Project Summary Important Problem. A critical event in human embryonic development is the elongation of the anterior-posterior axis. How the embryo achieves stable and sustained axial elongation remains unknown. Progenitor cells in the tailbud of the elongating embryo contribute to the future spinal cord and musculoskeletal system (1-4). How underlying gene regulatory networks and morphogen signals drive the fate decisions of these cells between self- renewal and differentiation in humans still needs to be determined. Addressing these questions will allow us to understand human axial elongation and the etiology of related diseases (5-11). We address two questions: How do morphogen signals break anterior-posterior symmetry and drive stable and self-sustained elongation along a single axis? And, how do the progenitors in the tail bud of the elongating embryo maintain a self-renewing pool even as they differentiate into neural and mesodermal fates? Proposed Research: To address these questions, we bring together a robust and validated in vitro organoid system, live imaging, validated pluripotent stem cell lines with multiple fluorescent reporters, and perturbation experiments. In Aim 1, we will determine 1A. the cell types and the spatiotemporal expression profiles of signaling ligands and receptors along the axially elongating organoids; 1B. the signaling pathways necessary for A-P symmetry breaking, driving cellular rearrangements, growth, and elongation; 1C. the endogenous spatiotemporal dynamics of these signaling pathways during axial elongation; 1D. the mechanisms for robustly breaking symmetry and activating self-sustaining elongation only at the posterior pole without being driven by noise to produce multiple tailbuds and branched axes. In Aim 2, we will determine 2A. The sequence of lineage decisions that tailbud progenitors make; 2B. The transcription factors (TFs) that control the fate decisions of these progenitors; 2C. the key signals are required to choose between the maintenance of progenitors and their differentiation into neural or mesodermal fates; and 2D. Mechanisms by which the signals and TFs maintain a pool of undifferentiated tailbud progenitors to allow continued elongation. Impact: Our work will uncover the dynamical system that governs human axial elongation and the fate decisions of tailbud progenitors. In doing so, it will find the feedback loops and concomitant bistable systems that lead to self-sustaining elongation, noise suppression mechanisms to prevent axis branching, which otherwise would lead to a multiple and branched axes, the mechanism that maintains a niche of tailbud progenitors that is essential for continued elongation, even while allowing these progenitors to differentiate into the developing tissues. We will thus achieve a quantitative understanding of human axial elongation and determine how the niche of tailbud progenitors is maintained. The research will reveal fundamental principles governing human de...