Discerning the Mechanisms of Compaction and Apical Domain Formation in Early Embryos. - How do individual cells self-organize into an epithelialized structure during early mammalian development, while simultaneously establishing distinct lineage identities? At the 8-cell stage, blastomeres undergo compaction and establish apical-basal polarity. During the transition to the 16-cell stage, polarized and apolar cells sort into distinct positions, forming the outer trophectoderm (TE) and inner cell mass (ICM). These processes rely on coordinated cell adhesion, cytoskeletal forces, and transcriptional programs activated during zygotic genome activation (ZGA). Despite extensive knowledge of mechanical signaling from other developmental contexts, how these cues interface with transcriptional programs during the earliest morphogenetic event of the mammalian embryo, compaction, remains incompletely understood. Our central hypothesis is that α-catenin functions as a mechanical gatekeeper during compaction, translating junctional tension into actomyosin polarization, while ZGA-activated transcription factors, such as Tfap2c and Tead4, coordinate with these mechanical cues to drive epithelial structure and lineage specification. Together, these systems couple mechanical and transcriptional signals to orchestrate the emergence of structure and cell fate. Aim 1 will define how junctional tension regulates actomyosin polarization during compaction. Using α-catenin conformational biosensors and mechanosensory- deficient mutants, we will test whether α-catenin senses mechanical forces at adherens junctions to modulate RhoA activity and apical contractility, revealing how cytoskeletal asymmetry and epithelial structure are established in response to force. Aim 2 will determine how α-catenin–mediated mechanosensation integrates spatial position with lineage identity. As blastomeres sort into inner and outer compartments, fate is directed by apical polarity, mechanical asymmetries, and Hippo signaling. This aim will assess how α-catenin conformation influences YAP localization and TE vs. ICM fate, using a combination of live imaging, FRET-based reporters, and computational modeling to infer and manipulate force landscapes. Aim 3 will determine how ZGA-induced transcription factors Tfap2c and Tead4 regulate compaction and apical domain formation. These factors are required and, with active RhoA, sufficient for precocious compaction and polarization. We will dissect their downstream effectors using gain- and loss-of-function approaches, and screen ~60 RNA-seq–identified targets for roles in cytoskeletal remodeling, adhesion, and polarity. Together, these studies will establish how gene expression and mechanical forces integrate to initiate epithelial morphogenesis and lineage segregation. By combining high-resolution imaging, biosensors, and targeted genetic perturbations, this work aims to clarify the earliest physical and transcriptional mechanisms underlying tissue organization in the early mammalian embryo. This research has broad relevance for developmental biology, cell biology, reproductive medicine, and stem cell engineering. Defects in compaction and polarization contribute to implantation failure and infertility; mechanistic insights may inform assisted reproductive technologies and improve stem cell–derived embryo models.
