Functional genomics of the enigmatic stellate cell

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Insects comprise the most species on earth, occupying almost all environmental niches, from arctic, desert to aquatic. Harmful insects incur enormous health and economic costs - crop damage, insect-borne plant and animal diseases, cause the loss of 20% of GDP worldwide. There is increasing pressure for insect control, given insect resistance to all insecticides and increasing environmental concerns, so understanding mechanisms of insect survival and environmental tolerance may be key to novel routes for insect control.
For all insects, survival depends on osmoregulation and fluid balance (homeostasis), which also allows them to withstand desiccation in low humidity environments, including that of the UK. The insect 'kidneys' - Malpighian tubules - are key tissues for fluid homeostasis. Over some years, we have investigated and established cell signalling, ion transport, functional genomics and integrative physiology in the genetically tractable D. melanogaster Malpighian tubule, which is an excellent model for insect tubules, especially Dipteran species. Examples of Dipteran insect pests relevant to the UK economy are the wheat bulb fly and the cabbage root fly (crop pests); the midge (blue-tongue, animal health); and increasingly, mosquitoes (human health).
Here, we plan to understand the mechanisms and neuroendocrine control of tubule fluid secretion and responses, specifically by the tubule stellate cell; and by chloride and water transport through the stellate cell under normal and desiccation conditions.
The proposed programme of work will include isolation of actively transcribed genes (the 'translatome') from specific tubule cell-types (principal and stellate cells) under normal and desiccation conditions, using a combination of a novel cell-specific translatome isolation method, as well as gene arrays (microarrays). This will allow us to assign genes to either principal or stellate cells and so understand the gene signature of each cell type; to identify cell-specific genes which are actively transcribed in response to desiccation stress; and to define those genes implicated in desiccation tolerance by also assessing the role of identified genes in whole fly desiccation tolerance assays.
We also plan to define the mechanism of chloride and water flux through the stellate cells for fluid secretion and fluid homeostasis under normal and desiccation conditions; and also in response to neurohormone control. In order to do this, we will define the role of chloride channels (CLCs) and aquaporins specific to the stellate cell at the molecular and physiological level using transgenic flies to knock-down specific CLC and aquaporin genes in only stellate cells; tubule fluid secretion assays; molecular methods; bioimaging using novel transgenic reporters; cell biology; physiological measurements (this in collaboration with a US group expert in ion channel electrophysiology); and whole fly desiccation tolerance assays.
Together, this will provide the first comprehensive understanding of chloride and water transport in fluid homeostasis, as well as insights into desiccation tolerance, which may in time, help identify, new, greener insecticides that target only a subset of insects.

Technical Summary

Insects comprise the most species on earth, occupying almost all environmental niches. However, harmful insects incur enormous health and economic costs - crop damage, insect-borne plant and animal diseases, cause the loss of 20% of GDP worldwide. Given insect resistance to all insecticides and environmental concerns about insecticide use, understanding mechanisms of insect survival and environmental tolerance may be key to novel routes for insect control. For all insects, survival depends on osmoregulation and fluid homeostasis by the Malpighian tubules, which also confer desiccation tolerance. Tubules of Dipteran insects (flies including disease vector mosquitoes) contain star-shaped stellate cells, which are essential for tubule function and fluid secretion.
We plan to uncover the mechanisms and neuroendocrine control of tubule fluid secretion and responses, specifically by the D.melanogaster tubule stellate cell under normal and desiccation conditions, by functional genomics of the stellate cell; and by defining stellate-specific chloride and water transport. Specifically, we will achieve:
- cell-specific 'translatomics', i.e., actively transcribed genes ('translatome') of the principal and stellate cell under normal and desiccation conditions, using a novel cell-specific translatome isolation method, and gene arrays (microarrays) - providing the gene 'signature' of each cell type, and candidate genes for desiccation tolerance.
- tubule fluid secretion data from stellate cell-specific chloride channel (CLC) and water channel (aquaporin) RNAi gene (transgenic) knock-downs and/or mutants
- molecular physiology of stellate-specific CLCs and aquaporins
- neuroendocrinology of CLCs/aquaporins
- bioimaging of chloride and pH in vivo reporters
- fly desiccation tolerance assays
Together, this will provide comprehensive understanding of stellate cell function in fluid homeostasis; and possibly provide new targets and/or routes for Dipteran pest control.

