c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Circadian coupling of mitochondria in a deep-diving mammal.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Hypothalamic tanycytes as mediators of maternally programmed seasonal plasticity.

In mammals, maternal photoperiodic programming (MPP) provides a means whereby juvenile development can be matched to forthcoming seasonal environmental conditions.1,2,3,4 This phenomenon is driven by in utero effects of maternal melatonin5,6,7 on the production of thyrotropin (TSH) in the fetal pars tuberalis (PT) and consequent TSH receptor-mediated effects on tanycytes lining the 3rd ventricle of the mediobasal hypothalamus (MBH).8,9,10 Here we use LASER capture microdissection and transcriptomic profiling to show that TSH-dependent MPP controls the attributes of the ependymal region of the MBH in juvenile animals. In Siberian hamster pups gestated and raised on a long photoperiod (LP) and thereby committed to a fast trajectory for growth and reproductive maturation, the ependymal region is enriched for tanycytes bearing sensory cilia and receptors implicated in metabolic sensing. Contrastingly, in pups gestated and raised on short photoperiod (SP) and therefore following an over-wintering developmental trajectory with delayed sexual maturation, the ependymal region has fewer sensory tanycytes. Post-weaning transfer of SP-gestated pups to an intermediate photoperiod (IP), which accelerates reproductive maturation, results in a pronounced shift toward a ciliated tanycytic profile and formation of tanycytic processes. We suggest that tanycytic plasticity constitutes a mechanism to tailor metabolic development for extended survival in variable overwintering environments.

Biological timekeeping in polar environments: lessons from terrestrial vertebrates.

The polar regions receive less solar energy than anywhere else on Earth, with the greatest year-round variation in daily light exposure; this produces highly seasonal environments, with short summers and long, cold winters. Polar environments are also characterised by a reduced daily amplitude of solar illumination. This is obvious around the solstices, when the Sun remains continuously above (polar 'day') or below (polar 'night') the horizon. Even at the solstices, however, light levels and spectral composition vary on a diel basis. These features raise interesting questions about polar biological timekeeping from the perspectives of function and causal mechanism. Functionally, to what extent are evolutionary drivers for circadian timekeeping maintained in polar environments, and how does this depend on physiology and life history? Mechanistically, how does polar solar illumination affect core daily or seasonal timekeeping and light entrainment? In birds and mammals, answers to these questions diverge widely between species, depending on physiology and bioenergetic constraints. In the high Arctic, photic cues can maintain circadian synchrony in some species, even in the polar summer. Under these conditions, timer systems may be refined to exploit polar cues. In other instances, temporal organisation may cease to be dominated by the circadian clock. Although the drive for seasonal synchronisation is strong in polar species, reliance on innate long-term (circannual) timer mechanisms varies. This variation reflects differing year-round access to photic cues. Polar chronobiology is a productive area for exploring the adaptive evolution of daily and seasonal timekeeping, with many outstanding areas for further investigation.

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics.

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.

A refined method to monitor arousal from hibernation in the European hamster.

BACKGROUND: Hibernation is a physiological and behavioural adaptation that permits survival during periods of reduced food availability and extreme environmental temperatures. This is achieved through cycles of metabolic depression and reduced body temperature (torpor) and rewarming (arousal). Rewarming from torpor is achieved through the activation of brown adipose tissue (BAT) associated with a rapid increase in ventilation frequency. Here, we studied the rate of rewarming in the European hamster (Cricetus cricetus) by measuring both BAT temperature, core body temperature and ventilation frequency. RESULTS: Temperature was monitored in parallel in the BAT (IPTT tags) and peritoneal cavity (iButtons) during hibernation torpor-arousal cycling. We found that increases in brown fat temperature preceded core body temperature rises by approximately 48 min, with a maximum re-warming rate of 20.9℃*h-1. Re-warming was accompanied by a significant increase in ventilation frequency. The rate of rewarming was slowed by the presence of a spontaneous thoracic mass in one of our animals. Core body temperature re-warming was reduced by 6.2℃*h-1 and BAT rewarming by 12℃*h-1. Ventilation frequency was increased by 77% during re-warming in the affected animal compared to a healthy animal. Inspection of the position and size of the mass indicated it was obstructing the lungs and heart. CONCLUSIONS: We have used a minimally invasive method to monitor BAT temperature during arousal from hibernation illustrating BAT re-warming significantly precedes core body temperature re-warming, informing future study design on arousal from hibernation. We also showed compromised re-warming from hibernation in an animal with a mass obstructing the lungs and heart, likely leading to inefficient ventilation and circulation.

