Periodic Reporting for period 5 - Clock Mechanics (Mechanosensation and the circadian clock: a reciprocal analysis)
Berichtszeitraum: 2021-09-01 bis 2022-12-31
General
In virtually all living organisms, so called circadian clocks pick up, and repeat, the fundamental beat of life, which is caused by the earth’s daily rotation about its own axis. Circadian clocks are autonomous agents, they keep ticking away without any external stimulus and thereby create their own nights and days. In order to synchronise, or entrain, themselves to the world they are living in, however, clocks require so called Zeitgeber, i.e. sensory stimuli, which are coupled to the underlying geophysical cycle, the most prominent one here being probably the continual succession of light and darkness; but also temperature oscillations serve as Zeitgeber and can synchronise the clock. In insects, such as the fruit fly Drosophila another sensory modality has been linked to circadian entrainment, namely mechanosensory pathways. In this project, we dedicate attention to the interface between insect mechanosensors and the circadian clock. We ask: How do mechanosensory systems affect the clock? And: How does the clock affect mechanosensory systems? We ask these questions with a focus on one specific mechanosensory system: hearing and acoustic communication (in fruit flies and disease-transmitting mosquitoes).
In virtually all living organisms, so called circadian clocks pick up, and repeat, the fundamental beat of life, which is caused by the earth’s daily rotation about its own axis. Circadian clocks are autonomous agents, they keep ticking away without any external stimulus and thereby create their own nights and days. In order to synchronise, or entrain, themselves to the world they are living in, however, clocks require so called Zeitgeber, i.e. sensory stimuli, which are coupled to the underlying geophysical cycle, the most prominent one here being probably the continual succession of light and darkness; but also temperature oscillations serve as Zeitgeber and can synchronise the clock. In insects, such as the fruit fly Drosophila another sensory modality has been linked to circadian entrainment, namely mechanosensory pathways. In this project, we dedicate attention to the interface between insect mechanosensors and the circadian clock. We ask: How do mechanosensory systems affect the clock? And: How does the clock affect mechanosensory systems? We ask these questions with a focus on one specific mechanosensory system: hearing and acoustic communication (in fruit flies and disease-transmitting mosquitoes).
The initial phase of the project involved designing new experimental paradigms in which mechanosensory entrainment - or circadian modulations of mechanosensory behaviour - could be tested – with one of the tested hypotheses being that mechanical vibrations do not necessarily entrain the clock (external ‘vibration entrainment’) but possibly the flies’ vibration-induced activity feeds back into the clock (internal ‘activity entrainment’). Vibrated flies were constantly monitored by video tracking and high-resolution activity monitoring. Mechanical stimulus paradigms were designed to minimise input from external stimuli and maximise stimulus-induced activity in order to test our hypothesis. Interestingly, preliminary results suggest that not all mechanically-evoked activity rhythms are associated with an entrainment of the flies’ clock, posing the question if activity alone is sufficient to set the clock.
The key molecular players that help maintain circadian rhythmicity are well documented, therefore we tested for their expression in tissues important for mechanosensation – one major model organ of mechanosensation in Drosophila is Johnston’s organ (JO), a multi-cellular chordotonal organ (ChO). Chordotonal neurons are stretch-receptors, which serve both as exteroceptors (sensing external vibrations) and as proprioceptors (sensing the animals’ own movements); most notably, however, ChOs have been implicated in both mechanosensory and thermosensory entrainment of the fly’s circadian clock. We dissected JO tissue at different time points across a day and quantified expression of core clock genes. We compared clock gene expression levels between time points in JO and a control tissue, the fly’s head (as the site of the ‘central clock’). Rhythmic oscillations of core clock genes were found in JO; oscillations in JO were in phase with oscillations in the fly’s head.
One significant question in circadian biology is how multiple entrainment stimuli are being processed when presented in conflict. When light and temperature oscillate in phase we observed seemingly ‘normal’ activity patterns; when light and temperature were opposing each other, with an ‘unnatural’ 180 phase difference in between the two of them, light dominated the entrainment, that means the temperature stimulus was basically neglected. However, when the phase of these two conditions was offset by a smaller margin, of only a few hours, then temperature became a much more prominent entrainment stimulus, at intermediate phase differences of 5-7 hours, finally, behavioural coherence, and circadian entrainment, broke down: a unique, aberrant locomotor activity pattern was observed and molecular oscillations of core clock collapsed (Harper et al. 2016).
This is clear evidence that these sensory inputs are weighted and integrated to elicit the appropriate behavioural (and molecular) responses. Overall it alludes to a complexity that may not be fully untangled by conventional experiments.
