Molecular dissection of the seasonal clock

Recent studies define thyrotrophs of the pituitary pars tuberalis (PT) as key mammalian calendar cells, which integrate melatonin signal duration to activate thyroid stimulating hormone (TSH) on long photoperiods, which in turn acts on hypothalamic targets in the ependymal cells to drive long-day neuroendocrine circuits. Long photoperiod (LP) activation is known to involve the developmental regulator, EYA3 in the PT, which operates as a co-transcription factor driving TSHB expression. We have previously shown that PT cells operate as binary switches driving the gradual accumulation of TSH positive cells on prolonged exposure to long photoperiod. In this model, PT thyrotroph cells exist in either a long or short-photoperiod state, with a gradual increase in LP-like cells over several weeks following exposure to long photoperiods (Wood et al, Current Biology, 2015). It is unknown how this binary switch operates at the single cell level or how the EYA3-TSH circuit is engaged by the circadian clockworks in individual PT cells. To address this we used single cell nuclei RNA sequencing of PT cells. PT tissues were collected at ZT4 from sheep housed in controlled artificial short photoperiods (SP day 84) and in transition to long photoperiods at LP +3, +10 and +35 days. This defined the molecular repertoire of individual PT thyrotroph cells as the tissue transitions from short to long photoperiods. This revealed separate populations of short-day like or long-day like cells with the same PT following exposure to LP. Our data confirm that individual PT thyrotroph cells operate as binary switches, driving dynamic changes in neuroendocrine responses over several weeks. We also reveal the circadian gene BMAL2 as a potent long-day activated regulator of EYA3, while on short photoperiods, DEC1 acts as a potent repressor of BMAL2-mediated drive on EYA3. Our data elucidate a complex set of dynamic cellular responses, mimicking rhythmic re-capitulation of developmental pathways in PT thyrotroph endocrine cells, and regulated by photoperiodic control of circadian gene elements. This work is supported by the BBSRC.