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The Transcriptomic and Biophysical Basis of Mechanosensory Submodality: A Drosophila Model Organ Study

Reference	BB/L02084X/1
Principal Investigator / Supervisor	Professor Joerg Albert
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Ear Institute
Funding type	Research
Value (£)	409,806
Status	Completed
Type	Research Grant
Start date	01/07/2014
End date	30/06/2017
Duration	36 months

Abstract

Previous work has found ~ 300 genes to be expressed in the Drosophila Johnston's Organ (JO).The fly's JO harbours discrete populations of mechanosensory neurons which have been linked to the submodalities of wind, gravity and sound. However, all neurons attach to the same receiver structure, the third antennal segment. Large parts of the submodality specific adaptations thus have to be implemented downstream of the external receiver on the level of the respective cellular and molecular components. Using the binary transcription regulation system Gal4/UAS to drive expression of FLAG-tagged PolyA binding proteins (UAS- PABP-FLAG) in different subsets of mechanosensory neurons we will create and sequence (by means of immuno-precipitation, RNA amplification and RNA-Seq) JO subset-specific transcriptomic libraries. Such cell-type-specific libraries will be analysed to reveal the molecular (and cellular) divisions of labour in JO. Several different strategies are being applied to identify the distinct roles of individual genes, and sets of genes, for subset-specific mechanotransduction and associated mechanosensory behaviours: (i) We will use i-cisTarget and iRegulon to predict the factors that are involved in the transcriptional regulation of the extracted, subset-specific libraries. (ii) We will then use UAS-RNAi-mediated knockdowns of discovered genes and predicted transcription factors (driven by ato-Gal4 or subset specific driver lines) in combination with biophysical analyses (laser-vibrometry, compound action potential recordings, compound receptor potential, sharp electrode and patch-clamp recordings of JO neurons) to characterise, and quantify, the genes' specific contributions to JO function and distinct mechanosensory submodalities. We will finally use newly devised experimental paradigms (employing electrostatic stimulation methods) to quantify a specific gene's requirement for behavioural responses to sound-like, wind-like or gravity-like stimuli.

Summary

Mechanosensory organs directly couple the mechanical energy of a stimulus to the open-state of an ion channel in the membrane of a mechanosensory cell. This process, known as mechanotransduction, lies at the heart of all mechanosensation. Conceptually, it is clear that the specific nature of this coupling will distinguish, and define, the various mechanosensory submodalities (such as sound, touch, balance, wind, pain, gravity or proprioception). But the concept is misleading in its clarity. It hides the fact that it is unclear how that coupling should differ between, say, a proprioceptive and tactile hair on a spider leg, a vestibular hair cell, or an auditory hair cell in the vertebrate cochlea. Adding darkness to short sight, the molecular identities of the actual transducer channels are still unknown for all of the above given examples. But even if we did know all the respective requirements, we would still not know how these are implemented molecularly for submodality-specific mechanotransduction, to occur. One of the most amenable mechanosensory model organs, the Drosophila Johnston Organ (JO), sits in the 2nd antennal segment and harbours discrete populations of mechanosensory neurons which have been linked to the submodalities of wind/gravity and sound. However, all neurons attach to the same receiver structure, the third antennal segment. Large parts of the submodality-specific adaptations thus have to be implemented downstream of the receiver on the level of the respective cellular and molecular components. We intend to carry out a comprehensive molecular, and functional, dissection of modality-specific mechanotransduction in JO. We will first profile the specific mRNA transcriptomes, (i.e. the complete sets of produced mRNAs) for different types of neuronal and non-neuronal cells. Such cell-type-specific transcriptomic analyses will unveil the molecular (and cellular) divisions of labour in JO and help identify the distinct roles of individual genes for subset-specific mechanotransduction and mechanosensory behaviours. Our behavioural analyses will use novel tools that we have specifically devised for this project. Combining molecular and biophysical analyses (laser vibrometry, extracellular and intracellular recordings) with predictive computational tools, this project will put particular emphasis on the identification of modality-specific differences in higher order regulatory genes, such as specific transcription factors. The identification of modality-specific transcription factors is expected to enable the identification of distinct modules of sensory transduction, which act as functional units within mechanosensory cells of different modalities. There is robust evidence that the sensory organs of different species, e.g. the ears of flies and the ears of humans, share multiple functional and molecular similarities. Likewise, different sensory organs within the same species, e.g. the ears and the eyes of flies display striking molecular and mechanistic overlap. Understanding how the underlying transcriptional pathways, which can be expected to be conserved across the animal kingdom, are being recombined during the process of evolution to create sensory systems of various modalities and submodalities will be of great value for better understanding of the myriad of human disease syndromes such as Usher syndrome Type IIA + IIIA or Bardet-Biedl syndrome, which simultaneously affect multiple sensory systems. Eventually, this research into the molecular and mechanistic fundaments of sensory modality will lead the way towards new therapeutic avenues.

