Analysis of FGF-signalling mechanisms in controlling cell migration in Drosophila

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

The position of cells within a multi-cellular organism is strictly controlled. The tissue environment provides information that tells the cells whether to divide, become motile or stay where they are. This plasticity enables the organism to react upon changing conditions, like pathogen attack. Consequently, a failure of this regulation may lead to severe diseases. During cancer, for example, cells leave their assigned position in a tissue, become motile, invade and eventually destroy other tissues. Our knowledge about the regulators of cell motility is therefore crucial for the cure of such diseases.

Research on genetic model organisms has demonstrated that the molecular regulation of cell motility is very similar between flies and man. However the way these regulators govern cell motility on a biochemical and genetic level is not known. We are using the fruit fly as a model with the aim to identify the important regulators and their modes of action. This information will provide a basis to see how these mechanisms relate to pathogenic situations in humans. These results will lead to a better understanding of human diseases like cancer, and will eventually be instructive for the generation of new medical treatments.

Technical Summary

Cell migration plays important roles in developing and adult organisms and failure to regulate migration events can result in severe diseases, including birth defects, immunological disorders and cancer. Great progress has been made in identifying signalling molecules and the cytoskeletal mechanisms that drive cell migration, but key questions remain open: how signals control migration in time and space and how the signal translate into cytoplasmic events that change the behaviour of the responding cells. We employ a highly tractable genetic model system to identify and analyse novel components of the molecular mechanism driving cell migration.

The specific goal of our research is to understand the molecular link between signalling and the local regulation of cell adhesion and the cytoskeleton. We are addressing this problem using a Drosophila embryo model and will dissect how Fibroblast Growth Factor (FGF) signalling controls mesoderm migration. We identified two novel FGF8-like growth factors and will now assess the cellular function of these factors. We will use genetics to investigate whether they act as permissive factors or as instructive cues, i.e. whether these molecules can act as chemo-attractants in vivo. The cell biological responses of living cells upon ectopic FGF expression will be monitored using high-resolution microscopy.

Our genetic approach identified a novel factor in mediating FGF-triggered cell shape changes: the fly orthologue of the human proto-oncogene ect2, called Pebble (Pbl). We now want to understand the mechanism by which Pbl links receptor-tyrosine kinase activation to changes in the cytoskeleton. We will perform structure-function analyses of Pbl with the aim of determining its functional interactions with other proteins in the pathway. Novel interacting proteins will be identified by genetic and proteomic approaches, followed by functional analyses of the respective genes. In addition to pbl, our previous genetic screens have already yielded additional candidate genes that we are currently analysing molecularly.

Migratory cells often derive from polarized epithelial tissues in a process called epithelial-mesenchymal transition (EMT). EMT occurs in many tissues of developing and adult organisms and its tight molecular control is crucial to prevent pathological situations, such as metastasis during carcinogenesis. We currently focus our research on molecules that have been implicated in cancer, e.g the proto-oncogene ect2. Our research using the Drosophila system will lead to a better understanding of the molecular control of EMT and cell migration and therefore help to address questions immediately relevant to biomedical applications.

Publications

10 25 50
 
Description MRC 4 year PhD programme
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Development of Portfolio agreement on PhD training and monitoring procedures and feedback with the funding bodies has generated PhD graduates with advanced skill sets not only covering their specialist research areas, but also in capability to apply transferable skills. This advanced and refined training allows our graduates to compete successfully for their future career.
URL http://www.lifesci.dundee.ac.uk/phdprog/phd-studentships/programmes/mrc-4-year-programme
 
Description Großgeräteantrag
Amount € 520,000 (EUR)
Funding ID INST 159/89-1 FUGG 
Organisation German Research Foundation 
Sector Charity/Non Profit
Country Germany
Start 04/2016 
End 04/2017
 
Description MRC Research Grant
Amount £325,769 (GBP)
Funding ID G0901020 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2010 
End 10/2013
 
Description MRC Research Grant
Amount £400,924 (GBP)
Funding ID MR/K018531/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2013 
End 06/2016
 
Description PhD studentship
Amount £80,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2007 
End 04/2010
 
Title Hexosamine flux and O-GlcNAcylation 
Description Work on the nesthocker mutation in the laboratory has established a link between the levels of Hexoamines and Fibroblast-growth factor signaling. Our research revealed that reduced cytoplasmic O-GlcNacylation is causative for the phenotype. We established that injection of OGA inhibitors can rescue the phenotype, thus providing a model for O-GlcNAcylation in cell signaling. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact Our results indicate that the Drosophila system can be used as a model for human disorders in which hexosamine flux is affected. This includes neurodegenerative diseases, cardio-vascular diseases, diabetes, cancer and inflammatory diseases. 
 