Planned Impact

This work will benefit the UK/international academic community and the wider public.

Academic community: in addition to that described in 'Academic Beneficiaries', our work will be disseminated via meetings, publications and collaborations. In 2012, we fulfilled >75 requests for transgenic flies and antibodies; and received many requests for information/discussion, so we are of real benefit to the community. We have existing collaborations with key groups in functional genomics, epithelial function, neuroendocrinology, signalling, stress resistance, and ion channel/transporter physiology, and so are well positioned to develop avenues of investigation of mutual interest during the course of the grant. We will produce highly trained researchers for the academic or industrial market. This extends beyond the researchers directly employed on the project, to other members of the lab, including PhD, Masters and undergraduate project students.

Industry/Economic impact: We have current significant investment from Pfizer Inc., recent investment from BASF Germany and previously from Syngenta. We have co-published with Syngenta and Pfizer. We have delivered reagents to all our industrial partners, to be further developed in-house. Thus, we have a significant track record of utility and benefit to major pharma. It is possible that this work will deliver IP (e.g., new screening targets), and if so, we have industrial partners in place who can be approached. At Glasgow, Knowledge Transfer/commercialisation is now managed at College level (Research and Business Development), and our College has prioritised development of economic impact of its research, so we are encouraged to exploit our findings where possible.

Public engagement: The PIs have had good engagement with the public via the media (BBC (Scientific Advisor, Dow), broadsheets, Radio 4). In 2012, Dow's work was covered by BBC news, Genetics Society of America and the American Physiological Society Podcast. Davies's collaborative work with Japan has been featured by Japan Society for Promotion of Science (2012). Our work has also been featured in BBSRC Business, International Innovations and Public Science Review; and is the subject of a BBSRC impact case study. Dow/Davies's BBSRC-funded research is also exhibited at the Glasgow Science Centre and has been presented to high school pupils at Hwa Chong Institute, Singapore, 2011, 2013 and local schools, 2013. We will continue to develop media interest, via University of Glasgow Corporate Communications. Also, Davies/Dow/Terhzaz regularly host final-year school pupils (Glasgow, Europe, Singapore) for a short courses in integrative biology, and co-supervise school research projects in Singapore. During the course of the grant, we will increase possible interactions with young people by running new projects for high school pupils (small sum requested for these activities) as well as mounting new interactive displays at e.g., Glasgow Science Week.

Project Management: For the Dow/Davies groups, Davies, Dow and Terhzaz play active roles in project management, essential to achieve measurable output/progress for all aspects of funded research. We utilise BaseCamp, a platform for project management which allows project-specific data display, discussion and planning, to which group members and company PIs have secure access. We also have regular 'one-to-one' meetings with each group member every week, with additional weekly group meetings as a forum for group discussion and presentation.

All the investigators have excellent, relevant track records in output; collaborations and exploitation; and communication and engagement - so can achieve the maximum from funded projects

Publications

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Cabrero P (2020) Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule. in Proceedings of the National Academy of Sciences of the United States of America

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Cabrero P (2014) Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. in Proceedings of the National Academy of Sciences of the United States of America

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Davies SA (2014) Cell signalling mechanisms for insect stress tolerance. in The Journal of experimental biology

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Dow JA (2015) What is an epitheliome, anyway? in Insect biochemistry and molecular biology

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Halberg KA (2015) Tracing the evolutionary origins of insect renal function. in Nature communications

