We use C. elegans as a model to study the biological roles of NAD+ biosynthetic pathways. Vitamin B3 precursors for NAD+ biosynthesis, such as nicotinamide riboside (NR), have garnered a lot of attention recently because of their ability to significantly raise NAD+ levels when supplemented to animals or cells, to apparent therapeutic benefit. To investigate the normal biological roles of this NR pathway, we have examined animals with mutations in the gene coding for nicotinamide riboside kinase (nmrk-1 / T27A3.6), an enzyme required to process NR for NAD+ biosynthesis. We frequently use UV-killed OP50 as a food source to prevent the bacterial food from metabolizing vitamin B3 supplements that we add to cultures. Upon growth of nmrk-1(ok2571) on UV-killed OP50, but not live OP50, we noted a surprising phenotype. Hatching is delayed by up to 10 days. The phenotype is specific to NAD+ biosynthesis from NR and is not a feature of mutants that affect other NAD+ biosynthesis pathways or combinations of pathways. The phenotype is leaky, with approximately 25% of the brood hatching in the expected time frame. 25% of the brood hatches between 3 and 10 days after egg laying, and about half of the brood never hatches. Embryogenesis is not delayed and animals maintain normal embryonic movements over the course of their time in the eggshell. By switching conditions of the mothers or the eggs from live to UV-killed OP50, we found that the mother must experience the UV-killed environment for the embryos to manifest the phenotype. Using global metabolomics profiling to compare populations of animals grown on live versus UV-killed OP50, we have identified metabolic signatures, including increased oxidative stress, associated with growth on UV-killed food. Thus, we tested if this feature was relevant to the delayed hatching phenotype. We find that treatment with paraquat can substitute for the requirement for UV-killed food to reveal the delayed hatching phenotype of the nmrk-1 mutants. Our observations are consistent with a hypothesis that the animals are physically unable to hatch, perhaps because of an altered eggshell.

Affiliation:
- The Pennsylvania State University, University Park, PA