To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year, we have made great progress in two major fronts. First we dissected the role of a key family of miRNAs expressed in early mammalian embryos. These miRNAs are expressed from two different locations in the genome, which are expressed at different times and places. Removal of one of these two loci results in a neural tube defect, a common developmental disorder of humans. We have dissected the mechanisms of this defect providing broad insights into how genes and cellular processes function together to form a proper neural tube. These insights provide a major leap forward in our understanding how neural tube defects in humans may occur. Second, by dissecting the regulation of expression of the two miRNA locations and more broadly the cell types that they mark, we have made a major advance in the understanding of how the developmental potential of cells are retained during the early stages of embryonic development. We have learned how epigenetic factors and transcription factors work together to ensure that genes, which must be activated at later developmental stages, are brought to the starting line prior to the opening of the gates. Without such “priming” of the genes, they cannot be activated and thus development cannot precede. It is very likely similar mechanisms are associated with failed pregnancies. This work concludes the funding period of this grant. The many discoveries we have made and published over the length of this grant should have a profound impact on how we can manipulate cells to understand and treat a variety of diseases.