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

Lead Research Organisation: University College London
Department Name: Ear Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Albert JT (2015) Hearing in Drosophila. in Current opinion in neurobiology

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Kavlie RG (2015) Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing. in Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology

 
Description This project made a first attempt to answer the question which molecular specializations are responsible for conferring different response properties to different mechanosensory cells. In other words, what makes one neuron respond preferentially to slow, more static, stimuli such as e.g. wind or gravity and others to fast, more dynamic stimuli, such as e.g. sound?
To answer this question we created a molecular inventory of the differences (and overlaps) between neurons of different mechanosensory submodality. This work involved state-of-the art techniques for profiling the specific genes, which are expressed in a distinct type of cell at distinct points in time. During the project's execution we started a collaboration with one of the leading experts in Drosophila neuro-transcriptomics, Dr Tony Southall from Imperial College.
The first major outcome of this project is thus (i) the generation of a molecular inventory of gene sets which mark the differences between sound, wind and gravity sensitive neurons. This gene list is right now being curated and prepared for publication.

A second outcome is the finding that one molecular key player of Drosophila mechanotransduction, the ion channel NompC, the functional contributions of which we had been studying in biophysical detail before, also contributes to conferring differential identities to different mechanosensory neurons, some of them express one type of NompC (isoform a) and others another type (isoform b). Isoform a and b differ in their adaptation behaviour in touch-receptive bristles, specifically in the speed at which their response to a stimulus decays. This is in line with one of the project's main hypotheses, namely that submodality-specific behaviour is partly caused by a differential adaptation behaviour. A fascinating recent result from this work is the finding that the two isoforms seem to mediate different types of behaviour. One of them is required for sensitive hearing (mediated by the fly's antennal Johnston's Organ) but does not seem to contribute to locomotor control functions, such as proprioception, whereas the other isoform is required for wildtype-like locomotion but does not rescue auditory function. This aspect will be followed up in the immediate future.
This work has been carried out in collaboration with Maurice Kernan (Stony Brook University, New York). The respective manuscript is currently revised for publication and has been resubmitted to Current Biology (Huang et al).


A third outcome relates to the developmental or regulatory genes, which kick-start, and orchestrate, the development of (not only mechano-) sensory organs: so called proneural genes. In the sensory domain, the most prominent proneural gene is arguably 'atonal'.
Atonal is member of a family of master transcription factors, those are genes that bind to other genes and regulate their function. In case of atonal, this involves the development of ears and eyes across the animal kingdom (in both fruit flies and humans). In collaboration with Bassem Hassan (Leuven, Belgium/Paris, France) we tested to what extent atonal genes taken from a variety of evolutionary distant organisms (from sponges over worms to mice) can 'rescue' atonal function in Drosophila. To our great surprise, all atonal versions rescued some functionality in the fly's ear, even the copy of the most distantly related animal class, the sponge. The functional rescue was modular with specific aspects of mechanosensory function restored almost fully or not at all. Some atonal versions, e.g. produced ears, which were almost indistinguishable in their response to soft sounds but failed to rescue the responses to loud sounds. Other gene copies restored the molecular properties of a particular ion channel class but not their numbers. Taken together, this indicates first that the underlying information is contained in the coding sequence of the atonal gene and second, the specific functional aspects can be linked to distinct parts of this coding sequence. As the human atonal is currently a hot spot for gene-therapeutical interventions to restore hearing, our research is expected to have a considerable impact. The work was published in eLife (Weinberger et al. 2017).

We also continue to review the respective modulation-specific gene lists to probe them for their role in different behaviours (e.g. sound-induced locomotor responses, or graviceptive responses tested in climbing assays).
Exploitation Route The results of this project will be taken forward by the scientific community (including ourselves) in three major ways:

