Chick embryo mesoderm cells are various stages of differentiation were cultured in three-dimensional matrices of hydrated collagen. The tissues used were: stage 5 mesoderm from regions adjacent to the primitive streak; stage 12 mesoderm, comprising somitic, unsegmented (segmental plate) and lateral plate mesoderm; and stage 18 sclerotome. Explants were examined by phase contrast microscopy, including time-lapse, and scanning and transmission electron microscopy. The cells showed an increased ability to adhere to, and move in, the collagen gel with advancing stage. Of the stage 12 tissues, the unsegmented mesoderm was initially the slowest to grow out of the explant. Sclerotome cells showed by far the greatest ability to move within the gel. Where the collagen fibrils were randomly oriented, the cell morphology was polypodial and advancing lamellipodia showed clear undulations at their leading edges. A distinction was drawn between these undulations and the classical major ruffles which are seen in two-dimensional culture to uplift and pass back along the cell surface. The latter were not seen in the collagen matrix and were presumably suppressed by the three-dimensional culture configuration while the leading edge undulations were not. Ultrastructural examination showed that the cells possessed patches of amorphous material on their surface, which was sometimes interposed between the plasma membrane and collagen fibrils. Addition of hyaluronic acid (2 mg/ml) had an effect only the segmented mesoderm, where outgrowth was enhanced. Although the addition of plasma fibronectin (50 micrograms/ml) to the cultures did not affect any of the tissues, the removal of this substance, by antifibronectin antiserum or by the use of fibronectin depleted serum, inhibited outgrowth in most cases. The only tissue not reproducibly inhibited in this way was sclerotome. Alignment of the collagen fibres by the explants was observed, accompanied by an elongation of the outgrowing cells which, in bipolar form, preferentially moved up and down the aligned tracts. Scanning electron microscopy suggested that cell processes attached to, and presumably exerted tension on, bundles of fibrils thereby pulling them into line. Cell-to-cell contact was not accompanied by contact paralysis as judged by time-lapse micrography.

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Antibodies to channel proteins and specific peptide sequences have been previously used to localize voltage-activated sodium channels in the rat brain. Here we describe the first localization of sodium channels in an insect nervous system using a site-directed antibody. The mesothoracic ganglion of the cockroach was stained with an antibody to the highly conserved SP19 sequence. Antibody labelling was visualized by light microscopy using the avidin/biotin method on wax sections, and transmission electron microscopy of immunogold-labelled thin sections. Central ganglia of insects contain clearly separated regions of cell bodies, synaptic neuropil, axon tracts, and nerves. Antibody staining by light microscopy was limited to neurons, and was intense in axons throughout the ganglion and nerves. Staining was also strong in the cytoplasm, but not the nuclei, of many neuronal cell bodies. Neuropil regions were relatively lightly labelled. These findings can be correlated with the known electrophysiology of the ganglion. Electron microscopy detected sodium channels in areas surrounding axons, probably including axon membranes and enveloping glial cell membranes. Axonal mitochondria were also heavily labelled, suggesting a sodium channel transport function for these organelles.

We have examined the expression of TNF-alpha and its receptors, TNFR1 and TNFR2, during gastrulation in the chick embryo, and have investigated the possible role of this factor in the control of cell death at this early stage of development. TNF-alpha, immunoreactive at approximately 17 kD, was found both in vivo and in vitro, most intensely associated with the cell surface and cytoskeleton of endoderm cells. TNFR2 was especially immunoreactive in endoderm cells of the marginal zone. TNFR1 was found in nuclei throughout the embryo. Embryos also showed widespread expression of both the bcl-2 and Bax gene products, which are both associated with cell death pathways. Intact embryos in culture were sensitive to the addition of TNF-alpha (approx. 110 ng/ml), responding by significantly increasing the incidence of DNA fragmentation in cells from all tissues of the embryo. This effect was abrogated by immunological pre-absorption of the cytokine. Cultured cells from these embryos also responded to the addition of agonistic antibodies to TNF-alpha receptors by increasing DNA fragmentation. A similar response to TNF-alpha antiserum by cultured cells appeared to be related to a concomitant decrease in cell-substratum adhesion caused by the antibody. Decreased cell adhesion, induced non-specifically with anti-integrin antiserum, also resulted in increased DNA fragmentation. TNF-alpha, synthesized and secreted by the embryo itself, may be able to exert a paracrine effect on the incidence of cell death in tissues of the embryo, and the cell death process may be related to the expression of bcl-2 and Bax gene products. The influence of TNF-alpha may be exerted by the activation of cell death signalling pathways directly, or indirectly through perturbation of the cytoskeleton or of integrin-mediated cell adhesion.

We examined the effects of transforming growth factor-beta 1 (TGF beta 1) and a neutralizing monoclonal antibody on two phases of early chick embryo development: gastrulation and chondrogenesis. We carried out experiments in vivo and in vitro on mesoderm cells from the gastrulating embryo at day 1, and on sclerotome cells from day 3 embryos, having previously shown that this factor is present among these cells at these stages of development. Addition of the antibody to cultures of these cells produced a dose-dependent decrease in cell out-growth and spreading and concomitantly reduced fibronectin deposition. In vivo studies of the effects of TGF beta 1 on mesoderm during gastrulation were carried out by grafting beads carrying this agent into gastrulating embryos. We used beads of ion-exchange resin as well as hydrolysed polyacrylamide, and found that the grafts produced an accumulation of mesoderm cells around the implant and, at later stages, the formation of enlarged somites. There was no effect on embryonic axis formation. Studies of bromodeoxyuridine (BrdU) incorporation indicated that the mesoderm accumulation was due, at least in part, to an increase in cell proliferation. However, examination of the effect of TGF beta 1 on BrdU incorporation by mesoderm during gastrulation and sclerotome cells in vitro indicated in inhibition of cell proliferation, an inconsistency explained in terms of the variation between the in vivo and in vitro conditions. We conclude that TGF beta 1 is both appropriately located, and is able, to influence cell proliferation among the mesodermal cell populations during early development, and that this effect contributes to the overall control of mesodermal morphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)