Title Rho-GEF pebble transgenic strains 
Description An array of transgenic pebble constructs have been established in flies for structure function analysis. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Fly strains are being distributed to other research laboratories studying various aspects of Rho GTPase signaling. Results obtained with this lines will further our understanding of the function of the mammalian homologue Ect-2, which represents a proto-oncogene. 
 
Title live imaging of mesoderm tissue in early embryos using two-photon microscopy 
Description The difficulty to selectively live-image the mesoderm tissue in early embryos has provided a major obstacle in studying the cell shape changes that occur during epithelial mesenchymal transition. We have developed protocols for live imaging early embryos which allows selective visualization of cell shape changes and protrusive activity. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This method is going to be made available to the scientific community after publication and will improve visualization of cell behavior of all mesodermal lineages in the embryo. 
 
Title rabbit anti DPAGM antibody 
Description An antibody was raised against the Drosophila homologue of human phospho-acetylglucosamine mutase. The antibody detects the protein on Western blot and immunostaining and was used to characterise the nst mutation and the localisation of the protein. 
Type Of Material Antibody 
Provided To Others? No  
Impact proof that mutation in the nst mutation affected Drosphila PAGM 
 
Title transgenic fly lines 
Description Several transgenic Drosophila stocks have been established in which the enzymes that control O-GlcNAcylation can be manipulated in vivo, creating either gain of function or loss of function mutant animals and tissues. 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact These tools provide invertebrate animal models to study the nutrient and metabolism dependent connection to cell signaling pathways that are relevant for human disease. The fly models contribute to the 3R's as those pathways are evolutionarily conserved and much more straightforward to study in this model. 
 
Description AGM1/PGM3 knock out mice 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Department Cancer and Haematology
Country Australia 
Sector Academic/University 
PI Contribution The aim is to determine the conservation of the critical signaling node of the FGF pathway and the Hexosamine biosynthetic pathway. We are in the process of monitoring expression of FGF target gene expression in those mouse embryos.
Collaborator Contribution generation and fixation of mouse E6.5 stage AGM1/PGM3 knock out embryos
Impact The result of this study will contribute to our current data on the conservation of the Hexosamine/FGF signaling node and promote the publication of a manuscript, which is under preparation. The conservation of this mechanism will help to translate our findings in Drosophila to human and raise the impact of our findings and will make the project more amenable to obtaining biomedical and translational research funding.
Start Year 2010
 
Description ETD mass spectrometry 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My group produced and purified protein from cultured cells for the analysis.
Collaborator Contribution Assistance in establishing ETD (electron transfer dissociation) mass spectrometry to determine amino acids sites that are modified by O-GlcNAc.
Impact ongoing - The Dof protein can be detected by MS, but the amount is currently insufficient to obtain reliable ETD data yet.
Start Year 2011
 
Description Guanine nucleotide exchange assays 
Organisation Heidelberg University
Department Center for Molecular Biology (ZMBH)
Country Germany 
Sector Academic/University 
PI Contribution Co-worker visited the partner laboratory and performed practical work with the help of a co-worked in the partner lab.
Collaborator Contribution providing tools, reagents and knowledge on biochemical exchange assays. Providing lab space for the research to perform assays within the partner laboratory.
Impact The results from this collaboration were used to address reviewers' comments to our publication in March 2009 in Development (19176590). The partner were included as co-authors.
 
