Individual hepatocytes in the mammalian liver harbor cell-autonomous circadian oscillators, yet across the liver lobule their clock-gene expression oscillates in synchrony with a period near 24 hours. What intercellular signal carries phase information between hepatocytes, and how it acts on the core clock network, is unknown: TGF-β (transforming growth factor β, a secreted paracrine signaling protein), identified as a peripheral coupling factor only in cell culture, remains an unverified candidate alongside gap junctions, pannexin hemichannels, FGF21, and mechanical signaling. The problem, posed by a 2024 multiscale model of the liver clock (Marri et al., bioRxiv) that had to assume a generic coupling ligand, is to identify the coupling factor(s) operating in the intact liver and establish their mechanism of action on the clock gene network. A September 2025 review by the field's leading group (Schibler et al., PNAS) confirms the mechanism remains elusive.

Background

Circadian (~24-hour) rhythms in mammals are generated by a transcriptional-translational feedback loop (TTFL) operating in nearly every cell type: the heterodimeric transcription factors CLOCK and BMAL1 bind E-box DNA motifs to activate transcription of the Period (Per) and Cryptochrome (Cry) genes, whose protein products PER and CRY feed back to repress their own synthesis, while nuclear receptors ROR (activator) and REV-ERB (repressor) compete for binding sites regulating Bmal1 transcription. In the brain's suprachiasmatic nucleus (SCN), the master circadian pacemaker, thousands of neuronal oscillators are phase-coupled to each other by neurotransmitters such as vasoactive intestinal peptide (VIP) and by gap junctions (direct aqueous channels connecting the cytoplasm of neighboring cells). Clocks in peripheral organs such as the liver are synchronized by systemic cues (zeitgebers, i.e., time-giving signals), prominently daily feeding-fasting cycles driven by the SCN. The liver's functional unit is the lobule: hepatocytes, its parenchymal cells, are arranged along a portal-to-central-vein axis along which metabolic functions are spatially graded (zonation), for example gluconeogenesis near the portal end and lipogenesis near the central end. Whether hepatocytes also couple to one another locally, like SCN neurons, was long controversial. Three observations define the problem. First, single-nuclei RNA sequencing (transcriptome profiling of individual isolated nuclei, here combined with single-molecule fluorescence in situ hybridization, smFISH, which visualizes individual mRNA molecules in tissue) showed that core clock-gene expression is non-zonated and temporally synchronized across the lobule despite cell-to-cell variability (Droin et al. 2021). Second, long-term in-vivo bioluminescence recordings from freely moving mice showed that circadian liver oscillations and their phase coherence persist even in animals lacking an SCN or any oscillators outside hepatocytes, implying hepatocytes exchange phase information locally (Sinturel et al. 2021); however, isolated hepatocytes in culture are only weakly coupled and cannot synchronize cells with differing periods (Guenthner et al. 2014). Third, the molecular identity of the coupling signal has been approached in vitro: TGF-β (transforming growth factor β, a secreted paracrine growth factor acting through SMAD transcription factors) was shown to mediate paracrine phase adjustment of circadian oscillators in two- and three-dimensional cultures, mainly of human U-2 OS cells (Finger et al. 2021); TGF-β/activin can reset the clock through rapid induction of the Dec1 transcription factor (Kon et al. 2008); and TGF-β/SMAD signaling itself oscillates in mouse liver (Sato et al. 2012). But none of this establishes which signal synchronizes hepatocytes in the intact liver. Against this background, Marri et al. (2024, bioRxiv) built a multiscale computational model: an ordinary-differential-equation clock network for six core clock genes (Clock, Bmal1, Per, Cry, Ror, Rev-erb) with 23 species per cell, embedded in a 435-hepatocyte spatial lobule, coupled through the spatially averaged concentration of an unnamed 'coupling ligand' acting through unspecified receptors. Simulations showed coupling was needed to lock oscillation periods near 24 hours (without it, simulated periods drifted over 21-28 hours), parameters were fitted to single-nuclei mRNA data only, and one-parameter bifurcation analysis (tracking how a system's limit-cycle oscillations appear or vanish as a parameter crosses a Hopf bifurcation point) predicted that reducing Cry or Ror transcription rates abolishes oscillations. The model thereby quantifies why an intercellular coupling mechanism must exist in the liver, while leaving the mechanism itself generic and unidentified.

Problem Statement

Identify the intercellular coupling mechanism that synchronizes autonomous hepatocyte circadian oscillators across the mammalian liver lobule in vivo, and establish it quantitatively. That local coupling exists is a premise of the question: circadian liver oscillations and their phase coherence persist even in animals lacking an SCN or any oscillators outside hepatocytes (Sinturel et al. 2021), and the source model's simulations require intercellular coupling to lock oscillation periods near 24 hours. Concretely: (a) determine the molecular identity of the signal(s) through which hepatocytes exchange circadian phase information in the intact liver - the candidates currently in play are TGF-β, FGF21 (fibroblast growth factor 21, a hormone secreted by hepatocytes in a circadian fashion), gap junctions and pannexin hemichannels (large membrane channels that release signaling molecules into the extracellular space), mechanical/actin-mediated signaling, or an as-yet-unnamed factor, and it is open whether a single factor or several partially redundant mechanisms are involved; and (b) characterize the mechanism of action on the clock gene network (e.g. the coupling conduit or receptor, the downstream signaling mediators and transcription factors, the target clock genes such as Per2, and the kinetics of phase shifts). Current multicellular models of the liver clock, including the source model, must assume a generic 'putative coupling ligand' acting on clock genes through unspecified receptor molecules. The mechanism of action on the clock network to be characterized comprises the coupling conduit or receptor, the downstream signaling mediators and transcription factors, the target clock genes such as Per2, and the quantitative phase-shift parameters the cited work reports.

References

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