Diversified regulation of circadian clock gene expression following whole genome duplication.

Across taxa, circadian control of physiology and behavior arises from cell-autonomous oscillations in gene expression, governed by a networks of so-called 'clock genes', collectively forming transcription-translation feedback loops. In modern vertebrates, these networks contain multiple copies of clock gene family members, which arose through whole genome duplication (WGD) events during evolutionary history. It remains unclear to what extent multiple copies of clock gene family members are functionally redundant or have allowed for functional diversification. We addressed this problem through an analysis of clock gene expression in the Atlantic salmon, a representative of the salmonids, a group which has undergone at least 4 rounds of WGD since the base of the vertebrate lineage, giving an unusually large complement of clock genes. By comparing expression patterns across multiple tissues, and during development, we present evidence for gene- and tissue-specific divergence in expression patterns, consistent with functional diversification of clock gene duplicates. In contrast to mammals, we found no evidence for coupling between cortisol and circadian gene expression, but cortisol mediated non-circadian regulated expression of a subset of clock genes in the salmon gill was evident. This regulation is linked to changes in gill function necessary for the transition from fresh- to sea-water in anadromous fish. Overall, this analysis emphasises the potential for a richly diversified clock gene network to serve a mixture of circadian and non-circadian functions in vertebrate groups with complex genomes.

An integrative view of mammalian seasonal neuroendocrinology.

Seasonal neuroendocrine cycles that govern annual changes in reproductive activity, energy metabolism and hair growth are almost ubiquitous in mammals that have evolved at temperate and polar latitudes. Changes in nocturnal melatonin secretion regulating gene expression in the pars tuberalis (PT) of the pituitary stalk are a critical common feature in seasonal mammals. The PT sends signal(s) to the pars distalis of the pituitary to regulate prolactin secretion and thus the annual moult cycle. The PT also signals in a retrograde manner via thyroid-stimulating hormone to tanycytes, which line the ventral wall of the third ventricle in the hypothalamus. Tanycytes show seasonal plasticity in gene expression and play a pivotal role in regulating local thyroid hormone (TH) availability. Within the mediobasal hypothalamus, the cellular and molecular targets of TH remain elusive. However, two populations of hypothalamic neurones, which produce the RF-amide neuropeptides kisspeptin and RFRP3 (RF-amide related peptide 3), are plausible relays between TH and the gonadotrophin-releasing hormone-pituitary-gonadal axis. By contrast, the ways by which TH also impinges on hypothalamic systems regulating energy intake and expenditure remain unknown. Here, we review the neuroendocrine underpinnings of seasonality and identify several areas that warrant further research.

Maternal Photoperiodic Programming: Melatonin and Seasonal Synchronization Before Birth.

This mini-review considers the phenomenon of maternal photoperiodic programming (MPP). In order to match neonatal development to environmental conditions at the time of birth, mammals use melatonin produced by the maternal pineal gland as a transplacental signal representing ambient photoperiod. Melatonin acts via receptors in the fetal pituitary gland, exerting actions on the developing medio-basal hypothalamus. Within this structure, a central role for specialized ependymal cells known as tanycytes has emerged, linking melatonin to control of hypothalamic thyroid metabolism and in turn to pup development. This review summarizes current knowledge of this programming mechanism, and its relevance in an eco-evolutionary context. Maternal photoperiodic programming emerges as a useful paradigm for understanding how in utero programing of hypothalamic function leads to life-long effects on growth, reproduction, health and disease in mammals, including humans.

How can a binary switch within the pars tuberalis control seasonal timing of reproduction?

