Project Summary To be a productive member of a collective, a cell must precisely and dynamically partition its own contents. Therefore, pathways that generate asymmetry in the cell, commonly referred to as polarity, are essential for development and homeostasis. How cells create de novo polarity and harness asymmetry to diversify cellular populations during morphogenesis remain important and open areas of study. The molecular logic that generates and sustains highly conserved polarity domains has been thoroughly studied in animals and yeast and has led to the creation of synthetic circuits capable of generating polarity in vitro. However, there are still significant challenges to interrogating these pathways in situ, where technical limitations make it difficult to track individual cells over days in developing animals as they transit through multiple identities. To overcome this hurdle, the proposed work will interrogate polarity pathways within developing plant tissues, where observation of subcellular dynamics can be paired to long-term tracking of full developmental decisions at single-cell resolution. Plants harness polarity for many of the same functions as animal cells, including regulation of asymmetric cell division and organelle positioning. Importantly, our recent progress investigating the formation and functions for cell polarity in developing Arabidopsis leaves highlights that these polarity circuits have both commonalities with and differences from canonical polarity pathways in animals. Therefore, our investigations will advance understanding of polarity mechanisms broadly and introduce an experimentally tractable and independently evolved system to test the generality of polarity models. Specifically, our aims are to 1) determine the molecular interactions that create polarity within leaf progenitors, 2) delineate the pathways that couple polarity to organelle topography for tissue formation, and 3) identify the control points where extrinsic signals regulate polarity pathways. We have developed imaging platforms, new genetic tools, and analysis pipelines that will allow us to make rapid progress on our aims. Taken together, we expect that we will identify novel means of harnessing polarity in cells, with potential future applications as tools to exert spatial control for bioengineering purposes and human health.