Dissecting circadian chronoarchitecture of the intestine - Project Summary The circadian clock is the fundamental timing system that regulates daily rhythms of physiology and behavior, enabling organisms to anticipate and adapt to the day-night cycle. Although the molecular machinery of cellular clocks is well characterized, how these clocks are organized within peripheral tissues remains largely unexplored. The intestine provides an ideal system to address this gap due to its remarkable cellular diversity and physiological complexity: it comprises enteric neurons, glial cells, smooth muscle cells, macrophages, and interstitial cells of Cajal (ICCs), each performing distinct roles in digestive, motility, and immune functions. Furthermore, intestinal physiology is closely linked to feeding-fasting cycles, a potent environmental cue that can influence the timing and coordination of circadian clocks within peripheral organs. Clinical studies underscore the importance of circadian regulation in intestinal health. Circadian disruption, such as that experienced by shift workers or individuals with irregular sleep-wake schedules, has been associated with increased risk of gut motility disorders, inflammatory bowel disease, and other gastrointestinal dysfunctions. These observations highlight the need to understand how intestinal clock networks maintain synchrony under normal conditions and how their disruption contributes to disease. To dissect the organization of gut clocks, we generated a novel reporter mouse in which click beetle green luciferase is expressed as a fusion with the endogenous PER2 protein upon Cre-loxP–mediated recombination, allowing cell-type–specific monitoring of circadian gene oscillations. Using ex vivo gut explants, we demonstrated that all major intestinal cell types harbor robust, self-sustaining circadian oscillators. Strikingly, in vivo feeding manipulations revealed heterogeneous entrainment properties: under daytime-restricted feeding, the circadian phase of enteric neurons shifted rapidly, while the circadian phase of ICCs remained unchanged. These findings indicate that gut clocks are not uniformly regulated and suggest that certain cell types, particularly enteric neurons, may function as circadian pacemakers within the intestinal tissue networks. Building on this foundation, the proposed studies aim to elucidate the hierarchical organization of the gut clocks, determine how these clocks adapt to environmental perturbations such as shifted light-dark cycles, and investigate how heterogeneous phase responses among gut clocks influence organ-level physiology and intestinal disease susceptibility. By linking cellular clock dynamics to gut physiology and disease outcomes, this work will provide critical insights into the circadian regulation of the intestine. Successful completion of these studies will not only advance fundamental understanding of peripheral clock networks but also inform novel strategies to preserve circadian homeostasis, improve gastrointestinal health, and reduce the burden of circadian-related intestinal disorders in vulnerable populations, including shift workers and patients with chronic gastrointestinal disease.
