Endogenous Expression and Subcellular Localization of Core Apoptosis Regulators Reveal Key Differences Between Embryonic and Germline Apoptosis in C. elegans

Apoptosis is a highly conserved form of programmed cell death controlled by a core molecular pathway that was first defined in Caenorhabditis elegans and is conserved in mammals. This pathway is composed of egl-1/ BH3-only, ced-9 /Bcl-2, ced-4 /Apaf-1, and ced-3/ Caspase. Despite being discovered more than 20 years ago, tissue-specific apoptosis induction as well as endogenous expression pattern and dynamic subcellular localization of apoptosis proteins remain incompletely defined. Here, we generated a complete set of CRISPR/Cas9-engineered transcriptional and translational reporters for all four apoptosis genes and systematically analyzed their expression and subcellular localization in the C. elegans germline and embryo. We show that somatic apoptosis is driven by precise, lineage-specific activation of egl-1 , whereas ced-9 , ced-4 , and ced-3 are ubiquitously expressed. In contrast, DNA-damage triggers a robust CEP-1/p53-dependent-induction of egl-1 throughout the germline, yet apoptosis occurs only in late pachytene cells. We also identify intron1 of egl-1 as essential for CEP-1–dependent transcriptional activation. Analysis of brc-1 and syp-2 mutants demonstrates that distinct meiotic surveillance pathways converge on egl-1 induction. Analysis of the subcellular localization of the downstream regulators CED-9, CED-4, and CED-3 reveals dynamic, tissue-specific localizations that refine the classical apoptosis model. CED-4 transitions from a perinuclear distribution in the germline and early embryos to a predominantly mitochondrial localization later in embryogenesis, while CED-3 changes its subcellular localization depending on developmental stage and apoptotic status. CED-9 localizes to distinct mitochondrial foci in both embryo and germline. Together, these reporters reveal that C. elegans apoptosis is governed by two mechanistically distinct programs: (1) lineage-specific egl-1 activation in embryos and (2) checkpoint-mediated activation of egl-1 in the germline, where additional, yet unidentified pathways restrict apoptotic execution. These reporters also provide a comprehensive toolbox for dissecting apoptotic and non-apoptotic functions of the conserved apoptotic machinery in vivo .