A Novel Cell-Type Specific Circadian Reporter Mouse Reveals Self-Sustained Food Entrainable Nature in Enteric Neurons

Global luciferase reporter gene technology is an important real-time readout for the analysis of circadian oscillations. However, because nearly all cells in the body possess cell-autonomous circadian oscillators, developing cell-type–specific reporter systems is essential to dissect how these oscillators interact within complex multicellular tissues and are modulated by brain-body circadian signals. The intestine is a complex organ composed of diverse cell types with distinct origins and functions, and it exhibits robust daily rhythms in physiological activities driven by intrinsic circadian clocks. While the circadian regulation of the mucosal compartment, including the intestinal epithelium, has been relatively well characterized, the mechanisms governing the circadian rhythms in the cells of the muscularis externa remain largely unexplored. Here, we report a novel Cre-dependent Per2-luciferase reporter mouse that enables precise measurement of circadian oscillations in specific peripheral cell populations ex vivo and demonstrate its utility in revealing the hierarchical circadian chrono-architecture within the intestine. Ex vivo gut explants from mice expressing the reporter in one of five major cell types of the muscularis externa—enteric neurons (ENs), enteric glial cells (EGCs), interstitial cells of Cajal (ICCs), smooth muscle cells (SMCs), and muscularis macrophages (MMs)—exhibited robust, self-sustained circadian bioluminescence rhythms, indicating that all of these cell types possess cell-autonomous circadian oscillators. Notably, ENs in the small intestine entrained more rapidly to feeding schedules than did those in the colon, revealing regional differences in food entrainment among the gut clocks. Moreover, the clocks of ENs, SMCs, and MMs shifted their phase in response to daytime-restricted feeding, whereas ICC clocks remained unaffected even after three weeks of restricted feeding. These findings demonstrate that distinct intestinal cell types possess unique entrainment properties and that feeding rhythms can induce heterogeneous phase shifts within the gut clocks. Given that circadian disruption, such as that caused by shift work, contributes to intestinal disorders, this reporter system could provide a powerful platform to dissect the mechanisms linking intercellular circadian desynchrony to intestinal homeostasis.