Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states.

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.

Expression of an epidermal antigen used to study tissue induction in the early Xenopus laevis embryo.

A monoclonal antibody (Epi 1) has been produced that recognizes an antigen expressed in epidermal cells of Xenopus laevis embryos. The Epi 1 antigen appears in embryonic epidermis at the end of gastrulation and is not expressed in nonepidermal structures derived from ectoderm (for example, neural tube or cement gland). The capacity to express the Epi 1 antigen is restricted to cells of the animal hemisphere prior to the midblastula stage of development (stage 8), and tissue interactions during gastrulation inhibit the expression of the Epi 1 antigen in neural ectoderm. This epidermal antigen will be a valuable marker for studies of ectodermal commitment.

Morphogenetic rearrangement of injected collagen in developing chicken limb buds.

A fundamental question concerning the development of the extracellular matrix is what factors control the arrangement of collagen fibrils within a tissue and at the same time allow for the great diversity of geometric forms exhibited by collagen. In this report, we test the possibility that physical forces within the embryo serve to organize collagen fibers into regular patterns. In particular, we test the prediction that patterns of stress having this morphogenetic function are generated by cell traction, the contractile force exerted by cells to propel themselves. To study the effects of these mechanical forces on the extracellular matrix, type I collagen was fluorescently labeled and injected into developing chicken wing buds. When the injected limbs were allowed to develop and then examined histologically, the exogenous collagen was found incorporated within normal connective tissues of the wing. The labeled collagen became arranged according to its site of injection, forming parts of tendons, perichondria, cartilages, perineuria, and blood vessels. Since the injected collagen formed a gel within minutes of its injection, the subsequent incorporation of this performed collagen within organized structures cannot be explained in terms of molecular self-assembly or other mechanisms occurring during collagen deposition. These results demonstrate that, within developing tissues, patterns of forces exist that are capable of physically rearranging collagen and determining its long-range order.

The role of cell proliferation and cellular shape change in branching morphogenesis of the embryonic mouse lung: analysis using aphidicolin and cytochalasins.

The formation of induced supernumerary buds in the embryonic mouse tracheal epithelium has been used as a model system to analyse the respective roles of cell proliferation and microfilament-mediated cell shape change during branching morphogenesis. In order to analyse the mitotic events associated with the formation of epithelial buds, the induction of supernumerary tracheal buds by mesenchymal grafts was carried out with the inhibitor of DNA synthesis, aphidicolin, present in the culture medium for varying intervals of time during the 16-hour inductive process. The presence of aphidicolin for 10 to 16 hours of the inductive period blocks the formation of induced tracheal buds, whereas the presence of the inhibitor for half of that time (either the first 8 hours or the last 8 hours) does not prevent this morphogenetic event from taking place, although smaller buds resulted from induction under these conditions. Both the inhibition of DNA synthesis and the recovery from 10 microM aphidicolin treatment, as measured by 3H-thymidine incorporation, were found to occur rapidly. The addition of 2 microM dihydrocytochalasin B (or cytochalasin B) together with aphidicolin during the second half of the inductive period inhibits the formation of supernumerary buds and upon removal of the cytochalasin rapid formation of buds takes place. We conclude that the formation of epithelial buds during branching morphogenesis occurs as a result of enhanced localized cell proliferation coupled with epithelial cell shape change (or preservation of cell morphology) mediated by microfilaments, which have been observed in both the apical and basal cytoplasm of the epithelial cells in the region where branching of the trachea is taking place.

The early development of mystacial vibrissae in the mouse.

The initial generation of the pattern of mystacial vibrissae (whiskers) in the mouse is described. The maxillary process is present in 10-day embryos but has a relatively flat surface. Beginning at approximately 11.5 days, the first sign of vibrissal development is the formation of ridges and grooves on the maxillary and lateral nasal processes. The first vibrissal rudiment to form subsequently appears posterior to the most ventral groove on the maxillary process. It is the most ventral whisker of the posterior, vertical row. The next few rudiments appear: dorsal to the first, also in the vertical row; and anterior to the first, on the ventral-most ridge and in the groove beneath it. Formation of vibrissal rudiments continues in a dorsal and anterior progression usually by an apparent partitioning of the ridges into vibrissal units. The hypothesis that this patterning of mystacial vibrissae might be determined by the pattern of innervation in the early mouse snout was investigated. Nerve trunks and branches are present in the maxillary process well before any sign of vibrissal formation. Because innervation is so widespread there appears to be no immediate temporal correlation between the outgrowth of a nerve branch to a site and the generation of a vibrissa there. Furthermore, at the time just prior to the formation of the first follicle rudiment, there is little or no nerve branching to the presumptive site of that first follicle while branches are found more dorsally where vibrissae will not form until later. Thus, a one-to-one spatial correlation between nerve and follicle sites also appears to be lacking. The developmental changes in ultrastructure within the neurites of the trunks and branches as well as the apparent rearrangements of the nerve trunks suggest that early innervation of the snout is a labile phenomenon and that the vibrissal pattern begins to be established before the neural pattern is completely developed. The results indicate that vibrissal pattern formation is likely to be a complex process relying on the interplay of the cells and tissues involved, rather than on unidirectional instructions from neurons to other cell types.

