The C. elegans germline contains an adult stem cell niche in which germ stem cells divide continuously during the larval and adult stages. We created a primary culture system for C. elegans germ cells. The tissue culture media we developed allows germ cells isolated from tumorous germline mutants to remain alive in culture for over four weeks. This culture system was used to identify two novel regulators of germ stem cell proliferation: dafachronic acid, and bacteria-derived folates. Dafachronic acid is a sterol-derived hormone that is a ligand for the nuclear hormone receptor DAF-12. We found that the addition of dafachronic acid inhibits DNA replication in isolated germ cells. Further, the addition of dafachronic acid inhibits the growth of germ cell tumors in germline tumor mutants, and reduces the number of mitotic germ cells in wild-type animals. Our results indicate that dafachronic acid negatively affects germ cells in a cell-autonomous and DAF-12-dependent manner.
We discovered that the addition of bacterial extract increases the rate of DNA replication in isolated germ cells. Stimulating bacteria to increase the production of folates prior to creating the bacterial extract enhances its activity. Feeding germline tumor mutants a diet of bacteria with increased folate production increases tumor growth. This diet also increases the percentage of mitotic germ cells in wild-type animals. Conversely, inhibiting folate production in bacteria reduces the efficacy of bacterial extract and inhibits germ cell proliferation in vivo. Folates are a family of structurally-related B-group vitamins that are required for one-carbon metabolism. Purified folates isolated from bacteria stimulate DNA replication in vitro and germ cell proliferation in vivo. The E. coli bacteria strains OP50 and HT115(DE3) and the Comamonas aquatica strain DA1877 have notable differences in their distributions of folate species, and in the abilities of the folates to stimulate germ cell proliferation. Our work therefore provides evidence for an exogenous signal linked to C. elegans’ diet that stimulates the proliferation of an adult stem cell population.
In the context of other published work, our results allow the key insight that three signals, dafachronic acid, bacteria-derived folates, and insulin, mediate a critical binary decision between lifespan extension and germ cell proliferation.
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