Autonomous and non-autonomous roles of DNase II during cell death in XXXX embryos.

@entity70071 @entity2199 @entity70072 @entity70070 @entity70073
UNASSIGNED: Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulfing cells (non-autonomous). The TUNEL method is used as an in situ assay of apoptosis by labeling DNA fragments generated by caspase associated DNase (CAD), but not those by the downstream DNase II. Here, we report a method of ToLFP (topoisomerase ligation fluorescence probes) for directly visualizing DNA fragments generated by DNase II in @entity2199 embryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells in @entity70070 embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals between @entity70070 and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was about 3 to 7. Among three DNase II mutant embryos ( @entity70071 , @entity70072 , and @entity70073 ), @entity70071 embryos exhibited the least number of ToLFP. The ToLFP results confirmed the previous findings that @entity70071 is the major DNase II for degrading apoptotic DNA. To further elucidate @entity70071 's mode of action, @entity70071 -rescuing transgenic worms that ectopically express free or membrane-bound forms of @entity70071 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed @entity70071 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA.