The key molecular players that help maintain circadian rhythmicity are well documented, therefore we tested for their expression in tissues important for mechanosensation – one major model organ of mechanosensation in Drosophila is Johnston’s organ (JO), a multi-cellular chordotonal organ (ChO). Chordotonal neurons are stretch-receptors, which serve both as exteroceptors (sensing external vibrations) and as proprioceptors (sensing the animals’ own movements); most notably, however, ChOs have been implicated in both mechanosensory and thermosensory entrainment of the fly’s circadian clock. We dissected JO tissue at different time points across a day and quantified expression of core clock genes. We compared clock gene expression levels between time points in JO and a control tissue, the fly’s head (as the site of the ‘central clock’). Rhythmic oscillations of core clock genes were found in JO; oscillations in JO were in phase with oscillations in the fly’s head.
One significant question in circadian biology is how multiple entrainment stimuli are being processed when presented in conflict. When light and temperature oscillate in phase we observed seemingly ‘normal’ activity patterns; when light and temperature were opposing each other, with an ‘unnatural’ 180 phase difference in between the two of them, light dominated the entrainment, that means the temperature stimulus was basically neglected. However, when the phase of these two conditions was offset by a smaller margin, of only a few hours, then temperature became a much more prominent entrainment stimulus, at intermediate phase differences of 5-7 hours, finally, behavioural coherence, and circadian entrainment, broke down: a unique, aberrant locomotor activity pattern was observed and molecular oscillations of core clock collapsed (Harper et al. 2016).
This is clear evidence that these sensory inputs are weighted and integrated to elicit the appropriate behavioural (and molecular) responses. Overall it alludes to a complexity that may not be fully untangled by conventional experiments.
The project activities have been heavily focused on developing novel computational and experimental tools to explore the interrelation of circadian clocks and mechanosensory systems. We have realized early on that understanding the interrelation between circadian and mechanosensory systems will require novel theoretical and experimental approaches and will also require to widen the scope to other sensory modalities (cross-modal approach). In a nutshell, the circadian clock has to be taken more seriously as a minibrain in its own right, which carries out complex sensory integrations when computing its various output signals. In Harper et al. 2016, we gave a first appreciation of this complexity. A second publication (Harper et al. 2017) highlighted the differential operation between the central and peripheral clocks, adding a further layer of complexity to circadian biology. The advances achieved during the first reporting period have helped us here greatly to build a novel experimental and theoretical platform for the study of circadian systems, which, when completed, will be of great value for the wider field of chronobiology and neurobiology. As our approach aims at a more comprehensive and ‘holistic’ approach to ‘the clock’ we have extended our scope towards the sensory integration in between different Zeitgeber stimuli (e.g. light and temperature) and the potential role of mechanosensory signals therein (e.g. through mediating activity feedback). We published an invited review to discuss these challenges faced by an organism’s circadian system (Somers et al. 2018). Also, we have discovered a second powerful insect model, which allows for testing the interrelation between circadian behaviour and mechanosensory systems in molecular and neurobiological detail. These are disease-bearing mosquito species, the behaviour of which displays an extreme form of circadian rhythmicity and is heavily dependent on mechanosensory systems. We will continue to use mosquitoes as secondary model system next to Drosophila. It is a benefit of our current methodological landscape that (in an associated PhD project) we have managed to successfully transfer virtually all experimental paradigms for the study of mechanosensory behaviour, and mechanotransduction, from Drosophila to mosquitoes (Su et al. 2018). We expect this aspect of our project to allow for a more detailed analysis of the circadian basis of mechanosensation and the mechanosensory basis of clock function.
In order to extend work on circadian modulations of mechanosensory behaviour, we investigated a behaviour that has both a clear circadian and mechanosensory component i.e. acoustic communication in mating swarms of Anopheline (‘malaria’) mosquitoes. Mating swarms occur at dusk (circadian) and involve a large number of male mosquitoes that must identify a female entering the swarm by their flight tones (mechanosensory). Furthermore, this behaviour encompasses all facets of this project as a gradual light transition period seems to be important for robust behaviour (sensory integration). We expect that by providing tools and data that makes mosquitoes available as circadian research models, this project will leave a lasting legacy.
In order to extend work on circadian modulations of mechanosensory behaviour, we investigated a behaviour that has both a clear circadian and mechanosensory component i.e. acoustic communication in mating swarms of Anopheline (‘malaria’) mosquitoes. Mating swarms occur at dusk (circadian) and involve a large number of male mosquitoes that must identify a female entering the swarm by their flight tones (mechanosensory). Furthermore, this behaviour encompasses all facets of this project as a gradual light transition period seems to be important for robust behaviour (sensory integration). We expect that by providing tools and data that makes mosquitoes available as circadian research models, this project will leave a lasting legacy.