Developmental activation of the capability to undergo checkpoint-induced apoptosis in the early zebrafish embryo

In this study, we demonstrate the developmental activation, in the zebrafis h embryo, of a surveillance mechanism which triggers apoptosis to remove da maged cells. We determine the time course of activation of this mechanism b y exposing embryos to camptothecin, an agent which specifically inhibits to poisomerase I within the DNA replication complex and which, as a consequenc e of this inhibition, also produces strand breaks in the genomic DNA. In re sponse to an early (pre-gastrula) treatment wi th camptothecin, apoptosis i s induced at a time corresponding approximately to mid-gastrula stage in co ntrols. This apoptotic response to a block of DNA replication can also be i nduced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydro xyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this re sponse is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which invo lves cell membrane blebbing, "margination" and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but n ot by the caspase-2,3,7[,1] inhibitor Ac-DEVD-CHO. This suggests a function al requirement for caspase-4 or caspase-5 in the apoptotic response to camp tothecin. Recently, Xenopus has been shown to display a developmental activ ation of the capability for stress- or damaged-induced apoptosis at early g astrula stage. En masse, our experiments suggest that the apoptotic respons es in zebrafish and Xenopus are fundamentally similar. Thus, as for mammals , embryos of the lower vertebrates exhibit the activation of surveillance m echanisms, early in development, to produce the selective apoptosis of dama ged cells. (C) 1999 Academic Press.