Wingless trafficking and epithelial biology in Drosophila

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

It is now clear that signalling must be tightly regulated, both during embryonic development and adult life. Indeed, many cancers are associated with an excess of signalling. For example, the large majority of colon cancers are due to excess signalling by beta-catenin, a key effector of Wnt signalling. Wingless is the fly homolog of Wnt 1 and our work on Wingless trafficking will provide important cues on the mechanisms that dampen or modulate signalling by this important pathway. Our work on epithelial organisation is particularly relevant to issues of tissue homeostasis. Again, there is a direct relevance to cancer. Normally, a defective cell is extruded from the epithelium and subsequently eliminated by apoptosis. If defective cells escape this process, they would continue to grow and could contribute to cancer progression.

Technical Summary

Overall aims: There are two strands to our programme. In one, we study the trafficking of an important developmental signal, the secreted protein encoded by wingless in Drosophila. In particular, we investigate the mechanisms that modulate the distribution of this signal in developing epithelia. This includes secretion, release from secreting cells, transport along the epithelium, and endocytosis/degradation in receiving cells. With respect to secretion, we are devising an RNAi-based screen to identify genes that are involved in this process. For transport, we are particularly interested in the role of heparan sulfate proteoglycans and the possible role of their sulfation state. Finally, we devote a lot of energy to understand the mechanisms that degrade Wingless in receiving cells after it has initiated signal transduction. This work involves genetic screens, cell based assays and the development of methods to track Wingless after it has been internalised. In a second strand of work, we study the maintenance of epithelial organisation during development. In one project, we track the cells of the epidermis during embryogenesis and record parameters like the orientation of cell divisions and tissue deformation. From this analysis, we found that oriented cell divisions contribute to tissue elongation. One long-term aim is to track the behaviour of all epidermal cells. We are also interested in developmental boundaries. In particular, we would like to define the cell biological basis of boundary formation, to identify the genetic regulators as well as the effectors. In a recently initiated project, we have begun to investigate the mechanisms that ensure the survival of epidermal cells. This is spurred by the observation that loss of adhesion (following cell dissociation) leads to apoptosis, as if maintenance of adhesion or epithelial organisation is required for cell survival. Methodological aspects are listed below: For Wingless secretion, we are devising a sensitive assay in S2 cells and will screen a genome-wide RNAi library for genes that modulates Wingless-specific and general secretion. Candidate genes, such as vps 35 are also being investigated in imaginal discs. For Wingless transport, we are studying a mutation in sulf, which encodes an extracellular sulfatase.We are also using transgenic flies to assess the role of post-translational modifications of Wingless (glycosylation and palmitoylation) in transport.With respect to Wingless degradation, we are devising degradation assays in S2 cells using either imaging or a sensitive ELISA machine. We also have an assay for Wingless degradation in imaginal disks. These assays will be used to test the role of various molecules and to identify new regulators.To study the behaviour of epithelial cells we are developing 4D microscopy of fly embryos with a spinning disk and a 2 photon confocal microscope. We are also using standard genetic techniques to investigate boundary formation. Finally, to assess cell survival/apoptosis. We are devising a fluorescence-based reporter of apoptosis. This will be used to follow the pattern of apoptosis following loss of adhesion (either in adhesion mutants or following mechanical cell dissociation).

Publications

10 25 50
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Alexandre C (2008) Cuticle preparation of Drosophila embryos and larvae. in Methods in molecular biology (Clifton, N.J.)

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Baena-Lopez L (2012) Integration of morphogen signalling within the growth regulatory network in Current Opinion in Cell Biology

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Bardet PL (2008) A fluorescent reporter of caspase activity for live imaging. in Proceedings of the National Academy of Sciences of the United States of America

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Bardet PL (2009) [Developmental control of organ growth by the Hippo pathway]. in Medecine sciences : M/S

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Da Silva SM (2007) Oriented cell divisions in the extending germband of Drosophila. in Development (Cambridge, England)

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Franch-Marro X (2005) Glypicans shunt the Wingless signal between local signalling and further transport. in Development (Cambridge, England)

 
Description EMBO Long-Term Fellowship
Amount £25,210 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 02/2013 
End 01/2015
 
Description EMBO funding (The role of cellular tension in the induction of apoptosis by axin mutant cells)
Amount £52,022 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 03/2010 
End 02/2012
 
Description ERC Advanced Grant
Amount € 1,938,845 (EUR)
Funding ID 294523-WNTEXPORT 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 05/2012 
End 04/2017
 
