Project Summary The broad, long-term objectives of this application are to characterize how age-related intrinsic changes in gene expression and age-related extrinsic changes in the niche microenvironment impact the process of dedifferentiation. This proposal will examine these issues using spermatogonial dedifferentiation - in which differentiating germ cells revert to become spermatogonial stem cells - in the Drosophila testis. State of the art techniques will be used to detect genome-wide binding of RNA Polymerase II in dedifferentiating spermatogonia. The proposal will capitalize upon the powerful genetics and lineage tracing available in Drosophila, as well as the ability to unequivocally identify the niche, spermatogonial stem cells, spermatogonia and somatic support cells in the Drosophila testis. This proposal is supported by published results demonstrating that spermatogonial dedifferentiation is required to maintain a robust pool of spermatogonial stem cells over a lifetime and that this process requires Jun N-terminal kinase (JNK) signaling. This work is also supported by unpublished preliminary data showing that the rate of spermatogonial dedifferentiation declines steeply during aging. Additionally, this proposal is supported by unpublished results showing that the niche microenvironment changes substantially during aging: in old males, an ectopic extracellular matrix accumulates around the niche. In the first aim, targeted DamID will be used to obtain the genome-wide binding of RNA Pol II only in reverting spermatogonia in testes from young and old males. Analyses of these data sets will reveal genes that are activated in young but not old spermatogonia. In the second aim, one or two validated genes from Aim 1 will be used to test the model that candidate gene depletion in young testes should decrease dedifferentiation rates and its overexpression should increase reversion rates in old males. Experiments in the second aim will also test the hypothesis that decreased dedifferentiation in old males is a result of reverting cells not upregulating extracellular matrix-binding proteins. The studies in this proposal will increase the knowledge base about how aging affects dedifferentiation in more complex system and will foster new avenues of research into mechanisms and treatments for age-related male infertility.