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Abstract

Abstract This project focuses attention on the input and output pathways of the circadian clock using zebrafish as a genetic model system. In particular, by establishing a new per2 loss of function (KO) zebrafish line we have revealed that the per2 gene plays a tissue-specific function in setting the phase of rhythmic expression of certain core clock genes under light dark cycle (LD) conditions. Specifically, we have observed rhythmic expression patterns with 6 hours phase delay for both the clock1 and cry1a core clock genes in per2 KO adult heart, liver, gut and muscle, all internal organs that experience a reduced light exposure. In contrast, no differences were detected in the core clock gene expression patterns in brain, eyes and fin tissues as well as in embryos, that all represent superficial organs/tissues and so are exposed to higher light intensities. Interestingly, we also observed an absence or strongly reduced amplitude of rhythmic core clock gene expression in embryonic and adult fin fibroblast cell lines that we generated from the per2 KO line, possibly suggesting that systemic signals might override local Per2 function in the entrainment of peripheral tissue clocks during light exposure. However, in explanted fin and heart tissue cultures prepared from the per2 KO fish and maintained under LD conditions, rhythmic clock gene expression was equivalent to that in cultures derived from wild type (WT) fish, thus demonstrating that the per2 gene function is not essential for the direct light entrainment mechanism of peripheral clocks, even in the absence of systemic signals in zebrafish. Subsequently, we have demonstrated that the rhythmic expression of several tissue-specific, clock-controlled genes, involved in the regulation of various aspects of heart, liver and muscle physiology shows a reduction of the amplitude or a phase shift of the rhythmic pattern in per2 KO adult fish thus implicating per2 in the circadian regulation of clock outputs and tissue-specific physiology in zebrafish. Therefore, we analyzed rhythmic locomotor activity of per2 KO larvae under different light conditions and revealed an alteration of the robustness and precision of rhythmic locomotor output in free running conditions. However, we also showed that under time restricted feeding regimes, food anticipatory activity (FAA) is equivalent to that of wild type fish in the per2 KO line suggesting that per2 function is linked with the light rather than the food entrainable clock. Importantly, we have also demonstrated that the per2 gene mutation has a strong effect on the circadian regulation of the cell cycle in vivo and in vitro in zebrafish, although it does not appear to impact on the rate of tissue regeneration following fin amputation. Together these results imply that the per2 clock gene plays multiple roles in both clock input and output pathways in zebrafish. In parallel, we have revealed the existence of a functional interaction between the circadian clock and the TGF- β signaling pathway in zebrafish. In particular, we have demonstrated that pharmacological alteration of TGF-β signaling interferes with the molecular circadian clock in zebrafish PAC-2 cells. Subsequently, we showed that TGF-β inhibition generates a phase delay of per1b mRNA rhythms in zebrafish larvae. Finally, we demonstrated that the inhibition of the TGF-β signaling pathway reversibly disrupts clock-controlled rhythmic locomotor activity in zebrafish larvae maintained under different lighting conditions. Therefore, we reveal complexity and overlap of the clock input and output mechanisms in zebrafish.