SUMMARY The maternal to zygotic transition is a universal step in animal development characterized by the clearance of the maternally provided mRNAs and the activation of the zygotic genome. These two processes are intimately interconnected, as maternal factors drive the activation of the zygotic genes, and zygotic products actively target maternal mRNAs for deadenylation, repression and clearance. While recent studies have begun to identify individual factors that regulate mRNA stability and activation of the zygotic genome, we lack major understanding of 1) the regulatory code (sequences, structures and readers) that shapes genome activation and post-transcriptional regulation, 2) the mechanisms that regulate genome activation and protein output, and 3) how different regulatory mechanisms are integrated to instruct mRNA turnover, translation regulation and genome activation in the embryo. The overarching project combines massive parallel reporter assays to determine the regulatory activity of different sequences in the early embryo, protein interaction maps (at the level of the DNA and RNA) to define the factors that mediate transcriptional and post-transcriptional regulation, and novel imaging approaches to determine how pioneer factors shape chromatin structure and, in turn, genome activation. Together these experiments will define the mechanisms that trigger each of these steps in vivo and the gene regulatory network that controls early vertebrate development. This project is relevant for public health across different contexts. First, from the standpoint of human disease and cancer, pathways that control mRNA stability play an important role in aberrant oncogene activation in cancer and are relevant to changes in cell fate where the cells transition to a new program and remove the previous one through post-transcriptional regulation. Second, from the standpoint of reproductive health, infertility is estimated to affect 15% of reproductive aged women, and early pregnancy loss occurs in 25% of all pregnancies and up to 70% of pregnancies after in vitro fertilization. Understanding the mechanisms of zygotic genome activation and maternal mRNA decay can provide fundamental insights into human infertility and the development of tools to evaluate early loss of fertilized eggs. The results from this project will help us understand how gene expression is regulated in the early embryo to trigger the activation of different developmental pathways during embryogenesis, and more generally, will have broad implications in the study of genome activation and changes in cell fate.