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Terhzaz S (2015) Insect capa neuropeptides impact desiccation and cold tolerance. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Introduction and grant objectives: The specialization of the insect Malpighian 'kidney' tubule (MT) stellate cell may explain the unique transporting proficiency of MTs from Dipteran insects (which include both the genetic model, D. melanogaster, and some serious crop pests and disease vectors e.g., mosquitoes), to withstand desiccation and other environmental stress. We planned to provide the first principal and stellate cell translatome; and link this to functional understanding of stellate cell-specific chloride channels (CLCs) and aquaporins (AQPs), under normal and desiccation conditions; and in response to specific neuropeptides e.g. kinin. Providing a detailed molecular, functional and genetic understanding of the roles of the Drosophila stellate cell will also provide a clear model that can be tested in other Dipteran pest insects that have stellate cells, e.g., mosquitoes.

Aims:
Aim 1. A stellate-specific translatome under normal conditions and desiccation stress.
Aim 2. Understanding stellate cell-specific Chloride Channels (CLCs).
Aim 3. Investigating stellate cell aquaporins - a stellate-cell specific route for water flux?

The project has been successful in delivering its objectives so far, and has produced impactful outputs (including several 4* publications including Nature Comms. and PNAS), which have also contributed to the success of securing other major awards (e.g. EU H2020 RIA project).

Based on neuroendocrinology, molecular physiology and insect genetics/transgenics, Aims 2 and 3 are almost completely fulfilled (evidenced by Cabrero, et al., PNAS, 2014; Terhzaz et al., PNAS, 2015; Cannell et al., Peptides, in press, 2016; Cabrero et al., in prep, 2016).
Aim 1 (A principal- and stellate-specific translatome under normal conditions) has taken longer than anticipated, as previously published methods from other groups haven't been entirely reliable, resulting in a long process of optimisation by our team. This has now been successful, and we are expecting the first translatome dataset from Edinburgh Genomics by March 2016. We will now progress to Aim 1B (cell-specific translatome under desiccation conditions), which will be achievable.
The team also contributed to development of fluorescently-labelled neuropeptides for in vivo receptor binding analysis across multiple insect orders and species including mosquitoes (Halberg et al., Nature Comms. 2015). This approach allows evolutionarily 'barcoding' of insect neuropeptide G-protein coupled receptors (GPCRs), towards intervention in these pathways in pest species for novel biocontrol.

Overall, this project has allowed us to determine the role of cell-specific neuropeptide signalling in insect environmental stress tolerance. It has also significantly contributed to our understanding in this area, allowing us to successfully bid for and coordinate a H2020 programme targeted to crop protection in Food Security (nEUROSTRESSPEP: Novel biocontrol agents for insect pests from neuroendocrinology, grant no: 634361, 7M Euros, 2015 -19).
Exploitation Route We have run BB/L002647/1 judiciously, and have saved money where possible. Factoring in ongoing project costs, we are requesting a 4-month no-cost extension to BB/L002647/1 (Submitted March 4th 2016), to capitalise on the project findings towards 'proof of concept' data for novel, neuroendocrinology-based biocontrol of insect vectors of disease. This is particular timely given the current negative impact of the Zika virus, spread by mosquito species e.g., A. aegypti; and together with Dengue fever, also spread by Aedes, are rapidly increasing global threats.
We plan to test novel compounds against neuropeptide GPCRs associated with environmental stress i.e., desiccation, as mosquito survival is known to be temperature and moisture dependent.

