Closed Projects

Chronobiology is the study of biological rhythms. The best understood cycles are those with circadian 24 hour periods that modulate the temporal dynamics of physiology and behaviour of all higher organisms and some bacteria. Four model organisms have been widely used to study the underlying biology of circadian clocks, Cyanobacteria, the fungus Neurospora, the fruitfly Drosophila, and the mouse. CINCHRON is an integrated European centre of excellence for the research training of researchers in the emerging multidisciplinary field of Comparative INsect CHRONobiology. The 2017 Nobel Prize for Medicine or Physiology was awarded jointly to three of our colleagues, Jeffrey Hall and Michael Rosbash (both at Brandeis University, Boston, USA) and Michael Young (Rockefeller University, New York, USA) for their work on the molecular dissection of the circadian clock in Drosophila.  Our warmest congratulations to these outstanding fly molecular geneticists for their achievements, which is particularly heartfelt because seven of the principle investigators of CINCHRON have worked and published with Hall and Rosbash. Insect clocks, both circadian and seasonal, are vital for adaptation to the environment and recent global patterns of climate change mean that insect pests and vectors of disease are expanding their ranges into Europe. Consequently there is an urgent need to study the circadian clocks of these insects and how they synchronise to the environment. CINCHRON will contribute to the integration and cohesion of future European research efforts in solving pure and applied biological problems.

Host Institution: Wuerzburg University
Supervisor: Prof Charlotte Helfrich-Foerster

Project 1: Seasonal clock in D. littoralis
Objectives: To discover the role played by the circadian clock in night-length measurement and induction of diapause in the northern European fruifly D. littoralis. The role and distribution of clock proteins and relevant clock-related neuropeptides will be studied under different photoperiodic and temperature conditions and mutagenesis (CRISPR/Cas9) of canonical clock genes will be used to examine whether clock genes measure night-length (which mediates diapause).

Project 2: Seasonal clock in pea aphids
Objectives: To discover the role played by the circadian clock in the induction of diapause in the pea aphid Acyrthosiphon pisum. Circadian behaviour will be characterised as will neuronal clock gene expression both temporally and spatially. CRISPR/Cas9 mutagenesis will be used to investigate whether clock mutations disrupt diapause.

The ability to synchronize to the cyclical changes in the environment is a fundamental property of circadian clocks. To do so, they use the light-dark cycles as most important Zeitgeber and temperature cycles as the second Zeitgeber. However it is largely unknown how the two informations are integrated by the clock. The clock of the fruit fly consists of different interacting clock neurons that give rise to two activity peaks per day - one in the morning and the other in the evening. Light accelerates the oscillations of one group of the clock neurons whereas it slows down that of the others, so that the morning activity is advanced and the evening activity delayed under long summer days. This response is mediated by rhodopsins in the compound eyes. In addition the compound eyes mediate direct light responses of the flies, such as an immediate activity increase when lights are shut on. In this project we want to clarify the roles of the 6 different rhodopsins in the eyes (Rh1, Rh3, Rh4, Rh5, Rh6 und Rh7) for the different responses. Furthermore, we investigate how temperature cycles and light-dark cycles are integrated by the clock neurons.

The fruit fly Drosophila melanogaster – is successfully used as model system to understand the function of endogenous clocks. As in mammals, several clock neurons interact in a neuronal network with neuropeptides as main communication signals. The best investigated neuropeptide in Drosophila’s clock is the "pigment-dispersing-factor" (PDF). PDF is expressed in 4 small and 4 large neurons per brain hemisphere and serves as coupling signal between the different clock neurons as well as as output signal to downstream neurons. Besides the PDF-positive neurons, neurons that express the short or long form of Neuropeptid F (NPF) and neurons expressing the Ion Transport Peptide (ITP) are important for the function of endogenous clock. The aim of the project is to classify their role in the circadian system and to unravel their action on other neurons by in vivo Ca2+ and cAMP-Imaging (Details see project of Christiane Hermann).

