Functional genomics of the enigmatic stellate cell

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.

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.

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