The promise of embryonic stem (ES) cells in regenerative medicine is based on their potential to make every cell in the body, a property coined pluripotency. We still do not fully understand the molecules that underlie pluripotency. It is essential for us to do so, in order to improve on the generation and quality control testing of the embryonic stem cells. Exciting recent work has shown that modifications to the genome that do not change the actual DNA sequence, but do change how that sequence is presented, is a central component of pluripotency. These modifications have been coined epigenetic modifications because they are not altering the underlying genetic code. Specifically, it was recently shown that these epigenetic modifications maintain the stem cell’s capacity to proliferate while poising them to differentiate into all tissues of body. They do so by keeping the programs required for differentiation into adult tissues off, but still accessible to activation. Failure in establishment and/or maintenance of the correct epigenetic program leads to diminished pluripotency and even tumor risk. Unfortunately, very little is known about how the epigenetic program of embryonic stem cells is established and maintained. The purpose of this grant is to understand the role of different classes of small non-coding RNAs in early embryonic development and the establishment and maintenance of the epigenetic program of ES cells. Last year we discovered a diversity of classes of small RNAs in embryonic stem cells including canonical microRNAs, short hairpin RNAs, mirtrons and endogenous siRNAs (endo-siRNAs). The first three appear to be subclasses of microRNAs and likely act a similar fashion to repress the production of proteins that would alter the properties of ES cells. In contrast, how the endo-siRNAs act is unknown. In non-mammalian species they have been showed to regulate the epigenetic status of the cell. To determine their function in ES cells, we are deleting the genetic loci that produce them. Soon we will test what roles they have in ES cell function, the molecular constitution of cells, and the epigenetic modifications of the genome. At the same time as we published our discovery of endo-siRNAs in ES cells, two other groups found a large population of endo-siRNAs in unfertilized eggs (oocytes). To test the role of endo-siRNAs in oocytes, we made two mutants, one that removes all microRNAs and another that removes microRNAs and endo-siRNAs. By comparing these two mutants we can infer the role of endo-siRNAs. To our surprise, endo-siRNAs appear to be the central small RNA players in the oocytes. Even more shocking, the entire microRNA functional pathway appears to be silenced in oocytes and the very early embryo. We predict that this silencing of miRNA function may be central to vast potential of early embryonic cells. We are now working to see how miRNA function is silenced and will then determine how such suppression influences developmental potential.