Chronic psychosocial stress and dys-regulation of the circadian clock share many common features. Both have dramatic consequences on health and life span, both prominently affect the HPA axis influencing glucocorticoid (GC) levels and immune responses, and both employ similar neuropeptides as signalling molecules. Anatomically, the HPA axis and the circadian clock in the suprachiasmatic nuclei (SCN) share a common interface - the paraventricular nucleus (PVN). Within the PVN, corticotrophin releasing factor (CRF) synthesizing neurons are stress responsive and trigger adrenal GC secretion. The PVN also receives inputs from the SCN, which regulates the circadian rhythm of GC secretion independent of stress. The daily GC peak prepares the animal for its active phase. This project will investigate the mutual interactions between psychosocial stress and the circadian clock in the SCN. We will determine (i) whether the circadian clock modulates the capability to cope with stress in a daytime dependent manner and (ii) whether psychosocial stress affects the circadian clock in dependence on the time of stressor exposure. To do so, we will subject mice to repeated social defeat (SD) stress in the morning or evening and investigate the physiological stress responses as well as the consequences on rhythmic behaviour and molecular circadian oscillations in the SCN.

The homeostasis of transmitters is essential for normal brain function and glia cells play an essential role in it. Disturbances of this homeostasis result for example in an abnormal sleep-wake pattern. This project will investigate the regulation of monamine release (specially of dopamine and serotonin) by postulated tripartite synapses between monaminergic neurons, glia cells and circadian clock neurons of the fruit fly Dosophila melanogaster.

In fruit flies, a mutation in the alanyltransferase "Ebony" that is exclusively expressed in glia cells results in disturbed sleep-wake patterns. Morphological investigations (confocal and EM) will show whether the postulated tripartite synapses exist. Sleep-wake studies of mutants with disturbed monamine transport and processing are planned to unravel the processes at synapses that lead to normal sleep. Ca++-imaging and period-luciferase imaging on cultivated brains will be performed to study the response of circadian clock neurons and glia cells to monamine transmitters as well as drugs that influence monaminergic signalling.

Animals living at high latitudes have to cope with prominent seasonal changes in their environment. In summer, they are exposed to long days and short nights with pleasant temperatures that allow reproduction, whereas the short days and low temperatures in winter require special adaptations to survive such as frost resistance and reproduction arrest.

In contrast, animals living close to the equator experience very little seasonal changes allowing reproduction throughout the year.

The circadian clock in the brain is known to control daily activity-rest rhythms and to provide an internal time reference for measuring day length. We found that the daily activity-rest rhythms and the neurochemistry of the clock network in the brain differs significantly in fruit fly species living at high and low latitudes and that these differences are causally related.

In order to understand circadian clock evolution we will investigate the clock network, the daily activity patterns of further fruit fly species living at different latitudes.

To understand the role of the circadian clock in day length measurement, we will use strains of Drosophila melanogaster caught at different latitudes. Our investigations will contribute to the understanding of circadian clock evolution by investigating fruit fly species adapted for a life at different latitudes.

FO 207/16-1

The circadian clock enables animals to be prepared in advance for the regular changes between day and night. Furthermore, some animals use their circadian clock for the memory of time, the measurement of day length (to anticipate seasonal changes) and for time-compensated sky compass orientation. The circadian clock network in the brain of the fruit fly belongs to the best-investigated. It consists of interconnected lateral and dorsal clock neurons, which generate circadian molecular oscillations and mediate these to downstream interneurons and neurosecretory centres in the dorsal protocerebrum. During the last years the fruit fly became a model in
sleep research; the first connections from the circadian clock to metabolism have been established, it was demonstrated that fruit flies have a time memory and that the clock is involved in diapause induction. Even the first hints exist that fruit flies are able to perform time-compensated sky compass orientation. The dorsal clock neurons appear to be involved in all these clock functions. While the lateral neurons represent “master oscillators”, the dorsal neurons are multimodal integrators that are essential for transferring the circadian rhythms to downstream interneurons and neurosecretory cells. Nevertheless, recent studies are controversial concerning the exact role of the dorsal neurons. Some authors claim that a special group of dorsal neurons (the DN1p) is responsible for sleep, whereas others postulate a role of the same neurons in arousal and elevated metabolism. The reason for these contradicting views lies most probably in the diverse neuronal projections of individual DN1p neurons. We could show that the dorsal neurons represent extremely heterogeneous groups of cells, but that their neuronal projections are still insufficiently characterized. 