Impact Summary

This project is important for understanding the molecular mechanisms that underlie submodality specific mechanosensation in the Drosophila melanogaster Johnston's Organ (JO). Alongside direct benefits to the scientific community, this project will have considerable benefit for non-academic communities interested in health and agriculture. Defects in mechanotransduction can lead to serious sensory impairments. Similar to mammalian ears, the JO has neuronal submodalities that allow for sound detection and the detection of wind and gravity. Because mammals and insect sound receivers share common evolutionary origins, direct hypotheses can be made regarding molecular composition of auditory (sound sensing) and vestibular organs (gravity sensing) based on data from Drosophila sensory structures. In addition to deafness this project has the potential to aid the efforts to treat those that suffer other sensory impairments. This project explores genetic and molecular links between the visual system as well as auditory and wind/gravity sensory systems. As a result, we expect this research will not only aid in the treatment of deafness but also those that suffer from both impaired hearing and vision. Auditory defects in particular are a serious health problem in the United Kingdom. The data on deafness and other sensory impairments are alarming. According to Deafness Research UK (http://www.deafnessresearch.org.uk/) Almost 9 million people in the UK, (1 in 7) suffer from deafness or experience significant hearing difficulty. The charity deafblind UK (http://deafblind.org.uk/) reports that 356,000 people suffer from both hearing loss and vision impairment. We expect that, as the population ages, the need for importance for treatments for sensory impairments (deafness, blindness and others) will only increase. This project will aid in understanding the genetic basis of deafness by further exploring the Drosophila homologues to specific to genetic disease genes and identifying their cell-specific role in the JO and how these genes might contribute to mechanosensory function and downstream behaviour. It will also aid the understanding of genetic diseases that can cause both deafness and blindness such as Usher Syndrome Type IIA and IIIA and Bardet Biedl syndrome by further identifying which cells have proteins with dual vision and auditory roles. Acute adult onset deafness will also benefit as the project explores seeks to understand normal auditory and general mechanosensory function and may therefore aid in the creating on bio-inspired hearing aids or other related treatments. While the healthcare industry is interested in the similarities between Drosophila and humans, the agricultural industries are more interested in exploiting the differences between insects and humans. Agrochemical companies e.g. seek to develop insecticides (such as the chordotonal toxins pymetrozine) with reduced human toxicity by targeting those proteins, not found in humans (e.g components of insect mechanotransducer machineries). Novel chordotonal targets (as likely to result from this study) will be of particular interest. Chordotonal organs (such as JO) are stretch receptors found in all insects but not in humans. Existing differences between humans and insects can be exploited to identify novel compounds that are less toxic for humans and vertebrates. Finally, this research will be of great interest to global efforts to controlling mosquitoes and combat the spread of mosquito borne illnesses such as malaria by increasing our knowledge of insect biology with the continued goal of better, less toxic and more environmentally friendly methods of pest control.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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