Genetic dissection of seasonal timing in Drosophila

Seasonal timing is a key process for survival for most organisms, especially in temperate regions. In broad ranges of species, from plants to mammals, the annual change in day-length is monitored the so-called a photoperiodic clock, allowing the accurate timing of the seasonal response. Many insects for example, including numerous agricultural pests, detect the shortening of the day during the autumn and switch to diapause (a developmental arrest), which allow them to survive the winter. Despite intensive study of the photoperiodic clock for the last 80 years, the underlying molecular mechanism is still largely unknown. This is in marked contrast to our understanding of the circadian clock that regulates daily rhythms, where studies in various model organisms, particularly Drosophila, have established firm principles and rich mechanistic detail, including genes conserved across diverse phyla.
In Drosophila, the genetic basis of the seasonal clock has focused on female diapause, manifested as a developmental arrest of the ovaries induced by short days and low temperatures. Unfortunately, this phenotype is not very robust, and practically is not amenable for large genetic screens. Recently, research in our laboratory has corroborated previous observations that flies developed under short days become significantly cold-resistant compared with flies raised in long-days. The difference in cold response can be easily quantified using the chill-coma recovery (CCR) assay, in which flies exposed to freezing temperatures enter reversible narcosis . The recovery time reflects how cold-adaptive the flies are, and our recent work has demonstrated that this response is largely regulated by the photoperiod (i.e. flies exposed to short photoperiods during development exhibit shorter recovery times). We have devised an automated system, allowing the monitoring of hundreds of flies, and here we propose to use this system for high-throughput genetic screen for genes involved in the photoperiodicresponse.

In Drosophila, low temperatures short day-lengths induce developmental arrest of the ovaries, serving as an adaptive response that allows females to overwinter. To date, this form of reproduce diapause has been the sole phenotype in Drosophila for studying the genetics of seasonal timing. However, this phenotype involves the manual dissection of large number of female ovaries and consequently is not easily amenable for large-scale genetic screens. Here, we describe a new approach for studying the molecular basis for seasonal timing, based on the chill-coma recovery (CCR) phenotype of flies. Using a commercial automated locomotory monitoring system we have recently found that the CCR can be analysed in hundreds of flies within a few hours, paving the way for large-scale genetic screens for genes involved in the photoperiodic clock.
We will carry out a genome-wide screen using the Cambridge Protein Trap Insertion (CPTI) strain collection (Objective i). Around 400 hundreds strains of this collection, which has been created using the piggyBac method to insert an EYFP construct, will be tested for their CCR following development in short and long days. (ii) Building on previous global profiling experiments that we have performedusing microarrays to search for photoperiodically regulated transcripts (and micro-RNAs), we will test the role of 40 candidate genes that show photoperiodic differential expression, in photoperiodic timing of CCR, by dsRNAi knockdown and GAL4-UAS misexpression. We will also test the role of candidate microRNAs in the CCR response. The role of various brain neurons in photoperodic measurements will tested by genetic ablation of these cells using the GAL4-UAS system (Objective iii). In another set of experiments (Objective iv), we will investigate which photoreceptors are involved in the photoperiodic response, by establishing the action spectrum of this response, and by testing the response of various photoreceptor knockdown strains.

We all notice the passing of the seasons, and marvel at how the plants and animals around us time their migrations, flowering or breeding with the changing of the seasons. Our study of seasonal timing and its underlying genetic, molecular and anatomical substrates will provide a novel perspective and theoretical framework by which to study seasonality in terms of both humans, and the organisms around us.

The appropriate daily and seasonal timing of physiological processes is essential to health. Disruption of the circadian clock or the seasonal timer results in illness. For example, in the USA alone, it is estimated that six of every 100 people suffer depressive disorders with seasonal pattern (seasonal affective disorder, SAD). Our work may have some direct implications for remedial action by shedding light (!) on the underlying molecular mechanisms. Based on the well established similarities between insects and mammals in the genetic basis of their circadian system, we predict that any new mechanism that we identify that underlie insect photoperiodic response, may have interesting mammalian homologues (or analogues), with similar functions.

While the research is 'pure', there are further downstream implications for our research, even if these are not immediate. Other beneficiaries apart from the academic beneficiaries would thus be
1. The medical profession and the pharmaceutical industry who might be interested in our genes as potential targets for developing therapies for those with seasonal problems.
2. Agricultural and medical entomologists interested in (i) controlling problem insects using diapause as a target or (ii) for manipulating diapause to improve storage of beneficial insects such as biocontrol agents ("shelf-life" is an issue for breeders and distributors of beneficial insects).
3. Policymakers who are concerned about the effects of global climate change and the impact on wild-populations and human health.
4. The public, who is always very curious and responsive to issues concerning "body clocks".

As with all basic research, it could be many years down the line before any of the discoveries we will make might be translated into drugs, treatments and policies. However, the work of many chronobiologists (including ourselves) has produced, through the years, a tangible effect: nowadays everybody is aware of the existence of a body clock and of its tremendous power in regulating our lives and our health. Moreover, we have ourselves a great deal of experience in talking to schoolchildren, their teachers, the general public in open lectures, to artists and to the press about how the days and the seasons influence our lives. We also communicate with the medical profession and with applied researches in the form of publications, and by dissemination at conferences. We also maintain websites, open to anyone, so that any informed citizen will have the opportunity to understand the problems we are concerned with in our research and to echo them to policy makers. Furthermore, we are open to collaborations with any interested groups, academic, or commercial.

Our project will also contribute to the cultural and economic growth of the UK by enhancing the skills of the PDRA and students that will be working on the proposed research. Typically, our labs host over 15 visiting students every year (including 3rd year undergraduate projects, MSc projects, Erasmus exchange students, and school work-experience placements). Students will gain specialised and much-needed skills through performing advanced laboratory techniques and data analysis. These are generally and widely applicable to subsequent career progression in molecular biology. In addition, transferable skills that will be acquired in the form of public speaking, computing and writing are again widely relevant to any professional forum.