Description Hexosamine measurements 
Organisation University of Dundee
Department Division of Biological Chemistry and Drug Discovery
Country United Kingdom 
Sector Academic/University 
PI Contribution Discussed genetics of hexoasmine deficient phenotypes in flies. Produced mutant and wild type embryos for amino sugar nucleotide measurements.
Collaborator Contribution Electrospray, tandem mass spectrometry based quantitative measurements of amino-sugar nucleotide concentrations in Drosophila embryos.
Impact Resulted in an ongoing multi-disciplinary collaboration of Biochemists and Geneticists. Together we developed a protocol that allows the exact quantification of aminosugar-nucleotides in Drosophila tissues which turns out to be important for the scientific community in the field of metabolomics and proteomics as weill as genetics. A manuscript has been accepted at Science Signaling and will be published in the 20.Dec 2011 issue of the Journal. Partners are coauthors on this manuscript entitled: 'O-GlcNAcylation is required for Fibroblast Growth Factor Signaling in Drosophila'.
Start Year 2007
 
Description Hypoxia in Drosophila 
Organisation Wellcome Trust
Department Wellcome Trust Centre for Gene Regulation and Expression
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Generated Drosophila mutants in the NF-KappaB pathway to determine the role of NF-KappaB in hypoxic response in flies. Generation of mutants and RNA preparation for q-RT-PCR.
Impact The results of this collaboration were pubished in PLoS Genetics: van Uden et al., "Evolutionary conserved regulation of HIF-1ß by NF-?B.' PMID:21298084 Dr. Rocha and myself are planning to submit a joint grant application in 2012 on this topic.
Start Year 2010
 
Description O-GlcNAcylation in stress responses 
Organisation University of Zurich
Department Institute of Molecular Life Sciences
Country Switzerland 
Sector Academic/University 
PI Contribution Measurements of aminosugar nucleotide amounts in embryos treated in hypothermic conditions.
Collaborator Contribution contribution of reagents, unpublished genetically modified Drosophila strains and sharing unpublished scientific data on O-GlcNAcylation.
Impact This collaboration contributed to the PhD thesis of Pablo Radermacher at the Institute of Molecular Life Sciences, Zurich. These data are included in a primary research manuscript that is co-authored by my former PDRA Dr. Daniel Mariappa and myself and is currently under review.
Start Year 2010
 
Description O-GlcNAcylation of Dof protein 
Organisation European Molecular Biology Laboratory
Country Germany 
Sector Academic/University 
PI Contribution The tools provided by the Leptin group were used to establish that the FGF receptor adaptor protein Dof is a target for protein modification via O-GlcNAcylation and that this modification is essential for Fibroblast Growth Factor Signaling.
Collaborator Contribution Prof. Leptin's laboratory provided reagents such as transgenic fly strains, DNA constructs, and antibodies against the Dof protein.
Impact This collaboaration contributed to results presented in the manuscript 'Protein O-GlcNAcylation is required for Fibroblast Growth Factor Signaling in Drosopihla' which was accepted for publication in Science Signaling published in the issue of Dec 20, 2011.
Start Year 2009
 
Description O-GlcNAcylation of fiboblast growth factor receptors in mammalian models 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Our laboratory performs biochemical assays, which were optimised using Drosophila cultured cells to monitor protein O-GlcNAcylation of the FGF receptor. The human receptors contain a single conserved site which will be investigated using site directed mutagenesis.
Collaborator Contribution The laboratory of Professor Sabine Werner provided mammalian cell lines expressing human fibroblast growth factor receptors I and II and also provided us with antibodies that recognise FGFR1 and FGFR2.
Impact Despite positive data on O-GlcNAcylation of FGF pathway components (FGF receptor 1 and 2) in humans, we were not able to obtain funding to continue this interesting link, which would have helped translation of the data obtained in Drosophila into humans.
Start Year 2011
 
Description OGA inhibitor studies in vivo 
Organisation University of Dundee
Department Division of Molecular Microbiology
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team has used small molecule inhibitors produced by the partner to perform chemical genetics experiment in which we suppressed an FGF mutant phenotype by injection of an OGA inhibitor into phospho-acetylglucosamine mutant embryos.
Collaborator Contribution The partner produced a highly selective hexosaminidase inhibitor, GlcNAcstatin C, against O-GlcNAc Hydrolase, which blocks the activity of the enzyme in pico molar ranges. We used this inhibitor to show that O-GlcNAcylation is affected in phospho-actetylglucosamine mutase mutants.
Impact This collaboration has led to important data supporting our discovery that the hexosamine biosynthesis pathway is required for Fibroblast Growth Factor signaling. A paper has been published in Science Signaling in the 20.Dec 2011 issue of the Journal. Partners are coauthors on this manuscript entitled: 'O-GlcNAcylation is required for Fibroblast Growth Factor Signaling in Drosophila'.
Start Year 2009
 
