Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics

ACKNOWLEDGMENTS The authors thank all of the animal staff at Kårvik havbruksstasjonen for their expert care of the research animals, and the University of Manchester Genomics Technology core facility (UK) for performing chromium 10x library preparation for snRNAseq. We also thanks the reviewers for their constructive comments on the original manuscript FUNDING AW is supported by the Tromsø forskningsstiftelse (TFS) grant awarded to DH (TFS2016DH). The Sentinel North Transdisciplinary Research Program Université Laval and UiT awarded to DH supports this work. SW is supported a grant from the Tromsø forskningsstiftelse (TFS) starter grant TFS2016SW. Experimental costs were covered by HFSP grant “Evolution of seasonal timers” RGP0030/2015 awarded to AL and DH. Storage resources were provided by the Norwegian National Infrastructure for Research Data (NIRD, project NS9055K).Peer reviewedPublisher PD

Activation of AMPA Receptors in the Suprachiasmatic Nucleus Phase-Shifts the Mouse Circadian Clock In Vivo and In Vitro

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN

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

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

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

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