Functional genetics of trophic support in neuron-glia interactions in the central nervous system of Drosophila

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

One of the most devastating burdens to human health is spinal cord injury, which occurs primarily through car or sport accidents and it wrecks peoples?
lives with paralysis than can affect all four limbs. The medical challenge is to promote the regeneration of the spinal cord, to allow functional recovery. The efforts to promote regeneration have been partly hampered by the fact that upon injury the cellular environment becomes inhibitory to axonal growth. One challenge is thus to turn the cellular environment into one that will promote axonal growth. However, this requires knowledge of how axons interact with glia during growth, how to eliminate secondary death of neurons and glia and how to promote the re-establishment of myelinated, functional neural networks. Unfortunately, little is still understood of the underlying molecular mechanisms that while keeping neurons and glia alive contribute to the restoration of functional axonal connections. Similarly, in neurodegenerative diseases (i.e. Multiple Sclerosis, Parkinson?s disease, Alzheimer?s), neurons and glia die and the promotion of neuronal and glial survival is a strategy to alleviate these diseases. However, it is unknown how to implement the restoration of normal neuronal connections and function from cells that are maintained alive. For the last five years, I have been studying how neuron-glia interactions link the promotion of neuronal and glial survival with the formation of axonal trajectories. I study these processes during development of the nervous system, as this knowledge is absolutely essential to work out how they can be implemented or manipulated upon injury or disease. I use the Drosophila equivalent of the spinal cord as a model system because it is possible to study and manipulate individual neurons and glia with single cell resolution, and in vivo, something not yet possible in vertebrate models. Furthermore, working with Drosophila is very cheap, progress is fast because the fly life cycle is short and experimentation with flies does not raise ethical concerns. Findings in Drosophila are totally relevant to the understanding of the human spinal cord and brain. Drosophila has long been one of the most powerful genetical model organisms to work out molecular pathways underlying universal cellular functions. Many important molecules involved in the development of the human brain and disrupted in human cancers were discovered in Drosophila. The fruit-fly is also a powerful model organism to model neurodegeneration and neurotrophic interactions are known to occur in flies. With this proposal, I aim to gain from Drosophila information of how a homologue of a trophic factor already known to exist in humans functions in axon guidance and targeting during development.
Using the power of Drosophila genetics and of the sequenced genomes, I also aim to discover novel trophic factors that maintain neurons alive, which I anticipate ultimately will be relevant for humans too.

Technical Summary

Recovery of neuronal function following spinal cord injury requires the re-establishment of axonal trajectories, synaptic connections and glial populations and the maintenance of neuronal and glial survival. Demyelinating and neurodegenerative diseases are also characterised by glial and neuronal death. One approach to cure spinal cord injury and neurodegenerative diseases is to understand the mechanisms that maintain cells alive and that promote axon growth.
Neuron-glia interactions are needed for axon guidance and to regulate the number of neurons and glia necessary for function. Cell number is adjusted through the non-autonomous control of cell survival and cell proliferation. The same molecules may implement trophic support and axon guidance. Neurotrophins are produced by the target to promote neuronal survival, they are involved in neuron-glia interactions and in axon guidance. Thus, neurotrophins are ideal molecules to implement repair.
However, multiple trophic factors present in limiting amounts are thought to maintain cell survival in the central nervous system. Thus, most likely trophic factors remain to be discovered.
Analysis of neuron-glia interactions during axon guidance in the developing spinal cord is technically challenging: the use of a more amenable model system is compelling. Drosophila has been used as a powerful model system to study axon guidance and neurodegeneration. In the Drosophila equivalent of the spinal cord, the ventral nerve cord, the trophic neuron-glia interactions taking place during axon guidance are well known. I have means of manipulating neurons and glia with single cell resolution, non-invasively, in vivo and in real time.
I aim to: (1) Analyse the in vivo function of the Drosophila BDNF homologue. I will study its roles in the control of cell survival and proliferation, axon guidance and fasciculation. I will also study genetic interactions, e.g. with neuregulin. This will tell me whether neurotrophins function in comparable ways in vertebrates and in insects and I anticipate it will reveal in vivo roles not yet known from vertebrates. (2) I will search for novel trophic factors, using suppressor genetic screens. The trophic function of an unknown gene is tested by its ability to rescue a mutant phenotype of extensive neuronal or glial death. I will use the GAL4/EP and GS systems to drive selectively in neurons or glia the expression of genes downstream of UAS inserted randomly throughout the genome. The sequenced genome and material available from the BDGP will provide molecular access to the candidate genes.
 
