Defining the molecular basis of photperiodism in mammals

The perpetual motion of the Earth on its axis and the orbit around the sun generates a rhythmic environment for life on Earth. Organisms of all types have responded by evolving biological clocks and calendars that allow anticipation of day and night, and the changing seasons. Recent research into this 'chronobiology' has characterised a small number of genes, called clock genes, which generate the intrinsic daily rhythm. These genes show conserved features in their structure and function from insects to man, reflecting their ancient evolution. Clock genes act within most cells of the body to produce a near 24-hour rhythmic output, and cells communicate to regulate daily rhythms in sleep-activity, feeding, hormone secretion and many other characteristics. Much less is understood about the molecular basis of longer-term timers and the mechanism by which animals respond to changing day length to synchronise seasonal rhythms. In mammals, a key aspect of seasonal timekeeping is the production of melatonin by the pineal gland. Melatonin is produced only at night and the pattern directly reflects the length of the night, and it is the changes in this hormonal signal that conveys information about time of year around the body. Long daily bouts of melatonin act as the signal for winter and short bouts for summer. Our previous work has shown that the changing pattern of melatonin is decoded through the switching on and off of specific clock genes. Notably, the melatonin increase at dusk activates Cry1 gene expression, and the melatonin decline at dawn activates Per1 gene expression. Based on this we have proposed an 'internal coincidence hypothesis' for photoperiod time measurement. This states that the extent of interaction between CRY1 and PER1 proteins, which is dictated by the period from dusk to dawn - the melatonin signal, governs the seasonal response. The daily clock mechanism has thus been co-opted for seasonal timing in melatonin-responsive cells that are located in the brain and pituitary gland. Now we plan to test this hypothesis using transgenic sheep in which the Cry1 gene is selectively neutralised. This strategy depends on our recent demonstration that modified DNA sequences can be efficiently introduced into sheep embryos and are expressed in lambs. The sheep is used as a model because of its very well characterised seasonal biology. The aim is to use two different 'transgenes' to interfere with endogenous expression of the Cry1 gene. The prediction is that this will not affect normal development, but will block photoperiodic responsiveness. The biology of this 'sheep for all seasons' will be of major interest to chronobiologists, and the use of transgenesis in sheep opens a new era in the study of genetic control in a long-lived species.

In mammals, seasonal timing depends on the production of melatonin (MEL) by the pineal gland. MEL is only produced at night, in a temporal pattern reflecting the night-length, and acts on tissues expressing high affinity G-protein coupled receptors (MT1). Changes in melatonin signal duration over the year determine seasonal responses. Our work on the pars tuberalis (PT) of the anterior pituitary, led us to propose that photoperiodic interpretation of the MEL signal depends on rhythmical expression of clock genes. We have highlighted evening induction of Cry1 and morning induction of Per1, as key MEL-dependent events. Since the protein products of these genes have interdependent function, we advanced an 'internal coincidence hypothesis' suggesting that, by controlling the phase relationship between peaks of Cry1 and Per1 expression, MEL governs the extent of PER:CRY complex formation. This model predicts that breaking the regulatory link between MEL and Cry1 will block photoperiodic responses. We will test this prediction in transgenic sheep. The strategy is to direct Cry1 knockdown (shRNA) or nuclear accumulation (by over-expression of a dominant negative, nuclear export deficient PER1) to MT1 receptor expressing cells. We will achieve this using the promoter of the MT1 gene to direct transgene expression from a lentiviral construct. Reporter experiments will ensure that effective cellular specificity, leading to germ line and somatic transformation studies. Zygote injection and embryo transfer will be used to make lambs in which Cry1 is mis-regulated in MT1 expressing cells, while intracranial micro-injections will allow the study of Cry1 mis-regulation in the PT. Endocrine profiling will be used to compare photoperiodic responses with those of untransformed control animals. The outcome of this project will be of wide interest to chronobiologists and endocrinologists, and provides a focus for developing novel transgenic research methods in sheep.