1) The molecular inventory, once completed, will provide a rich list of genes, which can be exploited, experimentally by testing corresponding mutant alleles or other transgenic variations in behavioural, or biophysical studies.
2) The molecular inventory can also be probed bioinformatically by using predictive software packages, which help identify the upstream regulatory landscapes, asking which master genes are responsible (?), or also to identify further downstream genes, asking, which other genes might be affected (?).
3) The results of our (continuing) behavioural tests will also help to develop a better view of the idea of sensory specificity in the first place, as our preliminary data indicates that there is a considerable cellular and molecular overlap between the mechanosensory contributors to distinct behaviours. In other words, the behavioural responses to e.g. sound stimuli may not rely on specialised sound-sensitive neurons alone but also incorporate contributions from other mechanosensory cell types. Some other contributors, such as the two different isoforms of the ion channel NompC, appear to be specifically expressed in distinct subsets of neurons and contribute to distinct mechanosensory functions. The associated paradigm shifts have the potential to affect future sensory research beyond Drosophila.
4) The implication of particular parts of the atonal gene coding sequence for particular auditory functions, finally, will be of high interest for all researchers aiming to use atonal-based gene-therapeutical approaches to restore mechanosensory (e.g. auditory or vestibular), we are also planning to take this research directly forward ourselves (and merge it with the homeostasis framework of grant BB/M008533/1). this will take place in collaboration with Dr Bassem Hassan (Paris).
Sectors Education,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description ERC consolidator grant 2014
Amount € 1,899,549 (EUR)
Funding ID Grant Agreement number: 648709 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2015 
End 08/2020
 
Description Collaboration with Bassem Hassan Lab (Leuven/Paris) 
Organisation University of Leuven
Department VIB Switch Laboratory
Country Belgium 
Sector Academic/University 
PI Contribution Together with the Hassan Lab we explored how a coding sequence exchange of atonal, one of the major proneural (and prosensory) genes, affects the function of mechanosensory submodalities within the fly's Johnston's Organ (JO). We analysed the response behaviours of JOs in flies carrying different atonal knock-in (KI) constructs (from sponges, over annelids to mice). Our analyses covered electrophysiological responses and biomechanical signatures of transduction.
Collaborator Contribution The Hassan lab generated the various atonal knockin constructs and analysed other mechanosensory and visual organs neuroanatomically.
Impact A first publication is currently pending (resubmission of revised manuscript to eLife, currently under review), publication is expected to occur within the next two months.
Start Year 2014
 
Description Collaboration with Dr Chun-Hong Chen International Partnering Award (Taiwan Partnering Award: Mosquito Research - From Sensory Biology to Vector Control) 
Organisation National Health Research Institutes (NHRI) Taiwan
Country Taiwan, Province of China 
Sector Charity/Non Profit 
PI Contribution Together with Dr Chun-Hong Chen we will conduct joint UK/Taiwan workshops with international leaders in the field of mosquito sensory and circadian biology. We will also start proof-of principle experiments with novel mosquito mutants and we will conduct skill/knowledge transfer workshops between our labs. We hold the expertise in mosquito auditory and circadian biology.
Collaborator Contribution Together with Dr Chun-Hong Chen we will conduct joint UK/Taiwan workshops with international leaders in the field of mosquito sensory and circadian biology. We will also start proof-of principle experiments with novel mosquito mutants and we will conduct skill/knowledge transfer workshops between our labs. Dr Chen's lab holds the expertise in mosquito mutagenesis.
Impact none yet (still to start)
Start Year 2017
 
Description Collaboration with Dr Tony Southall (Imperial College London) 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan Kavlie, the postdoctoral research associate has visited the Southall lab to learn and conduct targeted DamID (TaDa) on neuronal submodailities of the fly's auditory organ.
Collaborator Contribution The Albert lab has prepared the necessary biological samples for the analyses and Ryan Kavlie, Albert lab member, is conducting the necessary experiments. The Southall lab is providing the tools and lab space as well as the technical knowledge in order to perform TaDa.
Impact The collaboration is still ongoing and sample processing and analyses are underway.
Start Year 2016
 
Description Collaboration with Maurice Kernan (Stony Brook, New York) 
Organisation Stony Brook University
Department Department of Neurobiology and Behavior
Country United States 
Sector Academic/University 
PI Contribution We analysed the submodal functions of mechanosensory neurons in JO in flies expressing different NompC isoforms.
Collaborator Contribution The Kernan lab identified the different and generated genomic 'rescue' lines. they also tested their function in bristles and studied the isoforms' expression patterns.
Impact the collaboration identified different isoforms of NompC, which contribute differently to submodality-specific response behaviours. the first publication of these efforts is currently under review with Current Biology. Its publication is expected to occur within the next 4 months.
Start Year 2014
 
