Cell death in the avian sclerotome

Abstract

In this study the occurrence of apoptotic cells in chick embryo trunk somites, between 2.5 and 4 days of development, has been examined using an in situ nick-end-labeling method (TUNEL) to identify nuclei in which DNA is undergoing fragmentation. At 9.5 days of development, apoptotic cells were found in the sclerotome with a distribution that depended on the rostrocaudal level in the trunk. At the most rostral levels (somites 1-18), dying cells were present primarily in the rostral haft of the ventral sclerotome; at midlevels (somites 19-26), they were present throughout the ventral sclerotome; and at caudal levels (somites 27-32), no dying cells were present. By 4 days of development, the number of dying cells in the sclerotome was sharply reduced, and those present were primarily distributed to the caudal side of the intrasclerotomal fissure. Double labeling of cells for both TUNEL and the HNK-1 epitope, at 2.5 days, indicated that the majority of the dying cells were not neural crest cells. Further, dying cells in the rostral somite half were present largely in regions of the sclerotome that labeled poorly for HNK-1. It was confirmed that apoptotic neural crest cells retain the HNK-1 epitope and therefore would have been observed if present. Neural crest cells only appeared to be apoptotic in relatively small numbers and only at the ventral border of the sclerotome. Examination of DiI- labeled neural crest cells confirmed that the dying cells in the body of the somite were not primarily neural crest cells. Two hypotheses regarding the TUNEL-positive cells in the sclerotome were experimentally tested. First, that they originate from the somitocoel compartment of the somite, because their distribution patterns at 4 days were similar to those of somitocoel cells. To test this, somitocoel cells were labeled with carboxyfluorescein and grafted into host embryos in ovo. Results showed that these cells did not become apoptotic and that the dying cells were therefore not derived from the somitocoel. Second, the hypothesis was tested that the distribution patterns of the dying cells in the sclerotome are determined by factors outside the somite itself. Somites and segmental plates were transplanted into hosts in ovo with reversed orientation, after which the patterns of dying cells were examined using nile blue sulfate staining. The results indicated that the patterns were unchanged alter a further 2 days incubation, suggesting that the patterns of cell death in the sclerotome are not determined solely from within the somite. The distribution of the cell death-associated gene products, bcl-2, bax, and interleukin-1β converting enzyme, indicates that although these proteins are segmentally distributed in the dermomyotome and in the rostrodorsal quadrant of the sclerotome, their patterns are not directly correlated with the distribution of dying cells.