Background and project plan : We recently successfully screened a 3200 compound library focussed against transporters and GPCRs (BioAscent) for insect lethality using larval Drosophila with a 2.7% 'hit' rate, via UoG Knowledge Exchange funding. Recently, a BBSRC IAA award (Jan - July 2016) has funded lethality screening of these compound 'actives' in insect crop pest species. We now plan to extend this approach to target desiccation stress- and fluid homeostasis-associated neuropeptide GPCRs conserved between Drosophila and Aedes (e.g., kinin GPCR, capa GPCR) using a subset of BioAscent's focussed GPCR compound library (2500 compounds, costing £3221.80, quotation #20160033). We will assay GPCR inhibition via previously established cell-based calcium assays (Terhzaz et al., PLoS One, 2013) in 96-well format including secondary testing of active compounds (e.g., different concentrations) between September - November 2016.
During December 2016, we will test the most promising 'actives' from the GPCR screen on mosquito larval lethality and adult mosquito survival under desiccation conditions, as proof of concept. If successful, we will subsequently develop the translational potential of this work via other funding streams during 2017.
Sectors Agriculture, Food and Drink,Environment,Healthcare

URL http://www.ncbi.nlm.nih.gov/pubmed/?term=Davies+SA
 
Description We have determined the complex role of cell-specific neuropeptide signalling in insect environmental stress tolerance, and have demonstrated this in Dipteran species including Drosophila and mosquitoes. This has also significantly advanced our knowledge of neuropeptide modulation of behaviour and environmental stress responses, and has allowed us to successfully bid for and coordinate an EC H2020 programme targeted to novel insect biocontrol for crop protection in Food Security (nEUROSTRESSPEP: Novel biocontrol agents for insect pests from neuroendocrinology, EC grant no: 634361, 7M Euros, 2015-19) which will result in societal, environmental, policy and economic impact. Also, the findings from our planned extension work should have impact in global health area, towards insect disease vector control.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Environment,Healthcare
Impact Types Societal,Economic,Policy & public services

 
Description BBSRC responsive mode
Amount £1,143,000 (GBP)
Funding ID BB/P008097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description H2020 Marie Curie Sklodowksa Action Innovative Training Network
Amount € 4,500,000 (EUR)
Funding ID 642937 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 06/2015 
End 05/2019
 
Description H2020 SFS-03a - 2014 Native and Alien pests in Agriculture, Horticulture and Forestry
Amount € 7,000,000 (EUR)
Funding ID 634361 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2015 
End 05/2019
 
Title Antibodies 
Description Antibodies to Drosophila proteins, e.g. chloride channels, aquaporins 
Type Of Material Antibody 
Provided To Others? No  
Impact Academic impact 
URL http://www.ncbi.nlm.nih.gov/pubmed/25228763
 
Title Drosophila transgenic lines 
Description Transgenic lines for multiple genes, including overexpressors and RNAi lines. Novel GAL4 driver lines 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact Used worldwide by other labs 
URL http://www.ncbi.nlm.nih.gov/pubmed/25730885
 
Title Novel in vivo chloride reporters 
Description we generated the first transgenic flies for a genetically encoded fluorescent combined Cl-/pH - biosensor (ClopHensor), for the monitoring of chloride and pH in an organotypic context (Cabrero et al., PNAS, 2014). 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Ability to measure Cl- and pH in real time in targeted cells in vivo, and can be exploited in other organisms for biomedical research. 
URL http://www.ncbi.nlm.nih.gov/pubmed/25228763
 
Title Peptide analogues 
Description Peptide mimetics to alter insect behaviour and response to environmental stress 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Our work showed that peptide analogues for the capa family of neuropeptides can modulate insect behaviour and stress responses, providing evidence towards development of neuropeptide analogs for novel insect biocontrol methods. This was published in PNAS (Terhzaz et al., PNAS 2015), and featured in Science Signalling ((http://stke.sciencemag.org/content/8/367/ec54). 
URL http://www.ncbi.nlm.nih.gov/pubmed/25730885
 
Description Chloride channels 
Organisation Marquette University
Country United States 
Sector Academic/University 
PI Contribution Aim 2 of grant - establishing functional in vivo role of the CLCs - generation, characterisation, validation and experimentation of transgenic (including RNAi) flies,plus in vivo chloride reporters, generation of CLC Abs
Collaborator Contribution Measurements of CLC electrophysiology
Impact Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Cabrero P1, Terhzaz S1, Romero MF2, Davies SA1, Blumenthal EM3, Dow JA4.
Start Year 2013
 