Life in seasonally changing environments is challenging. Biological systems have to not only respond directly to the environment, but also schedule life history events in anticipation of seasonal changes. The cellular and molecular basis of how these events are scheduled is unknown. Cellular decision-making processes in response to signals above certain thresholds regularly occur i.e. cellular fate determination, apoptosis and firing of action potentials. Binary switches, the result of cellular decision-making processes, are defined as a change in phenotype between two stable states. A recent study presents evidence of a binary switch operating in the pars tuberalis (PT) of the pituitary, seemingly timing seasonal reproduction in sheep. Though, how a binary switch would allow for anticipation of seasonal environmental changes, not just direct responsiveness, is unclear. The purpose of this review is to assess the evidence for a binary switching mechanism timing seasonal reproduction and to hypothesize how a binary switch would allow biological processes to be timed over weeks to years. I draw parallels with mechanisms used in development, cell fate determination and seasonal timing in plants. I propose that the adult PT is a plastic tissue, showing a seasonal cycle of cellular differentiation, and that the underlying processes are likely to be epigenetic. Therefore, considering the mechanisms behind adult cellular plasticity offers a framework to hypothesize how a long-term timer functions within the PT.

Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep.

This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how "Vegfaxxxb-like" PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR.

The pars tuberalis: The site of the circannual clock in mammals?

Accurate timing and physiological adaptation to anticipate seasonal changes are an essential requirement for an organism's survival. In contrast to all other environmental cues, photoperiod offers a highly predictive signal that can be reliably used to activate a seasonal adaptive programme at the correct time of year. Coupled to photoperiod sensing, it is apparent that many organisms have evolved innate long-term timekeeping systems, allowing reliable anticipation of forthcoming environmental changes. The fundamental biological processes giving rise to innate long-term timing, with which the photoperiod-sensing pathway engages, are not known for any organism. There is growing evidence that the pars tuberalis (PT) of the pituitary, which acts as a primary transducer of photoperiodic input, may be the site of the innate long-term timer or "circannual clock". Current research has led to the proposition that the PT-specific thyrotroph may act as a seasonal calendar cell, driving both hypothalamic and pituitary endocrine circuits. Based on this research we propose that the mechanistic basis for the circannual rhythm appears to be deeply conserved, driven by a binary switching cell based accumulator, analogous to that proposed for development. We review the apparent conservation of function and pathways to suggest that these broad principles may apply across the vertebrate lineage and even share characteristics with processes driving seasonal adaptation in plants.

Transcriptome analysis in calorie-restricted rats implicates epigenetic and post-translational mechanisms in neuroprotection and aging.

BACKGROUND: Caloric restriction (CR) can increase longevity in rodents and improve memory function in humans. α-Lipoic acid (LA) has been shown to improve memory function in rats, but not longevity. While studies have looked at survival in rodents after switching from one diet to another, the underlying mechanisms of the beneficial effects of CR and LA supplementation are unknown. Here, we use RNA-seq in cerebral cortex from rats subjected to CR and LA-supplemented rats to understand how changes in diet can affect aging, neurodegeneration and longevity. RESULTS: Gene expression changes during aging in ad libitum-fed rats are largely prevented by CR, and neuroprotective genes are overexpressed in response to both CR and LA diets with a strong overlap of differentially expressed genes between the two diets. Moreover, a number of genes are differentially expressed specifically in rat cohorts exhibiting diet-induced life extension. Finally, we observe that LA supplementation inhibits histone deacetylase (HDAC) protein activity in vitro in rat astrocytes. We find a single microRNA, miR-98-3p, that is overexpressed during CR feeding and LA dietary supplementation; this microRNA alters HDAC and histone acetyltransferase (HAT) activity, which suggests a role for HAT/HDAC homeostasis in neuroprotection. CONCLUSIONS: This study presents extensive data on the effects of diet and aging on the cerebral cortex transcriptome, and also emphasises the importance of epigenetics and post-translational modifications in longevity and neuroprotection.

Binary Switching of Calendar Cells in the Pituitary Defines the Phase of the Circannual Cycle in Mammals.

Persistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3(+)) or short (CHGA(+)) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation.

The Digital Ageing Atlas: integrating the diversity of age-related changes into a unified resource.

