Angiogenic mechanisms underlie seasonal adaptation to a changing environment

Adaptive responses to seasonal changes in the environment are a fundamental strategy for survival in species living in temperate zones. The resulting annual rhythms in physiological processes such as reproduction, metabolism, hair and hoof growth, etc., are controlled by seasonal cycles of hormones secreted by the pituitary gland, and synchronised by the predictable changes in day length (photoperiod). Photoperiodic information is decoded by the pattern of melatonin secreted by the pineal gland. As melatonin is only produced at night, the duration of the nocturnal peak tells the animal how long the night is; thus, the night production of melatonin is much longer during the winter than during the summer. Melatonin exerts its effects on the pituitary gland, in a region called the pars tuberalis (PT). The PT is strategically located between the brain and the rest of the pituitary and has been regarded as the site of the annual biological clock. However, most of the hormones that control seasonal changes in physiology are produced by the pars distalis (PD), an anatomically different region of the pituitary. Therefore, an intra-pituitary system of communication, to convey photoperiodic signals from the melatonin sensitive PT to the endocrine cells of the PD, is fundamental to ensure seasonal adaptation to the environment. We have recently shown that in sheep the blood vessels connecting the PT with the PD undergo seasonal remodelling in response to photoperiod. Vascular remodelling is governed by vascular endothelial growth factor (VEGF-A), a protein that controls the structure and permeability of blood vessels. Our previous work demonstrated that two forms of VEGF-A are produced, one that stimulates blood vessel growth, referred to as the pro-angiogenic form, and another one that inhibits it, referred to as the anti-angiogenic form. These two forms result from a slight modification of the same gene product and compete with each other in the target cells. We have revealed that the melatonin sensitive cells of the PT also produce VEGF-A, and that the duration of the melatonin signal controls the differential production of the two VEGF-A forms. In the summer, the short duration of melatonin stimulates the pro-angiogenic VEGF-A form, leading to an increase in the number of blood vessels and stimulation of prolactin (a hormone that regulates hair growth) from the PD; in contrast, in the winter, the long duration of melatonin stimulates the anti-angiogenic VEGF-A form, leading to a decrease in blood vessels and suppression of prolactin secretion. Additional pilot data have shown that a similar system operates in the equine pituitary, providing preliminary evidence that the melatonin-induced VEGF dependent control of pituitary function is a conserved mechanism of adaptation across species. How the duration of melatonin exposure can modify VEGF-A gene expression to produce pro- or anti-angiogenic variants is not known. The differential VEGF-A gene outputs result from a process known as alternative splicing, and depends on the actions of a factor called SRSF6. In rodents, alternative splicing of other genes can be regulated by external cues such as feeding/fasting, and is associated to the daily (circadian) biological clock machinery. Because melatonin is a major regulator of the circadian clockwork within the PT, a link between this daily time measuring system, the differential production of VEGF-A forms as seasonal time decoding messengers, and their regulation by another external cue, i.e. photoperiod, can be envisaged. Here, we will investigate how melatonin generates differential production of VEGF-A forms to induce adaptation to a changing environment using animal models that reproduce at opposite times of the year, and a variety of laboratory techniques and technical strategies which, when combined, will allow us to gain an in-depth understanding of this novel mechanism of adjustment that has evolved to maximise species survival.

We have recently shown that a mechanism regulating angiogenesis within the pituitary gland participates in the seasonal adjustment of photoperiodic species to a changing environment by: a) controlling the remodelling of the pituitary microvasculature; and b) acting as messenger signals from the melatonin sensitive pars tuberalis (PT) to the endocrine cells of the pars distalis involved in the regulation of seasonal fertility. Here we will investigate whether this is a conserved system of adaptation, operating in species that reproduce at different times of the year, and examine the intra- and inter-cellular pathways underlying this process. It is well established that the pattern of melatonin secretion from the pineal gland decodes day length information through a direct action in the PT. The overall hypothesis of this project is that angiogenic mechanisms within the pituitary participate in the melatonin signal readout to translate photoperiodic cues into an annual physiological response. Melatonin is known to affect the circadian molecular clockwork of the PT, but whether clock genes are involved in the referred melatonin-induced differential expression of VEGF isoforms, and whether a similar mechanism operates in both short and log day breeding species is not known. Differential expression of VEGF-A isoforms results from SRSF6-dependent alternative splicing of a single gene, and recent studies have revealed that the circadian clockwork can regulate alternative splicing of other genes in a tissue dependent manner, and that this can be modulated by external cues such as feeding/fasting; however, the possibility that a similar process can be triggered by another externally regulated cue, namely the melatonin signal, remains to be determined. We will therefore investigate whether melatonin induces VEGF-A alternative splicing and assess the implication of clock genes in the VEGF-A regulation of seasonal physiology using whole animal systems and in vitro strategies.

This work will investigate the processes underlying the adaptation of animals to a changing environment. The project is based on our recent discovery that angiogenic mechanisms within the pituitary gland respond to seasonal changes in day length, i.e. photoperiod, generating pro- or anti-agiogenic isoforms of VEGF-A at different times of year. The differential expression of VEGF-A isoforms is regulated by the duration of melatonin exposure at night, which is the translator of photoperiodic information, and is predicted to result from alternative splicing of the VEGF-A gene, leading to seasonal remodelling of the vascular connections within the pituitary and paracrine regulation of pituitary endocrine networks that control annual cycles in reproduction, hair growth and other functions. Therefore, these studies are applicable to all animal species that have developed annual physiological cycles as a strategic means to ensure survival in a changing environment. In particular, the project is directed towards farm animal and domestic species that display robust seasonal cycles in their physiology such as sheep and horses. A better understanding of their processes of adaption will ensure improved efficiency in the farming and equestrian industries. The impact of these studies extends to human health, in particular to the understanding, prevention and treatment of illnesses derived from day/night shifts in work schedules and trans-equatorial flying. Moreover, the impact can extend to the treatment of other diseases that show seasonal incidence such as certain types of depression and cancer. A key objective of this project is to unravel the way in which the photoperiodic decoder, i.e. the duration of nocturnal melatonin output throughout the year, can induce alternative splicing of the VEGF-A gene to generate the desired adaptive biological response. We will tackle this vital question using animal models that exhibit opposing phases in their annual physiological cycles at a given season, using in vitro, ex vivo and in vivo strategies. If the molecular and cellular signalling mechanisms underlying the adaptation to a changing environment can be more clearly understood, then both artificial and natural regulators of seasonal physiological cycles may be developed and controlled, and pathologies with seasonal incidence may be more effectively overcome. For instance, foodstuffs that regulate alternative splicing have been identified (e.g. broccoli, carrots, olive oil), and although much of the current work has focused on the effects of high fat diets, understanding the regulation of micro-vascular remodelling and intra-pituitary paracrine control by alternative splicing of VEGF may help us to recognise how, for example, different farming and breeding regimens could be implemented to improve the control of fertility in sheep and horses, how novel photoperiodic strategies could be used to enhance the welfare of horses subjected to trans-equatorial travel for breeding or competition purposes, and how the occurrence or recurrence of clinical conditions in humans could be better prevented and/or treated more effectively. The project benefits from the combined expertise of four scientists with different but complementary backgrounds and skills; this is highly beneficial not only for the successful completion of the studies, but also for the dissemination of the impact of the findings in different scenarios, communities and locations.