During the earliest stages of embryonic development, the totipotent zygote proliferates and differentiates, generating diverse populations of cells that will ultimately form the organs and tissues of a mature organism. These early events dictate the course and success of the developing organism. Thus, it is critical that the pathways regulating these early developmental transitions are elucidated in order to diagnose and understand early pregnancy loss, as well as to advance stem cell technologies and infertility therapies. Most studies have concentrated on the role of transcriptional regulation, which fails to account for the fact that gene expression is dictated as much by the rate of mRNA decay as by the rate of RNA synthesis. In this proposal, we investigate the role of a conserved and highly selective RNA degradation pathway - Nonsense-Mediated RNA Decay (NMD). The overarching hypothesis of this proposal is that NMD is critical for early embryo development because it influences specific differentiation events through its ability to regulate the decay rate of key RNA transcripts in a stage-specific manner. In support, NMD degrades RNAs encoding developmental regulators and mouse KO studies have demonstrated that global loss of several NMD factors leads to early embryonic lethality, with defects evident during pre- and peri-implantation stages. To date, no studies have examined the underlying mechanism. Another outstanding issue in the field is how NMD is regulated. This is critical to understand, as shifts in NMD magnitude during development are predicted to trigger alterations in the stability of scores of RNAs. A breakthrough is our recent discovery of a potent repressor of NMD – UPF3A. Undetectable in most adult tissues, UPF3A is highly expressed in the early embryo, and loss of UPF3A in mice leads to lethality during the peri- implantation stage of embryo development. The first Aim of this proposal is to use existing mouse KO models to elucidate the roles of NMD—including the necessity of its repression by UPF3A—in the developmental progression of early embryos in vivo. To pinpoint NMD's mechanism of action, we will use single-cell transcriptome analysis, as this will allow us to (i) identify the specific embryonic cell subsets acted upon by NMD and UPF3A, (ii) define the repertoire of mRNAs degraded by NMD in the cell subsets in which NMD acts, and (iii) identify shifts in NMD activity that occur within the embryo as development proceeds. The second Aim is to elucidate the molecular mechanisms underlying NMD's essential roles in early embryogenesis. Leveraging our discovery that NMD is critical for dictating germ layer cell fate in embryonic stem cells (ESCs), we will use “mimic” and “rescue” approaches to identify the specific mRNAs that must be degraded by NMD to drive hESC differentiation decisions. To understand how NMD regulation influences these events, we will study the developmental and molecular roles of the NMD repressor, UPF3A...