PROJECT SUMMARY Plants have exceptional capacity to regenerate adult body parts after injury, providing an excellent conceptual model for organ replacement. They can even rapidly reform their adult stem cell niches after complete excision. My lab has pioneered new systems and techniques to study plant regeneration. We developed methods for tracking cell identity of reprogramming cells using transcriptomic and live imaging techniques during regeneration. In connection with this work, I have led efforts to introduce new technologies like single-cell RNA-seq into plant research. Using these and other techniques developed under NIGMS R01GM078279, we found that regenerating roots appear to recapitulate embryonic programs during organ regeneration. The work has led to us to propose models of how this highly self-organizing system orchestrates the regeneration of an entire organ and its stem cell niche. However, while new tools like in single-cell RNA-seq provide powerful approaches to dissect developmental programs cell-by-cell, they are largely silent on the role of cell-cell communication in mediating tissue assembly. Hence, our current efforts are focused on developing new approaches to address this gap. In one proposed system, we inducibly block symplastic (direct) communication channels in a cell type-specific manner and use single- cell RNA-seq to test the consequences on neighboring and distant tissues during normal post-embryonic organogenesis and regeneration (Block-Seq). This is akin to observing the consequences of signaling from one cell to all its neighbors but not the signals themselves. One common mode of signaling in plants is the protein-level movement of transcription factors from one cell to its neighbors. To track protein movement comprehensively, we are developing a click chemistry approach to incorporate labeled amino acids in specific cell types followed by capture and mass spectrometry of the mobile proteins in neighboring cell types (the Mobilome). This approach is designed to reveal many of the signals that plant cells use to organize development. Overall, these approaches are motivated by a basic question that underpins a medically relevant question: how do positional signals generate a set of rules that allow the flawless reconstruction of organs in regeneration?