Project Summary Small molecules are critical for proper regulation of stem cell behavior in multicellular organisms. Consequently, defects in the biosynthesis, perception, or metabolism of these compounds can cause developmental abnormalities and disease. Despite the critical importance of small molecules, the vast majority of our understanding of their functions is derived from indirect measurements. Typically, studies of small molecule biology are limited to genetic or biochemical approaches that ascribe functional roles to compounds based on the properties of the genes or proteins that interact with these molecules. Alternatively, small molecules are studied using chemical analysis approaches that homogenize bulk tissue and destroy the native context of the signals. High-resolution spatial information is critical in development, where stem cells comprise only a small fraction of the tissue. To enable deeper investigations of chemical regulation of stem cell behavior, my lab will apply technologies capable of directly measuring the localization and activity of small molecules in their native developmental contexts. This work will be done using plant roots, which are a powerful developmental system. Roots store all of their stem cells at the root tip, which generates a developmental gradient that can be examined in a single slice of tissue. My lab will leverage this gradient to investigate the role of small molecules in stem cell decisions. We will map the developmental chemistry of plant roots using mass spectrometry imaging and visualize small molecule interactions with proteins using a synthetic fluorogenic reporter. Metabolite-driven developmental mechanisms will be explored in depth by investigating citrate and retinaldehyde (retinal), two highly conserved metabolites with novel roles in root stem cell divisions and identity. This research will generate: 1) high-spatial resolution atlases detailing the chemical profiles of stem cell decisions, from regeneration to differentiation 2) novel insight into pathways that promote proliferation in stress-resilient stem cell subpopulations and 3) elucidation of dynamic metabolite-driven signaling pathways that regulate stem cell patterning. Our results suggest that there are many small molecules with important developmental roles that await discovery. Conducting research at the intersection of chemistry and developmental biology will provide mechanistic insight into stem cell decisions that would not be possible using a single-disciplinary approach. Accordingly, this work will enrich our understanding of the conserved and divergent principles that govern stem cell patterning, maintenance, divisions, and fate acquisition.