Description EU Integrated Project-FP6-2006
Amount £277,246 (GBP)
Organisation Sixth Framework Programme (FP6) 
Sector Public
Country European Union (EU)
Start 02/2006 
End 01/2010
 
Description EU International Re-integration Grants- Marie Curie Fellowship-2008
Amount £79,269 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 04/2008 
End 03/2012
 
Description Fellowship for prospective researchers
Amount SFr. 26,840 (CHF)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 01/2012 
End 01/2013
 
Description Henry Wellcome fellowship
Amount £250,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2008 
End 03/2012
 
Description Wellcome Trust 4 year PhD studentship (Jorge Beira)
Amount £100,000 (GBP)
Funding ID 089593/Z/09/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2013
 
Description Wellcome Trust Grant
Amount £166,528 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 10/2014
 
Title Apoliner + Dronc activity sensor 
Description DNA encoding a fluorescent reporter of apoptosis. And also transgenic flies carrying this DNA 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact This reagent is used by numerous groups to study the control of apoptosis 
 
Title CD8-HRP 
Description Developed means of creating HRP fusion protein to track trafficking or to label cell membranes 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Many groups are currently using this material to study connectivity in the nervous system 
 
Title Reagents for homologous recombinations in Drosophila 
Description We are developing various transgenic lines which will greatly facilitate and speed up knock in technology and subsequent genome engineering in Drosophila 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact These reagents are still under development. When ready, we expect that many groups will use them to study gene function in Drosophila 
 
Description Characterisation of clinically relevant axin mutation 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We have made mutations in Drosophila axin that mimic mutations sen in human cancer patients. We have analysed the effects of such mutation on the growth in Drosophila
Collaborator Contribution They have provided us with the nature of the mutations and with the analysis of the mutant proteins by NMR and in mammalian cell culture
Impact We found that these mutations affect the folding and stability of axin raising the possibility that a treatment that stabilise the mutant protein could be effective. Anvarian, Z.*, Nojima, H.*, Madl, T., Kuper, I., Jordens, I., Gerlach, J., Vincent, J.-P., Rüdiger, S., Maurice, M. (2016) Cancer mutations convert the scaffold protein Axin into a molecular trap. Nature Structural and Molecular Biology, in Press Gerlach, J. P., Emmink, B. L., Nojima, H., Kranenburg, O. & Maurice, M. M. (2014) Wnt signalling induces accumulation of phosphorylated ß-catenin in two distinct cytosolic complexes. Open Biology 4, 140120. doi:10.1098/rsob.140120.
Start Year 2010
 
Description Design of apoptosis sensor 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution We design and construct new sensors
Collaborator Contribution To provide advice and reagents.
Impact PMID: 18779587
Start Year 2007
 
Description EM analysis of Wingless secretion 
Organisation Medical Research Council (MRC)
Department MRC Cell Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We have made the appropriate transgenic files and prepare the samples for analysis
Collaborator Contribution Help with sample preparation and analysis of tissue sections by electronmicroscopy
Impact Information was gained but no publication was produced
Start Year 2011
 
Description Functional analysis of Mouse Notum 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have obtained a mouse KO line
Collaborator Contribution They will help analysing metabolic and neural functions
Impact We have obtained the Notum mutants
Start Year 2014
 
Description Functional analysis of Mouse Notum 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have obtained a mouse KO line
Collaborator Contribution They will help analysing metabolic and neural functions
Impact We have obtained the Notum mutants
Start Year 2014
 
Description Functional analysis of RhoGap activity in cell delimitation 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution We have generated a RhoGap mutant in Drosophila and are assessing the phenotype
Collaborator Contribution They are assessing activity of RhoGap in MDCK cells They perform high resolution imaging
Impact None yet
Start Year 2012
 
Description Functional analysis of RhoGap activity in cell delimitation 
Organisation Imperial College London
Department National Heart & Lung Institute (NHLI)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated a RhoGap mutant in Drosophila and are assessing the phenotype
Collaborator Contribution They are assessing activity of RhoGap in MDCK cells They perform high resolution imaging
Impact None yet
Start Year 2012
 
Description Mass specrometry analysis of exosomes and protein complexes 
Organisation University of Birmingham
Department College of Life and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We prepare protein complexes by immuno-precipitation or density gradient centrifugation and our collaborator provides us with mass spec identification of the components
Collaborator Contribution He has provided mass spec analysis of various preparations generated in the lab
Impact We have obtained a list of exosome components and this is guiding further functional analysis
Start Year 2008
 
