Single Cell RNA Sequencing Defines Cellular Binary Switching Mechanism Driving Circadian Regulation of Mammalian Photoperiodism in Melatonin-Target Calendar Cells.

Background/Objectives
Recent studies define thyrotoph cells of the pituitary pars tuberalis (PT) as key mammalian seasonal calendar cells, driving photoperiodic responses to melatonin via thyroid stimulating hormone, TSH, which in turn regulates hypothalamic thyroid hormone conversion pathways. In the PT, long photoperiods activate the developmental regulator, EYA3 that drives TSH. We have shown previously that PT cells operate as binary switches driving accumulation of increasing numbers of TSH positive cells on long photoperiods. We do not know how this transcriptional switch drives cellular re-modelling within single thyrotroph cells nor do we know how the EYA3/TSH circuit is engaged by the circadian clockwork.
Methods/Results
We performed single nuclei RNA sequencing (iCELL8) of PT tissue collected from sheep housed in controlled artificial short photoperiods and in transition to long photoperiods (LP days 3, 10, 35). This revealed single PTs unique populations of endocrine cells exclusively expressing marker genes for short photoperiods (hormone processing chromogranin, CHGA) or long photoperiods (TSHB). Importantly, we show that the molecular repertoire characterising short or long photoperiods exist in one of 2 binary states (winter or summer-like). The proportion of cells in an LP state increases over several weeks of LP-exposure. We also identify BMAL2 as a critical LP-activated photoperiodic regulator of EYA3, thus coupling the circadian clock to mammalian photoperiodism.
Conclusions
Our data confirm that individual PT thyrotroph cells operate as binary switches, driving dynamic changes in neuroendocrine responses over several weeks, and we identify BMAL2 as a key up-stream circadian switch. Long-term timing is thus driven in a gradual digital-like accumulation of individual cells that switch phenotype in a stochastic manner. Single cell analysis is thus an essential platform to define complex dynamic tissue responses, and we demonstrate that seasonal timing is driven by rhythmic re-capitulation of developmental pathways in PT thyrotroph endocrine cells.