Multiple studies characterizing the human ageing phenotype have been conducted for decades. However, there is no centralized resource in which data on multiple age-related changes are collated. Currently, researchers must consult several sources, including primary publications, in order to obtain age-related data at various levels. To address this and facilitate integrative, system-level studies of ageing we developed the Digital Ageing Atlas (DAA). The DAA is a one-stop collection of human age-related data covering different biological levels (molecular, cellular, physiological, psychological and pathological) that is freely available online (http://ageing-map.org/). Each of the >3000 age-related changes is associated with a specific tissue and has its own page displaying a variety of information, including at least one reference. Age-related changes can also be linked to each other in hierarchical trees to represent different types of relationships. In addition, we developed an intuitive and user-friendly interface that allows searching, browsing and retrieving information in an integrated and interactive fashion. Overall, the DAA offers a new approach to systemizing ageing resources, providing a manually-curated and readily accessible source of age-related changes.

Clocks for all seasons: unwinding the roles and mechanisms of circadian and interval timers in the hypothalamus and pituitary.

Adaptation to the environment is essential for survival, in all wild animal species seasonal variation in temperature and food availability needs to be anticipated. This has led to the evolution of deep-rooted physiological cycles, driven by internal clocks, which can track seasonal time with remarkable precision. Evidence has now accumulated that a seasonal change in thyroid hormone (TH) availability within the brain is a crucial element. This is mediated by local control of TH-metabolising enzymes within specialised ependymal cells lining the third ventricle of the hypothalamus. Within these cells, deiodinase type 2 enzyme is activated in response to summer day lengths, converting metabolically inactive thyroxine (T4) to tri-iodothyronine (T3). The availability of TH in the hypothalamus appears to be an important factor in driving the physiological changes that occur with season. Remarkably, in both birds and mammals, the pars tuberalis (PT) of the pituitary gland plays an essential role. A specialised endocrine thyrotroph cell (TSH-expressing) is regulated by the changing day-length signal, leading to activation of TSH by long days. This acts on adjacent TSH-receptors expressed in the hypothalamic ependymal cells, causing local regulation of deiodinase enzymes and conversion of TH to the metabolically active T3. In mammals, the PT is regulated by the nocturnal melatonin signal. Summer-like melatonin signals activate a PT-expressed clock-regulated transcription regulator (EYA3), which in turn drives the expression of the TSHß sub-unit, leading to a sustained increase in TSH expression. In this manner, a local pituitary timer, driven by melatonin, initiates a cascade of molecular events, led by EYA3, which translates to seasonal changes of neuroendocrine activity in the hypothalamus. There are remarkable parallels between this PT circuit and the photoperiodic timing system used in plants, and while plants use different molecular signals (constans vs EYA3) it appears that widely divergent organisms probably obey a common set of design principles.

A-to-I RNA editing does not change with age in the healthy male rat brain.

RNA editing is a post-transcriptional process, which results in base substitution modifications to RNA. It is an important process in generating protein diversity through amino acid substitution and the modulation of splicing events. Previous studies have suggested a link between gene-specific reductions in adenosine to inosine RNA editing and aging in the human brain. Here we demonstrate that changes in RNA editing observed in humans with age are not observed during aging in healthy rats. Furthermore, we identify a conserved editing site in rats, in Cog3. We propose that either age-related changes in RNA editing are specific to primates or humans, or that they are the manifestation of disease pathology. Since rodents are often used as model organisms for studying aging, these findings demonstrate the importance of understanding species-specific differences in RNA biology during aging.

An analysis and validation pipeline for large-scale RNAi-based screens.

Large-scale RNAi-based screens are a major technology, but require adequate prioritization and validation of candidate genes from the primary screen. In this work, we performed a large-scale pooled shRNA screen in mouse embryonic stem cells (ESCs) to discover genes associated with oxidative stress resistance and found several candidates. We then developed a bioinformatics pipeline to prioritize these candidates incorporating effect sizes, functional enrichment analysis, interaction networks and gene expression information. To validate candidates, we mixed normal cells with cells expressing the shRNA coupled to a fluorescent protein, which allows control cells to be used as an internal standard, and thus we could detect shRNAs with subtle effects. Although we did not identify genes associated with oxidative stress resistance, as a proof-of-concept of our pipeline we demonstrate a detrimental role of Edd1 silencing in ESC growth. Our methods may be useful for candidate gene prioritization of large-scale RNAi-based screens.