DYNAMIC HETEROGENEITY IN THE FATEDECISIONS OF EARLY MAMMALIAN EMBRYOGENESIS - PROJECT SUMMARY THE MAMMALIAN EMBRYO IS A SELF-ORGANIZING SYSTEM THAT ROBUSTLY PATTERNS ITSELF, FORMING A BLASTOCYST WITH THREE TYPES OF CELLS ESSENTIAL FOR FUTURE STEPS OF DEVELOPMENT. FORMATION OF THE BLASTOCYST IS AN IMPORTANT AND CONSERVED MILESTONE IN THIS PROCESS. TWO SEQUENTIAL FATE DECISIONS GIVE RISE TO THE THREE CELL TYPES THAT COMPRISE THE BLASTOCYST: CELLS ON THE OUTSIDE ARE FIRST SEPARATED FROM THOSE ON THE INSIDE, THE INNER CELL MASS (ICM), THAT THEN DIFFERENTIATES INTO THE EPIBLAST (EPI) AND PRIMITIVE ENDODERM (PE). IDENTIFYING HOW AND WHEN THESE DECISIONS ARE MADE HAS BEEN DIFFICULT TO RESOLVE DUE TO THE HIGHLY STOCHASTIC AND DYNAMIC NATURE OF EARLY MAMMALIAN DEVELOPMENT. LIVE IMAGING APPROACHES HAVE SHED LIGHT ON THESE CELL TRAJECTORIES, WITH RECENT WORK SHOWING THAT THE SECOND FATE DECISION BEGINS WITH THE COMMITMENT OF A SINGLE EPI LINEAGE. THIS LINEAGE ITSELF BREAKS SYMMETRY OF THE BIPOTENT ICM BUT IS ALSO A SOURCE OF INTERCELLULAR SIGNALING THAT INDUCES PE FATE IN SURROUNDING CELLS. STRIKINGLY, THIS LINEAGE CAN BE TRACED BACK TO AN EARLIER HETEROGENEITY IN THE ICM BUT HOW THIS DIFFERENCE ARISES IS UNKNOWN. THE PROPOSED WORK SEEKS TO DETERMINE THE FEATURES OF THE EARLY BLASTOMERES THAT BIAS THEM TOWARDS THE EARLIEST EPI LINEAGE AND TO CHARACTERIZE THE FUNCTIONAL ROLE OF THIS PRE-PATTERNING. AIM 1 WILL USE LIVE IMAGING OF ENDOGENOUS REPORTERS THAT VISUALIZE BLASTOMERE VOLUMES, DIVISIONS AND CELL CYCLES ALONGSIDE TRANSCRIPTION FACTOR DYNAMICS TO TRAIN A STATISTICAL MODEL THAT PREDICTS GENE EXPRESSION FROM CELL HISTORIES. THIS AIM WILL IDENTIFY THE DYNAMIC INPUTS THAT TRIGGER SYMMETRY BREAKING WITHIN THE ICM. AIM 2 WILL PRECISELY TARGET THE EARLY MOLECULAR ASYMMETRY OF THE ICM TO DISRUPT THE FIRST EPIBLAST LINEAGE AND THE SEPARATION OF CELL FATES IT INDUCES. THE IMPACT OF THIS PERTURBATION WILL BE QUANTIFIED BY LIVE IMAGING MARKERS OF THE SECOND FATE DECISION OVER TIME. THE WORK DESCRIBED IN AIM 2 WILL SHOW HOW HETEROGENEITY IN THE EARLY ICM INITIATES AND PROPAGATES LATER CELL FATE COMMITMENT. THE PROPOSED STUDIES, WHICH ARE SUPPORTED BY NOVEL FLUORESCENT REPORTERS AND AN ESTABLISHED IMAGE ANALYSIS PIPELINE, WILL PROVIDE A DEEPER UNDERSTANDING OF THE DECISION CIRCUITS AND CELL SIGNALING NETWORKS THAT GOVERN THIS CRITICAL STAGE OF MAMMALIAN DEVELOPMENT.