PROJECT SUMMARY Over 1 in 4 American workers are shift workers with frequent disruptions in their circadian rhythms and increased risks of gastrointestinal (GI) diseases. Circadian rhythms influence fundamental GI processes, including digestion, immunity, and regeneration of the GI epithelium. Thus, disruption of circadian rhythms is associated with GI diseases ranging from peptic ulcers to cancer. Recent studies demonstrated that the circadian clock influences the cell cycle and vice versa, but the nature and consequences of this connection remain largely unknown. One major limitation is the lack of a tractable model system to investigate the molecular basis of these interactions. To address this, we developed 3D mouse enteroids, or an in vitro “mini-gut”, as a system to identify the molecular mechanisms and consequences of this coupling. This system allows manipulation and tracking of both cell cycle and circadian clock components as gut stem cells undergo differentiation into specialized cells. Importantly, we have demonstrated that mouse enteroids possess robust circadian rhythms, and mimic the response of gene ablation that is observed in mouse intestine. Specifically, we demonstrated that the circadian clock in Paneth cells regulate coordinated cell division cycles via intercellular WNT signaling in enteroids, and elimination of circadian rhythms results in disrupted development of intestinal crypts. In the proposed project, we will determine: (1) cell type-specific roles of circadian rhythms in the small intestine, (2) WNT signaling-mediated intercellular coupling between the GI circadian clock and cell cycle, (3) circadian clock-regulated epithelial barrier function, and (4) circadian clock and Paneth cell- mediated innate immune response to Salmonella infection. Our results will lay a foundation for identifying potential targets and temporal regimens to restore circadian clock-dependent adult stem cell regeneration, epithelial barrier function, and innate immune response for GI-related diseases.