Regulated RNA Decay in Totipotent State Transitions - PROJECT SUMMARY Totipotency is the earliest and most unrestricted developmental state, defined by the capacity of a single cell to generate all embryonic and extraembryonic tissues. A transient state, totipotency is limited to the zygote (1-cell) and 2-cell (2C) stages in mice and the 1-cell to 8-cell stages in humans. Failures during the transition from totipotency to pluripotency represent a major developmental bottleneck, with nearly half of human IVF embryos arresting before or during this shift, underscoring the need to understand the molecular pathways that govern it. Although pluripotent stem cell states have been well characterized, mechanisms controlling entry into, maintenance of, and exit from totipotency remain poorly understood. A powerful model for studying these transitions arises from the discovery that mouse embryonic stem cell (mESC) cultures contain rare, spontaneously cycling 2-cell–like cells (2CLCs) that transiently activate Zscan4, MERVL, and other zygotic genome activation (ZGA)-associated genes while adopting chromatin and transcriptional features characteristic of totipotent embryos. Preliminary data suggests that post-transcriptional RNA regulatory pathways, particularly those influencing RNA turnover and stability, play central roles in coordinating these transitions. Specifically, perturbing these pathways in vitro increases the fraction of cells entering the 2CLC state, while manipulation in 1-cell embryos permits cleavage but results in developmental arrest coupled with failure to initiate ZGA and transition to pluripotency, supporting a model in which regulated RNA clearance and stability shape the timing and fidelity of early totipotent transitions. This fellowship will define how RNA stability mechanisms govern totipotent–pluripotent transitions using two complementary systems. Experiments in Aim 1 will determine how shifts in RNA stability control 2CLC transitions in mESCs. These analyses will take advantage of new approaches that integrate metabolic labeling and single cell RNA sequencing to examine changes in RNA transcription and stability transcriptome wide during totipotency entry and exit. Experiments in Aim 2 will determine how shifts in RNA stability govern ZGA and totipotency exit in embryos. Taking advantage of highly sensitive pull-down approaches, these analyses will uncover how changes in RNA stability shape transcript clearance, ZGA onset, and early developmental progression. Together, these studies will reveal fundamental RNA-stability networks that govern cell identity during the first hours of mammalian development and provide key insights relevant to embryo arrest, infertility, cellular reprogramming, and regenerative medicine. Training will occur within an interdisciplinary environment with expertise in embryogenesis, RNA decay, and RNA/isoform-resolved analysis, 3D chromatin architecture. The fellowship provides focused training in RNA-stability, single-cell, and isoform- resolved approaches, supported by the genomics, imaging, and computing resources at the Sanford Consortium. Together, this setting provides the comprehensive scientific and professional development needed for an independent physician-scientist career.
