CELL-DEATH IN THE GASTRULATING CHICK-EMBRYO - POTENTIAL ROLES FOR TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA)

We have examined the expression of TNF-alpha and its receptors, TNFR1 and TNFR2, during gastrulation in the chick embryo, and have investiga ted the possible role of this factor in the control of cell death at t his early stage of development. TNF-alpha, immunoreactive at approxima tely 17 kD, was found both in vivo and in vitro, most intensely associ ated with the cell surface and cytoskeleton of endoderm cells. TNFR2 w as especially immunoreactive in endoderm cells of the marginal zone. T NFR1 was found in nuclei throughout the embryo. Embryos also showed wi despread expression of both the bcl-2 and Bar gene products, which are both associated with cell death pathways. Intact embryos in culture w ere sensitive to the addition of TNF-alpha (approx. 110 ng/ml), respon ding by significantly increasing the incidence of DNA fragmentation in cells from all tissues of the embryo. This effect was abrogated by im munological 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 con comitant decrease in cell-substratum adhesion caused by the antibody. Decreased cell adhesion, induced non specifically with antiintegrin an tiserum, also resulted in increased DMA fragmentation. TNF-alpha, synt hesized and secreted by the embryo itself, may be able to exert a para crine 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 a nd 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.