Description PhD studentship AK 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided lab space, materials and infrastructure for research. provided supervision of the project.
Collaborator Contribution The studentship allowed AK to continue PhD studies in my laboratory after moving from Germany.
Impact Research by AK resulted in two publications (PMID 19515694 and PMID21613323).
Start Year 2006
 
Description PhD studentship AvI 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided lab space, materials and infrastructure for research. provided supervision of the project.
Collaborator Contribution Allowed my student AvI to continue his PhD studies at the University of Dundee.
Impact Research by AvI resulted in a publication (19176590). Additional data from his thesis will contribute to another publication. The data from his thesis did provide preliminary data to support a successful grant application for a research grant at the MRC (MR/K018531/1)
Start Year 2006
 
Title mutant Drosophila lines 
Description Mutations in members of the highly conserved Fibroblast Growth Factor family of proteins in Drosophila melanogaster. These mutant fly lines are being distributed to researchers throughout the world and used to investigate the function of growth factors in various biological processes using Drosophila as a model organism. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2009
Development Status Under active development/distribution
Impact Mutations in genes encoding Fibroblast Growth Factors in Drosophila have revealed important functions of these factors in epithelial-mesenchymal transition (EMT) and cell migration. The much lower complexity of growth factor signaling in the fruit fly together with its high level of evolutionary conservation resulted in efficient discovery of novel pathway components and how signaling networks control biomedically important cellular processed such as EMT. 
 
Title transgenic Drosophila lines 
Description Transgenic Drosophila lines were established that express fluorescently tagged growth factors, signaling molecules (including homologues of human oncogenes and tumour suppressors), and subcellular reporter. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Under active development/distribution
Impact The tools are widely distributed throughout the scientific community worldwide and used by our laboratory and others for analysing signalling pathways and the cellular processes they affect with a high subcellular resolution. 
 
Description College of Life Sciences Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact about 50-60 members of the public passed by our science stand and participated in activities such as microscopy and a quiz. The strategy of using fruit flies as genetic model organisms for medical research was discussed.

Awareness of my lab members and myself of the interests of the general public in biomedical research and their expectations and fascination about science. Every public engagement activity led us to reconfigure our presentations and activities every year and integrate it with the presentation of the Division of Cell and Developmental Biology at the College of Life Sciences.
Year(s) Of Engagement Activity 2010,2011,2012
URL http://www.dundeesciencefestival.org/
 
Description Create and Inspire 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Training for public engagement activity in Dundee Sensations. Creation of a stand and posters for research demonstration to public.

Intense contact with public engangements staff at the Dundee Sensations Science Centre.
Year(s) Of Engagement Activity 2010
 
Description Dundee Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The aim of the activity was to make the public aware of the use of model organisms, in particular Drosophila melanogaster, in research of human diseases and genetic disorders. We provided a stand with research materials, posters, and living organisms to have people look at our model organsims. We organized a quiz and the winners were invited to visit the laboratory. We had informal discussions about our research with the interested public. The event lasted 7 hours and we obtained a great feedback.

Create awareness of the use of model organisms for medical research. The event was featured in all Tayside newspapers mentioning our presentation among others.
Year(s) Of Engagement Activity 2009,2011
URL http://www.dundeesciencefestival.org
 
Description Higher Biology Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact I presented two lectures entitled "Of Flies and Humans: the Contribution of Developmental Biology to Understanding Human Disease".

The event was very well attended (> 300 pupils from Tayside's and Central Scotland's secondary schools). The feedback was very good, with teachers mentioning that they will use some of the material presented in their genetics and cell biology classes.
Year(s) Of Engagement Activity 2009
 
Description School visit - Menzieshill High School Dundee 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A postdoctoral researcher visited science classes in the school and discussed his professional career with the students.

Provided information and interest towards those pupils intending to enter a higher education in science.
Year(s) Of Engagement Activity 2008