Description Mode of action of Notum 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide Notum protein and molecular genetics expertise
Collaborator Contribution They provide structural information, mass spectrometric assays and synthetic peptides
Impact Kakugawa, S.*, Langton, P.F.*, Zebisch, M.*, Howell, S., Chang, T.H., Liu, Y, Feizi, T., Bineva, G., O'Reilly, N., Snijder, A., Jones, Y. @, Vincent, J.P.@ (2015) Notum deacylates Wnt proteins to suppress signalling activity. Nature, 519, 187-192.
Start Year 2013
 
Description Mode of action of Notum 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We provide Notum protein and molecular genetics expertise
Collaborator Contribution They provide structural information, mass spectrometric assays and synthetic peptides
Impact Kakugawa, S.*, Langton, P.F.*, Zebisch, M.*, Howell, S., Chang, T.H., Liu, Y, Feizi, T., Bineva, G., O'Reilly, N., Snijder, A., Jones, Y. @, Vincent, J.P.@ (2015) Notum deacylates Wnt proteins to suppress signalling activity. Nature, 519, 187-192.
Start Year 2013
 
Description Purification of exosomes 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department UMR 6061 (Institute of Genetics and Development of Rennes (IGDR))
Country France 
Sector Public 
PI Contribution We provide the conditioned medium and they analyse the exosome preparation supplied by our collaborators
Collaborator Contribution They purify exosomes from culture medium conditioned by Wingless-expressing cells
Impact We now have a reliable methods for the purification of Wnt-containing exosomes. This will be described in print at some point. A paper has now been published: Beckett, K., Monier, S., Palmer, L., Alexandre, L., , Green, H., Bonneil, E., Raposo, G., Thibault, P., Le Borgne R., and Vincent, J.-P. (2012) Drosophila Wingless is loaded on exosome-like vesicles but forms a gradient in an exosome-independent manner. Traffic Ahead of Print http://www.ncbi.nlm.nih.gov/pubmed/23035643
Start Year 2008
 
Description Regulation of reaper expression 
Organisation Albert Ludwig University of Freiburg
Department Centre for Biological Signalling Studies (BIOSS)
Country Germany 
Sector Academic/University 
PI Contribution We perform genetic analysis
Collaborator Contribution They perform molecular biology experiments
Impact Beira, J.V., Springhorn, A., Gunther, S., Hufnagel, L., Pyrowolakis, G., and Vincent, J.-P. The Dpp/TGF?-dependent corepressor Schnurri protects epithelial cells from JNK-induced apoptosis in Drosophila embryos. Dev. Cell, 31, 1-8.
Start Year 2013
 
Description Role of ESCRT in autophagy and growth 
Organisation Oslo University Hospital
Department The Norwegian Radium Hospital
Country Norway 
Sector Hospitals 
PI Contribution We have generated transgenic tools that allow activation and repression of VPS4 activity
Collaborator Contribution Genetic analysis of VPS4 activity with our tools
Impact PMID: 19194501 + PMID: 17935992
Start Year 2006
 
Description Secretion in metazoans 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed and executed a genome-wide screen for new components of the secretory pathway
Collaborator Contribution They characterised proteins that we had identified as being required for secretion
Impact Wendler, F., Gillingham, A. K., Sinka, R., Rosa-Ferreira, C., Gordon, D.E., Franch-Marro, X., Peden, A.A., Vincent, J.-P. and Munro, S. (2010) A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway. EMBO Journal, 29, 304-314. PMID: 19942856
Start Year 2007
 
Description The interface between transformed and normal cells 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We obtained supporting evidence using Drosophila as a model system
Collaborator Contribution They performed most of the analysis of cell behaviour at the interface between transformed and normal cells
Impact Hogan, C., Dupré-Crochet, S., Norman, M., Kajita, M., Zimmermann, C., Pelling, A.E., Piddini, E., Baena-López, L.A., Vincent, J.-P., Itoh, Y., Hosoya, H., Pichaud, F., and Fujita, Y. (2009). Characterisation of the interface between normal and transformed epithelial cells. Nature Cell Biology, 11, 460-467. PMID: 19287376
Start Year 2008
 
Description HOSTING ST PAUL SCHOOL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Schools
Results and Impact About 50 A level students attended a talk on what can fruit flies tell us about human biology and disease

Students seemed interested and asked many questions
Year(s) Of Engagement Activity 2011,2012,2013