A functional light-entrainable molecular clock is revealed in gilthead seabream (Sparus aurata) from early developmental stages using an embryonic stem cell line

Abstract

Like other vertebrates, fish exhibit physiological and behavioural rhythms that are synchronized by the cyclic variations in the external environment, being photoperiod, i.e., the alternation of light and dark during the 24-h cycle, the most predictable and powerful environmental cue. Many of these rhythms are endogenous and controlled by a molecular clock present in cells from early developmental stages. In a previous study, we have developed and characterised a new monoclonal embryonic stem cell line, SAEC-H7, derived from the morula stage embryos of a marine teleost, the gilthead seabream (Sparus aurata), a species with high commercial value for European aquaculture. In the present study, we aimed to identify the main components from the positive (clock and bmal1) and negative (cry1a, per1, per2 and per3) feedback loops of the molecular clock in this embryonic cell line and characterise putative daily variations and circadian rhythms in their expression under different photoperiod regimes, as well as the effects of nocturnal light pulses on their transcriptional activation. Our results revealed that SAEC-H7 cells contain a molecular clock that is entrained by the daily light-dark (LD) cycle. All clock genes analysed showed daily rhythms under LD conditions and were able to re-entrain to a 12-h phase shift (DL) in the photocycle, some of them (clock, bmal1, per1, per3) exhibiting circadian rhythms that persisted over a certain period under constant conditions, although with affected amplitudes and acrophases. Both cry1a and per2 genes showed a statistically significant increase in expression after a nocturnal light pulse, confirming the light-inducible character of these clock genes. The results obtained reveal that seabream embryonic cells are highly sensitive to light and already contain the phototransduction pathways that entrain the molecular clock and overt rhythms from the morula stage, making this embryonic cell line a useful tool for developmental and chronobiological studies in fish. Our results also reinforce the importance of maintaining natural cyclic light conditions in in vitro studies performed with fish cell lines as well as in embryonic and larval rearing protocols in aquaculture practices.