ABSTRACTThe goal of the work outline in this proposal is to understand the fundamental signaling that controls cell fate tomaintain tissue homeostasis. Epithelial tissues demonstrate an intrinsic ability for their constituent cells toorganize and maintain a steady-state of form and function. Many disease states lack these intrinsic controls.Epithelial tissues of the human body are in a constant state of renewal. Our understanding of the signalingsystems that control how these complex epithelial tissues maintain robust organization is incomplete. Essentialfor progress we need a quantitative understanding of signaling at the single-cell level in the context ofphysiological conditions to reveal systems-level behaviors that can be targeted therapeutically. We focus onprotein kinases as critical mediators of signaling in the cell which are well known to play prominent roles intissue function and drivers of disease. Our kinome-wide studies have identified GSK3 and CLK3 as majortissue homeostasis regulators that govern the balance between proliferation and differentiation. In Project 1we hypothesize GSK3 requires multiple suppressive inputs that uniquely produce different fate outcomesranging from stem cells transit-amplifying and differentiated. We will provide the first systems-level mappingfor multiple inputs onto GSK3 dynamics and how these dynamics are decoded into distinct cellular outcomesof renewing epithelium. In Project 2 we hypothesize CLK3 is a gatekeeper controlling stem cell fate throughtranscriptome regulation. We will define the activity of CLK3 in the stem cell niche as a regulator of expressionand splicing of Wnt-target genes to promote stemness. Our approach uses high-throughput quantitativemicroscopy to measure single-cell behaviors in physiological organoid homeostatic culture models. Ourresearch will define the regulatory mechanisms for two critical kinases GSK3 and CLK3 and discover novelsignaling circuitry needed for the accurate organization of renewing and regenerative epithelia uncovering newstrategies for treating diseases of regenerative tissues.