Animals living at high latitudes have to cope with prominent seasonal changes in their environment. In summer, they are exposed to long days and short nights with pleasant temperatures that allow reproduction, whereas the short days and low temperatures in winter require special adaptations to survive such as frost resistance and reproduction arrest.

In contrast, animals living close to the equator experience very little seasonal changes allowing reproduction throughout the year. The circadian clock in the brain is known to control daily activity-rest rhythms and to provide an internal time reference for measuring day length. The latter is essential for a timely preparation for the winter.

We found that the molecular oscillations of the clock proteins Period (PER) and Timeless (TIM) in the brain of the fly differ under long summer and short winter days. Most interestingly, these flies also respond to short days with a change in the composition of their hydrocarbon (CHC) profile  on the surface of the.

In order to understand the role of the circadian clock in day length measurements, we will use lab and wild-caught strains D. melanogaster. We will especially test whether there is a causal correlation between day length, PER/TIM oscillations, and cold resistance by investigating clock mutants that cannot normally adapt their clock to the seasons but remain in a quasi-permanent clock-winter- or clock-summer- state.

Our investigations will provide the first basis in understanding the role of the circadian clock in seasonal adaptation using the well characterized model D. melanogaster.

The circadian clock enables animals to be prepared in advance for the regular changes between day and night. Furthermore, some animals use their circadian clock for the memory of time, the measurement of day length (to anticipate seasonal changes) and for time-compensated sky compass orientation. The circadian clock network in the brain of the fruit fly belongs to the best-investigated. It consists of interconnected lateral and dorsal clock neurons, which generate circadian molecular oscillations and mediate these to downstream interneurons and neurosecretory centres in the dorsal protocerebrum. During the last years the fruit fly became a model in
sleep research; the first connections from the circadian clock to metabolism have been established, it was demonstrated that fruit flies have a time memory and that the clock is involved in diapause induction. Even the first hints exist that fruit flies are able to perform time-compensated sky compass orientation. The dorsal clock neurons appear to be involved in all these clock functions. While the lateral neurons represent “master oscillators”, the dorsal neurons are multimodal integrators that are essential for transferring the circadian rhythms to downstream interneurons and neurosecretory cells. Nevertheless, recent studies are controversial concerning the exact role of the dorsal neurons. Some authors claim that a special group of dorsal neurons (the DN1p) is responsible for sleep, whereas others postulate a role of the same neurons in arousal and elevated metabolism. The reason for these contradicting views lies most probably in the diverse neuronal projections of individual DN1p neurons. We could show that the dorsal neurons represent extremely heterogeneous groups of cells, but that their neuronal projections are still insufficiently characterized. 

For animals, it is vitally important to time and synchronise development and physiological activity of the different body systems and to adjust behaviour accordingly. The timing and synchronisation of different body systems and behaviour requires both a timer (represented by central and peripheral endogenous clocks) and an integrating communication system (represented by the (neuro)endocrine system). Yet, we know astonishingly little about the complex neuronal and endocrine pathways and underlying molecular and cellular signalling mechanisms by which endogenous clocks and neuroendocrine systems interact with each other. Such knowledge could provide a handle to understand and treat associated developmental disorders and circadian dysfunctions including impaired fertility, sleep disturbances and psychiatric problems that can result from long-term disruption of this integrated timing. The aim of this project is to dissect the interactions between developmental and circadian timers and the neuroendocrine system, and to start characterising cellular and molecular signalling principles underlying timed behaviour. A main focus here is on the interplay between peptide and steroid hormone signalling and its circadian control. We also aim to find out where in the brain circadian and developmental signals are integrated to time a specific behaviour. This specific behaviour will be the eclosion of the fruit fly Drosophila. The fruit fly appears a highly suited model system for this project as it is genetically and experimentally well tractable, and possesses a relatively low number of individually identifiable neurons. This in the long range offers the possibility to in completeness decipher neuronal and hormonal connections between central and peripheral clocks and target tissues from the molecular to the systemic level. Eclosion is well suited as it is timed by developmental and circadian timers, and its timing is under control of both peptide and steroid hormones. From a zoological point of view, understanding the architecture of the neuronal-endocrine network timing eclosion is of great interest since correct eclosion timing is most critical for the survival and fitness of insects and other arthropods, which represent the vast majority of animals on our planet.