Collective organisation of the circadian clock in the intestinal epithelium: insights from an organoid-on-array model

The circadian clock orchestrates physiological and behavioral rhythms in living organisms over a period of approximately 24 hours. This vital adaptation allows mammals to optimize biological processes in response to the external environment and disruption of these rhythms has deleterious consequences on health. The circadian system consists of a complex network of oscillators present in nearly all cells; however, fundamental questions remain about how these circadian clocks collectively sustain organismal fitness. Growing evidence suggests that the mechanisms linking molecular clock, cell differentiation and cell-cycle are coupled during homeostasis and disease. However, most studies rely on population-level measurements, such as bulk sequencing or bulk recordings, without addressing single-cell or cell-type specific dynamics. Therefore, understanding circadian clock function at the organism-level first requires the characterization of how cells collectively oscillate at the tissue-level while differentiating and cycling.

An organ known to exquisitely recapitulate these processes daily is the intestine, which is characterized by high rhythmic turnover, absorption, motility and hormone secretion. To study the intestinal epithelium in culture, multicellular intestinal organoids represent a valuable asset for studying circadian interactions. Building on the fact that intestinal organoids display circadian oscillations in vitro and that circadian properties of different mammalian cell-types in the epithelium have not yet been addressed, we used mouse intestinal organoids to study the collective behaviour of circadian rhythms during proliferation and differentiation through qualitative and quantitative analysis. With the help of microfabricated hydrogels, genome-editing, bioluminescent and fluorescent live imaging, we developed a system to visualize, for the first time, circadian clock dynamics at cellular resolution in developing multicellular mouse intestinal organoids. In particular, microarrays of epithelial tissue support standardized bioluminescent recording of developing Per2::Luc organoids, revealing overall robust phase-coherent circadian oscillations. Microscopy revealed sparsity of PER2::LUC signals along the epithelium, highlighting variable mean levels and amplitudes. We identified that the phase and synchronization of the circadian clock depend on culture media composition. Additionally, we find that PER2::LUC levels correlate with enterocyte differentiation. Additionally, we inferred circadian properties for Paneth cells, LFABP+ enterocytes and cells in G1 phase, suggesting differences in circadian parameters between these cell-types and cell-state. Integration of an ASCL2 activity reporter for stem cells and progenitors revealed that these cells have distinct circadian clocks and oscillator properties. Similarly, homeostatic mini-guts exhibited sustained PER2::LUC rhythms along the epithelium, validating the circadian phenotypes obse