Hypoxic preconditioning (HP) is a phenomenon observed in various vertebrate tissues whereby a sub-lethal exposure to hypoxia protects cells from dying after a subsequent, normally lethal, hypoxic insult. We have found that C. elegans is also capable of HP. A 4-hour hypoxic preconditioning exposure results in an approximately 2-fold reduction in whole C. elegans death after a subsequent lethal hypoxic exposure; hypoxia-induced pathological changes in body wall myocytes and subsets of neurons are also significantly reduced by HP. This protection by HP is slow in onset, not appearing for at least 6 hrs, peaks by 16 hrs, and does not disappear completely until greater than 30 hrs later. This time course is consistent with delayed HP, a form of HP found in vertebrate cells to be dependent on new transcription and translation. Unlike immediate HP, delayed HP is poorly understood. To define the mechanisms of HP in C. elegans, we are testing the role of candidate genes along with performing DNA microarray experiments. The one set of genes that we have found to be required for HP are a subset of the C. elegans programmed cell death (PCD) genes. All loss-of-function
ced-4 alleles tested were HP defective with no significant protection conferred by HP incubation of adult animals. The HP-defective phenotype of
ced-4(
n1162) was rescued by transformation with
ced-4(+). During developmental PCD, CED-4 is negatively regulated by CED-9, which in turn is negatively regulated by EGL-1. This pathway regulates the activity of CED-3 caspase. Consistent with this pathway, both
egl-1(lf) and
ced-9(lf) alleles were HP defective. However distinct from the classical PCD pathway, multiple
ced-3(lf) alleles were capable of a normal HP response. Interestingly, all loss-of-function alleles of
egl-1,
ced-4, and
ced-3 along with
ced-9(gf) were similarly and significantly hypoxia resistant without HP, indicating that the PCD pathway is active in adult animals and is responsible for a portion of hypoxic death. Examination of microarray data comparing whole genome expression with and without HP indicates that global expression of the components of the PCD pathway is not altered by HP. Thus, transcription of
egl-1,
ced-9, and
ced-4 do not appear to be altered by HP; rather, a model where an upstream HP-induced signal that is mediated by these proteins acting independently of CED-3 is more likely.