Dissection of a novel molecular pathway involved in seasonal timing in a melatonin-target tissue using an experimental and systems-level approach

Most species of wild animal and many of man's domesticated species are adapted to live in seasonal environments and experience significant annual changes in food supply and temperature. It is essential that seasonal animals time the onset of breeding and lay down and store fat at the appropriate time of year. In order to achieve this, they operate a seasonal clock which controls timing of many hormone rhythms. A key hormone regulating this seasonal timer is called Melatonin, which is produced within the brain in the pineal gland. Melatonin is secreted at night and the pattern of secretion changes seasonally, with longer-duration profiles produced on the long winter nights. It is known that these changes in seasonal duration drive seasonal hormone rhythms and provide the brain with an internal representation of external photoperiod change, acting on physiology and behaviour. Melatonin acts on a specialised structure called the pars tuberalis (PT) located in the pituitary gland, in a region close to the hypothalamus in the base of the brain. The PT is thought to regulate seasonal rhythms of prolactin secretion by secreting a local factor which acts on prolactin-secreting cells in the distal pituitary tissue. It also produces a hormone locally, called thyroid stimulating hormone (TSH), which we now suspect acts on TSH receptors on cells called tanycytes in the immediate hypothalamus. Here it regulates activity of key enzymes controlling thyroid hormone activity. By this means, the PT may act both on the pituitary and also the hypothalamus. We have discovered a group of genes in the PT which become active when the PT is exposed to long-duration melatonin signals on short daylengths and are also directly responsive to melatonin. These genes act on pathways which are crucial for thermogenesis, and controlling the synthesis and use of stored fat reserves. Our work aims to establish how these 'metabolism' genes may be used in this seasonal timing structure to control annual hormone rhythms. In order to monitor output, we focus on the hormone prolactin, where we can measure activity by culturing with PT cells, and on TSH production, which we can measure by assay or measures of gene expression. The goal is to work out how the melatonin signal acting on the PT drives genetic pathways which result in activation of these two hormone pathways. Finally, we have discovered that another group of genes previously known to be involved in development of many tissues including hormone secreting cells are also activated in the PT in response to daylength change. We suspect that these 'developmental' genes are linked to the metabolic pathway genes above. Our study will use several different techniques, and for much of the work we will use sheep. The reason is that the sheep PT is easy to undertake anatomical studies and can be cultured in the laboratory, allowing us to test which metabolic pathway genes may be involved in hormone regulation. First, we will describe in detail changes in activity of the metabolic and developmental genes in the PT and how they change, both with season, and when animals are exposed to abrupt changes in daylength and melatonin. We will then go on to study how proteins in the PT interact with one another, and also with DNA. This will ultimately allow us to describe a 'circuit diagram' within a melatonin-target cell and describe how genes may be activated or suppressed by melatonin. We will use the culture system to see whether changes in the melatonin signal result in altered hormone output, by measuring TSH activity (direct measure) and action on prolactin-secreting cells (in-direct measure). We will also use laboratory rodents (hamsters and rats) as here we can more easily administer drugs which act on the 'metabolic' pathway genes and see whether we see changes in hormone secretion. A final advantage to using hamsters is that we will be able to check results from sheep in a different type of seasonal breeder.

The hormone melatonin plays a central role in seasonal time-keeping in mammals. It acts on the hypothalamus, and on the pars tuberalis (PT) of the pituitary gland to regulate prolactin secretion, via an intra-pituitary signalling pathway. Within the PT, melatonin regulates the expression of two clock genes (Per and Cry) and seasonal changes in the duration of melatonin tune this local circadian clockwork. Using arrays, we have discovered new genes in the PT directly regulated by melatonin, including a key activator of a de-acetylase SIRT1, which acts on PPARa & ? (peroxisome proliferator-activated nuclear receptors) pathways. Abrupt changes to long photoperiods (LP) also activates a gene involved in PPAR regulation (Lipin, up on Day 1 of LP), and 7 and 28 days later we observed discovered other genes known to be involved in cellular development and differentiation (EYA, NeuroD1). We will test the hypothesis that PPAR's have been co-opted from their conventional role regulating energy metabolism to the control of seasonal neuroendocrine pathways by melatonin signals. To define the action of melatonin on a target tissue, we will use a novel high-throughput screening method (SOLEXA) to define the seasonal transcriptome. We will examine the involvement of Lipin, EYA and NeuroD1, and whether acute activation by long photoperiods uses a molecular developmental pathway. Finally, we will use in-vitro and in-vivo models to test neuroendocrine outcomes, using both manipulation of PPAR's and shRN. Our current data provide important fresh insight into melatonin regulation of genes at a target site, and the characterisation of acute and long-term induction. Our proposed work aims to define how this gene network may be involved in seasonal regulation of neuroendocrine circuits. The outcome will be the first comprehensive description of melatonin-regulated genetic pathways linked to regulation of seasonal hormonal pathways, a problem of considerable significance.