Differential antigen adhesivity used to select spleen cells for the production of monoclonal antibodies to embryonic neurons.

Monoclonal antibodies specific for cell surface antigens on embryonic chick ciliary ganglion neurons (CG) have been obtained at high frequencies by fractionating spleen cells from immunized mice according to their adhesiveness for cell surfaces of the cultured neurons. Spleen cells from mice that had been immunized with live or lightly fixed (0.125% glutaraldehyde) CG neurons were selected for subsequent hybridization with myeloma cells after fractionation on lawns of CG neurons in tissue culture. Immunized spleen cells were cultured with the neurons for 4-7 days prior to fractionation. Three groups of spleen cells were selected for fusion with a myeloma cell line: a non-adherent population of spleen cells, a population of spleen cells that could be removed from the neuronal cells by shaking on a vibratory shaker for 1 h, and a population that could be removed from the neuronal cells only by treatment with low concentrations of trypsin. Of the 3 groups of spleen cells, the population that required trypsin treatment produced the greatest number of hybridomas specific for neurons and for neuronal cell surfaces. Fewer neuron-specific hybridomas resulted from fusion of the group of spleen cells that could be removed from the antigen lawn by shaking. None of these was specific for the CG neurons. No neuron-specific hybridomas resulted from the fusion of the cells that did not adhere to the neuronal cells, and at most only 1 neuron-specific hybridoma resulted from fusions of comparable groups of unselected spleen cells (spleen cells from immunized animals which were not selected on antigen lawns).

Neuronal motility: the ultrastructure of veils and microspikes correlates with their motile activities.

We have documented the ultrastructural characteristics that correlate with protrusion, adhesion and retraction of neuronal veils and microspikes, by filming individual neurons of the chick ciliary ganglion and examining the same cells with high-voltage electron microscopy. We find that new veils invariably contain only a cortical meshwork of filaments and are devoid of microtubules, groups of vesicles and other organelles. At sites of recent veil retraction a cortical meshwork on the substratum side underlies a filament-free space containing vesicle clusters and a complexly folded upper membrane. Areas without veil activity are smooth-surfaced and contain a three-dimensional lattice of filaments. We discuss the implications of these observations for the mechanisms of surface recruitment and retrieval during motile activity. We also find that the ultrastructure of moving and attached extensions of the cell surface differs dramatically. Unattached microspikes and actively extending veils have an open, criss-cross array of filaments, whereas attached microspikes contain more aligned filaments, which extend as a small bundle into the growth cone. These results suggest that cell surface protrusion is mediated by meshworks of loosely packed filaments. More compact bundles of filaments are probably generated only at adhesion points.

Responses to cell contacts between growth cones, neurites and ganglionic non-neuronal cells.

The motility of growth cones of embryonic peripheral neurons is not inhibited by contact with the surfaces of neurites or of non-neuronal cells. Rather, growth cones and microspikes adhere to other cell surfaces and often respond with forward movement and elongation in contact with other cells, as they do on adhesive surfaces in vitro. Furthermore, non-neuronal cells do not display contact inhibition when they contact growth cones or neurites. If anything, surface motility and ruffling is stimulated by contact with a neuronal cell surface and some non-neuronal cells prefer to migrate along neurites rather than on the surface of the culture dish. These observations on the contact behaviour of cells from peripheral nerve ganglia imply that the surfaces of embryonic neurons differ from those of non-neuronal cells in that the neuronal surfaces do no elicit the typical contact inhibition response.

Actin and cortical fiber reticulation in the siphonaceous alga Vaucheria sessilis.

Since light-induced organellae aggregation in the siphonaceous alga Vaucheria sessilis (Vauch.) D.C. is accompanied by the formation of a cortical fiber reticulum in the light, we proposed that this process of reticulation might be causally related to aggregation (Blatt and Briggs, 1980). In this paper we report the tentative identification of actin filaments and filament bundles in the cortical cytoplasm of V. sessilis, and present additional evidence, obtained using the inhibitors cytochalasin B and phalloidin and indicating that aggregation in response to low-intensity point irradiation with blue light is dependent upon the formation of a cortical fiber reticulum. Phalloidin stabilized the cortical fibers, preventing both reticulum formation and organelle aggregation in blue light. Cytochalasin B partially destabilized the cortical fibers to the extent of permitting light-induced reticulum formation and organelle aggregation in the light in the presence of phalloidin.

Differential labeling of the cell surface of single ciliary ganglion neurons in vitro.

Cationic ferritin binds in a time and concentration dependent manner to all surfaces of ciliary ganglion neurons in culture except "mounds" and "veils". In chase experiments, bound ferritin clears from the cells surfaces and forms larger and larger patches, even at low temperatures. Binding of cationic ferritin is inhibited by poly-L-lysine, potentiated by poly-L-glutamate, and not affected by neruaminidase (acylneuraminyl hydrolase, EC 3.2.1.18), hyaluronidase (hyaluronoglucosidase, hyaluronate 4-glycanhydrolase, EC 3.2.1.35), or chondroitin ABC lyase (EC 4.2.2.4).

Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study.

New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.