Description British Neuroscience Association (2015 Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Research was presented to the British Neuroscience Association. The intended purpose was to present current findings and receive feedback from others in related fields to improve upon research activities. The most significant result was from meeting members of other labs that has provided fruitful training for members of the lab of Dr Stefano Casalotti at the University of East London who were interested gaining experience with Drosophila model organism research and a new collaboration for the post-doc Ryan Kavlie with the same lab.
Year(s) Of Engagement Activity 2015
 
Description European Drosophila Research Conference (2015 Heidelberg) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The intended purpose was to present current research to professionals that use Drosophila melanogaster as the model organism in their research. Professional contacts were made including several requests for further information,
Year(s) Of Engagement Activity 2016
 
Description H3 symposium / Physiological Society / Sensory Transduction in Insects / 8th December 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This symposium was conceived by Dr Ben Warren and Prof Joerg Albert. It was motivated by work which bridged sensory modalities and highlighted the common principles of operation of superficially very different sensory neurons. The main aim is to bring together researchers studying sensory transduction in different modalities to promote new interactions, and new insights, in insect sensory biology. The whole day meeting continued until long after the last talks with vivid discussions about the interactions, differences and similarities between the senses and how those could be exploited scientifically. The Physiological Society funding made it possible to assemble a stellar line up of internationally renowned speakers.
Year(s) Of Engagement Activity 2017
 
Description Neurofly 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact To be added
Year(s) Of Engagement Activity 2016
URL http://www.neurofly2016.gr/
 
Description Public seminar 23/11/2017: What one can hear when listening to the ear of a fly University of Leicester (Sponsor: The Physiological Society) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Lecture to University of Leicester staff and students led to multiple new engagements and also fostered an ongoing interaction and collaboration with Drs Kyriacou and Matheson. Abstract: All sensation starts with the elementary act of sensory transduction. For the sense of hearing this involves ion channels that are directly (mechanically) gated by the forces of sound. Multiple properties of the entire auditory systems arise from, and have evolved around, these transducer channels; investigating their function, and the constraints that govern their operation, thus can reveal fundamental properties of sound sensation.
Year(s) Of Engagement Activity 2017
URL https://www2.le.ac.uk/departments/npb/news-1/invspk/seminar-abstracts/albert-231117
 
Description Royal Society Theo Murphy International Scientific Meeting: From sender to receiver: physics and sensory ecology of hearing in insects and vertebrates, 4th / 5th December 2017, Kavli Royal Society Centre, Chicheley Hall, Newport Pagnell, Buckinghamshire, MK16 9JJ 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The meeting was a prestigious Royal Society Theo Murphy international scientific meeting organised by Dr Andrei Kozlov and Dr Joerg Albert. It assembled colleagues and scientists from across the globe and from various fields (visual/auditory + insect/vertebrate) to hold a 2-day scientific exchange, which challenged and tried to change, some of the currently held views in order to establish a novel, more interdisciplinary approach to sensory biology. As judged by the following collaborations and the feedback from the audience, the meeting was a great success!
Year(s) Of Engagement Activity 2017
URL https://royalsociety.org/science-events-and-lectures/2017/12/physics-ecology-hearing/
 
Description collaborative, cross-project visit at the Ludwig Maximilian University (LMU) of Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact During a 4-day visit at the Ludwig Maximilian University of Munich, Prof Albert explored the collaborative potential across three ongoing (or recently ceased) grants:
Award References: BB/L02084X/1 + BB/M008533/1 + BB/R021007/1 + ERC-consolidator grant Clock mechanics (648709), S34, BB/R000549/1, 1206383, 1336457

The visit included a central seminar with the LMU's neurolunch series and meetings with various group leaders and internationally leading PIs in animal evolution, neuroscience and health (e.g. Prof Benedikt Grothe, Prof Axel Borst, Prof Nicolas Gompel, Prof Peter Becker, Prof Till Roenneberg and Prof Martha Merrow). The aim of this visit was to present the recent data of the involved projects just prior to submission for publication and to explore how the overlaps between the different projects can be harvested by new collabrations with experts in the respective fields. Examples include the exploitation of circadian clock function and auditory homeostasis (Profs Becker, Grothe and Merrow), or the evolution of sensory modality and submodality (Profs Borst + Gompel). The short-term goal will be to apply for larger national (e.g. Wellcome Trust collaborative awards) or international (e.g. European Research Council) follow-up grants! The LMU is a centre of German academic excellence, which is of strategic interest for UCL.
Year(s) Of Engagement Activity 2018