Characterisation of pituitary melatonin target cells under photoperiod changes by single cell RNAseq

Anticipation and adaptation of behaviour and physiology to the season changes are essential for animals to survive. Recent studies have defined that the melatonin rhythms responding to photoperiodic input acts on thyrotroph cells of pituitary pars tuberalis (PT), leading to seasonal activation of thyroid hormone converting pathways in the brain. In turn this activates reproductive and metabolic neurones driving fertility and metabolism. Action in the PT is mediated by activation of the developmental regulator EYA3, which is driven by the circadian clock in response to melatonin. It is unclear which downstream transcriptional switches drive these changes at the single cell level in the several complex cell types of the PT. To address this, we performed single cell RNA sequencing (using ICELL8 single-cell system) with PT tissue collected from a series of sheep housed over short (winter) photoperiods to long (summer) photoperiods. We observed in single PTs unique populations of cells exclusively expressing marker genes for short photoperiods (hormone processing chromogranin, CHGA) or long photoperiods (TSHB, EYA3), as well as the thyrotroph cell marker (aGSU) and folliculostellate cell marker (S100). These results indicate that single cell RNAseq offers the opportunity to define complex programmed switching mechanisms driving seasonal neuroendocrine responses in multiple cell types in the PT. Importantly, it suggests that timing mechanisms exist in one of 2 binary states (winter or summer-like), with rapid conversion of individual cells within the PT from one state to another. Single cell analysis is thus essential to define these mechanisms, as tissue based approaches can only provide an overall mean value for complex populations of timing cells.