Seasonal synchronization based on day length (photoperiod) allows organisms to anticipate environmental change. Photoperiodic decoding relies on circadian clocks, but the underlying molecular pathways have remained elusive [1]. In mammals and birds, photoperiodic responses depend crucially on expression of thyrotrophin β subunit RNA (TSHβ) in the pars tuberalis (PT) of the pituitary gland [2-4]. Now, using our well-characterized Soay sheep model [2], we describe a molecular switch governing TSHβ transcription through the circadian clock. Central to this is a conserved D element in the TSHβ promoter, controlled by the circadian transcription factor thyrotroph embryonic factor (Tef). In the PT, long-day exposure rapidly induces expression of the coactivator eyes absent 3 (Eya3), which synergizes with Tef to maximize TSHβ transcription. The pineal hormone melatonin, secreted nocturnally, sets the phase of rhythmic Eya3 expression in the PT to peak 12 hr after nightfall. Additionally, nocturnal melatonin levels directly suppress Eya3 expression. Together, these effects form a switch triggering a strong morning peak of Eya3 expression under long days. Species variability in the TSHβ D element influences sensitivity to TEF, reflecting species variability in photoperiodic responsiveness. Our findings define a molecular pathway linking the circadian clock to the evolution of seasonal timing in mammals.

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2 −/− mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2 −/− mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.circadian clock | suprachiasmatic nucleus | period protein | Tau mutation | pacemaking

Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinoic acid-binding protein 1 (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photoperiod. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster.

Ivanova EA, Bechtold DA, Dupré SM, Brennand J, Barrett P, Luckman SM, Loudon AS. Altered metabolism in the melatoninrelated receptor (GPR50) knock out mouse. Am J Physiol Endocrinol Metab 294: E176-E182, 2008. First published October 23, 2007 doi:10.1152/ajpendo.00199.2007.-The X-linked orphan receptor GPR50 shares 45% homology with the melatonin receptors, yet its ligand and physiological function remain unknown. Here we report that mice lacking functional GPR50 through insertion of a lacZ gene into the coding sequence of GPR50 exhibit an altered metabolic phenotype. GPR50 knockout mice maintained on normal chow exhibit lower body weight than age-matched wild-type littermates by 10 wk of age. Furthermore, knockout mice were partially resistant to dietinduced obesity. When placed on a high-energy diet (HED) for 5 wk, knockout mice consumed significantly more food per unit body weight yet exhibited an attenuated weight gain and reduced body fat content compared with wild-type mice. Wheel-running activity records revealed that, although GPR50 knockout mice showed no alteration of circadian period, the overall levels of activity were significantly increased over wild types in both nocturnal and diurnal phases. In line with this, basal metabolic rate (O 2 consumption, CO2 production, and respiratory quotient) was found to be elevated in knockout mice. Using in situ hybridization (wild-type mice) and ␤-galactosidase activity (from LacZ insertion element in knockout mice), brain expression of GPR50 was found to be restricted to the ependymal layer of the third ventricle and dorsomedial nucleus of the hypothalamus. GPR50 expression was highly responsive to energy status, showing a significantly reduced expression following both fasting and 5 wk of HED. These data implicate GPR50 as an important regulator of energy metabolism. dorsomedial hypothalamus; metabolic rate; obesity; tanycyte THE ORPHAN MELATONIN RELATED receptor (GPR50) shares ϳ45% homology with melatonin receptors MT 1 and MT 2 (20). However, the receptor does not bind melatonin (13,20) despite the presence of a conserved histidine residue in transmembrane domain 5 of the receptor, which is a key amino acid for binding of melatonin to MT 1 (9). Although the physiological function of GPR50 remains unknown, in situ hybridization studies on mouse, rat, and hamster brain sections have demonstrated an expression of the receptor in several areas associated with energy metabolism, namely the dorsomedial hypothalamic nucleus (DMN), lateral hypothalamus, and arcuate nucleus (12). Although the extent and distribution of GPR50 exhibits some degree of species specificity, high levels of expression are consistently observed in the ependymal layer of the third ventricle (12, 25).Recently, we have shown that ependymal GPR50 expression in the seasonally breeding Siberian hamster is strongly modulated by photoperiod, in which expression is greatly reduced following exposure to short photoperiods (1). Prolonged exposure to short photoperiods leads to a dramatic reduction in ...

Summary Seasonally breeding mammals such as sheep use photoperiod, encoded by the nocturnal secretion of the pineal hormone melatonin, as a critical cue to drive hormone rhythms and synchronise reproduction to the most optimal time of year [1, 2]. Melatonin acts directly on the pars tuberalis (PT) of the pituitary, regulating expression of thyrotropin (TSH) which then relays messages back to the hypothalamus to control reproductive circuits [3, 4]. In addition, a second local intra-pituitary circuit controls seasonal prolactin (PRL) release via a currently uncharacterised low molecular weight peptide(s) termed tuberalin(s) of PT origin [5–7]. Studies in birds identified the transcription factor Eya3 as the first molecular response activated by long photoperiods (LP) [8]. Using arrays and in situ hybridization studies, we show Eya3 as the strongest LP activated gene in sheep, revealing a common photoperiodic molecular response in birds and mammals. We also identified TAC1 (encoding the tachykinins Substance P and Neurokinin A; NKA) to be strongly activated by LP within the sheep PT. We show that these PRL secretagogues act on primary pituitary cells, and are thus candidates for the elusive PT-expressed “tuberalin” seasonal hormone regulator.