Ablation of axial structures activates apoptotic pathways in somite cells of the chick embryo

It has been known for some time that ablation of the neural tube and/or the notochord in the chick embryo leads to a massive wave of cell death in the adjacent somites. It is postulated that in the normal embryo, survival signals emanate from the neural tube and/or notochord that suppress apoptosis in the cells of the somites, except for a small population of sclerotome cells that are programmed to die naturally. In this study we show that axial ablation results in the death of sclerotome and not somitic neural crest cells, and we have examined the apoptotic response of these cells to the ablation. We show that several elements of the apoptotic cascade become detectable in somite cells in response to the withdrawal of survival signals. We demonstrate the down-regulation of bcl-2 protein in the somites adjacent to, and caudal to, the site of ablation, corresponding to the region that displays an elevated level of cell death. Although caspase-9 appeared to be activated in somites at all levels of the trunk, caspase-2 showed a clear response to the ablation of the axial structures. Removal of the neural tube and notochord produced an up-regulation of caspase-2 activity in somites in the region of the operation. Cleavage of two down-stream substrates of these caspases was examined. The cleavage of poly (ADP-ribose) polymerase (PARP) was apparent in somites at all levels of the trunk, and showed only a modest up-regulation after ablation. By contrast, the cleavage of DNA fragmentation factor (DFF45) showed a marked up-regulation in response to ablation, suggesting that this is a primary substrate for a caspase-dependent apoptotic mechanism. Evidence was also found for a caspase-independent mechanism, since the expression of apoptosis-inducing factor (AIF) was found to be very sensitive to, and up-regulated in somites by, axial ablation. Because the wave of apoptosis that is precipitated in somites by removal of the axial structures may be mediated by BMP-4, we examined the levels of BMP-4 in somites in response to axial ablation. BMP-4 expression was clearly up-regulated in somites adjacent to, or close to, the site of operation.