Description Chloride reporters 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution Joint development of in vivo chloride reporters and also joint development of kidney stone work (funded by NIH).
Collaborator Contribution Joint development of in vivo chloride reporters
Impact Outputs associated with this grant: Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Cabrero P, Terhzaz S, Romero MF, Davies SA, Blumenthal EM, Dow JA. Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14301-6. doi: 10.1073/pnas.1412706111. Epub 2014 Sep 16.
Start Year 2014
 
Description Cold tolerance 
Organisation Ohio State University
Country United States 
Sector Academic/University 
PI Contribution Availability of insects, peptide analogues and assays
Collaborator Contribution Assay development and testing
Impact Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2882-7. doi: 10.1073/pnas.1501518112. Epub 2015 Feb 17. Insect capa neuropeptides impact desiccation and cold tolerance. Terhzaz S1, Teets NM2, Cabrero P3, Henderson L3, Ritchie MG4, Nachman RJ5, Dow JA3, Denlinger DL6, Davies SA1.
Start Year 2013
 
Description Cold tolerance and ion transport 
Organisation Aarhus University
Country Denmark 
Sector Academic/University 
PI Contribution Malpighian tubule functional assays; training and expertise
Collaborator Contribution Functional assays and expertise
Impact Sci Rep. 2015 Dec 18;5:18607. doi: 10.1038/srep18607. The capacity to maintain ion and water homeostasis underlies interspecific variation in Drosophila cold tolerance. MacMillan HA1, Andersen JL1, Davies SA2, Overgaard J1.
Start Year 2013
 
Description Kenneth 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution Provision of expertise, facilities, reagents, know how
Collaborator Contribution Expertise, technique and reagent co-development
Impact 3: Overend G, Cabrero P, Halberg KA, Ranford-Cartwright LC, Woods DJ, Davies SA, Dow JA. A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae. Insect Biochem Mol Biol. 2015 Dec;67:47-58. doi: 10.1016/j.ibmb.2015.05.007. Epub 2015 May 21. PubMed PMID: 26003916. 4: Halberg KA, Terhzaz S, Cabrero P, Davies SA, Dow JA. Tracing the evolutionary origins of insect renal function. Nat Commun. 2015 Apr 21;6:6800. doi: 10.1038/ncomms7800. PubMed PMID: 25896425; PubMed Central PMCID: PMC4410669.
Start Year 2012
 
Description Peptide analogues 
Organisation U.S. Department of Agriculture USDA
Country United States 
Sector Public 
PI Contribution Devised physiological and functional assays for neuropeptide analogues developed by USDA.
Collaborator Contribution Provision of neuropeptide analogues.
Impact Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2882-7. doi: 10.1073/pnas.1501518112. Epub 2015 Feb 17. Insect capa neuropeptides impact desiccation and cold tolerance. Terhzaz S1, Teets NM2, Cabrero P3, Henderson L3, Ritchie MG4, Nachman RJ5, Dow JA3, Denlinger DL6, Davies SA1.
Start Year 2013
 
Description Images on the Clyde 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact BBSRC and Glasgow Science Centre supported permanent exhibition on the River Clyde, with research images, text and online referencing.
Our exhibit is 'Glowing flies'
Year(s) Of Engagement Activity 2015
URL http://www.gla.ac.uk/colleges/mvls/researchimpact/imagesontheclyde/
 
Description Singapore schools research placement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 1 week research placement using Drosophila melanogaster. Remote supervision of A level projects.
Participation (and awards gained) in Singapore Science and Engineering Fair
Year(s) Of Engagement Activity 2010,2011,2012,2013
 
Description Sixth form school research visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 1 week lab placement for sixth form pupils from Moreton House School, Shropshire.
Provision and supervision of 6th form projects using Drosophila to model human disease.
Year(s) Of Engagement Activity 2013,2014,2015