermis, and muscle as key regulators of regeneration. Among higher vertebrates, the digit tip in mammals, including humans, is regeneration-competent and offers a unique mammalian model for regeneration. Recent genetic studies in mice identify the Msx1 gene as playing a critical role in the injury response leading to digit tip regeneration. The results from regeneration studies ranging from amphibians to mammals can be integrated to develop a roadmap for mammalian regeneration that has as its focus understanding the phenomenon of dedifferentiation.

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Abstract. An extracellular matrix molecule has been purified from sea urchin (Lytechinus variegatus) embryos. Based on its functional properties and on its origin, this glycoprotein has been given the name "echinonectinY Echinonectin is a 230-kD dimer with a unique bow tie shape when viewed by electron microscopy. The molecule is 12 nm long, 8 nm wide at the ends, and narrows to ,o4 nm at the middle. It is composed of two ll6-kD U-shaped subunits that are attached to each other by disulfide bonds at their respective apices. Polyclonal antibodies were used to localize echinonectin in paraffin-embedded, sectioned specimens by indirect immunofluorescence. The protein is stored in vesicles or granules in unfertilized eggs, is released after fertilization, and later becomes localized on the apical surface of ectoderm cells in the embryo. When used as a substrate in a quantitative in vitro assay, echinonectin is highly effective as an adhesive substrate for dissociated embryonic cells. Because of the quantity, pattern of appearance, distribution, and adhesive characteristics of this protein, we suggest that echinonectin serves as a substrate adhesion molecule during sea urchin development.

We adapted an assay that has been used to estimate the strength of eukaryotic cell-cell and cell-extracellular matrix adhesive interactions (McClay et al., 1981) to quantify the strength of bacterial (streptococci, fusobacteria, actinomyces) interactions with salivary receptors. Bacteria are centrifuged onto human submandibular/sublingual or parotid-saliva-coated microtiter wells. Plates are sealed with pressure-sensitive, double-sided tape which allows them to be inverted and centrifuged again. The force required to remove the bacteria from the coated wells is a direct measure of the adhesive strength of the interaction(s) being disrupted. The bacteria-saliva adhesive forces we detected ranged from 1.6 x 10(-8) dynes (Streptococcus sanguis 72-40) to > 1.1 x 10(-7) dynes (Actinomyces viscosus T14 V). These forces were in the range to withstand the shear stress produced by salivary flow, which we calculated as approximately 6.1 x 10(-7) dynes.

No abstract