Description BBSRC Pool of Experts
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description British Society for Developmental Biology Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description EU FP7 Grant Evaluator
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
 
Description Evaluation committee of Neuroscience Institute Fer a Moulin in Paris, France
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Fellowship Evaluator for the Spanish "Agencia Nacional de Evaluacion y Prospectiva" from the Spanish Ministry for Science and Education
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description Member of the European Science Foundation Pool of experts
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Nominated Specialist Advisor for RAE 2008
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC PhD Studentship Simon Bishop
Amount £22,500 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2013
 
Description BBSRC Project Grant
Amount £417,781 (GBP)
Funding ID BB/H002278/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2010 
End 09/2013
 
Description BBSRC Scholarship
Amount £1,400 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2008 
End 08/2008
 
Description BBSRC Scholarship
Amount £1,400 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2006 
End 08/2006
 
Description EU Marie Curie IIF-NPN
Amount € 176,011 (EUR)
Funding ID 274393 
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 09/2011 
End 08/2013
 
Description EU Marie Curie International Incoming Fellowship
Amount £127,095 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 02/2007 
End 01/2010
 
Description MRC PhD Studentship
Amount £30,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2006 
End 09/2010
 
Description MRC PhD Studentship 2011
Amount £30,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 09/2011
 
Description MRC Studentship Jenny Pennack
Amount £30,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2004 
End 09/2008
 
Description Nuffield Scholarship
Amount £1,400 (GBP)
Organisation Nuffield Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2008 
End 08/2008
 
Description Royal Society Short Visit Fellowship
Amount £3,982 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 01/2006 
End 03/2006
 
Description Wellcome Trust Project Grant
Amount £231,076 (GBP)
Funding ID 088583/Z/09/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2010 
End 10/2013
 
Description Wellcome Trust Project Grant
Amount £301,453 (GBP)
Funding ID 094175/Z/10Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2011 
End 07/2015
 
Description Wellcome Trust Scholarship
Amount £1,360 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2007 
End 08/2007
 
Title BALM confocal microscopy 
Description During the course of my MRC-CEG, I coordinated the successful application of an Equipment Grant from the Wellcome Trust, that enabled us to purchase a new confocal microscope, employ an imaging specialist and create the Biosciences Advanced Light Microscopy Facility. 
Type Of Material Improvements to research infrastructure 
Year Produced 2006 
Provided To Others? Yes  
Impact The Leica SP2 confocal microscope has been essential for all the projects carried out by my research team. Thjs confocal and the general management and access to BALM has been open to everyone in our University, and has helped many other research groups in the University of Birmingham (a total of 67 users). 
 
Title DeadEasy Synapse software 
Description This is a programme to count automatically the number of active zones (ie synapses) at the neuromuscular junction of the ruit-fly. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? No  
Impact This programme is freely available through my webpage, to be used as an ImageJ plug-in. 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title DeadEasy software 
Description We have written a package of six programmes called DeadEasy for the automatic quantification in vivo of apoptotic cells, mitotic cells, neurons and glia in the nervous system of Drosophila. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact So far, we have used these programmes for multiple projects that have resulted in 3 publications, and more manuscripts are now in preparation. The programmes are publicly and freely available as ImageJ plug-ins through my lab web-page (after publication). 
 
Title FlyTracker software 
Description Software tool for tracking moving Drosophila flies to measure their speed, trajectory and wobbling (loss of balance). This programme is a modification of an existing programme called MTRACK-2. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact This programme will be made freely available after publication in a journal. 
 
Title Generation of DNT reagents 
Description We generated strains for Drosophila fruit-flies mutant for the DNTs, as well as strains of transgenic flies bearing over-expression of knock-down constructs (RNAi) for the DNTs. We are the only lab in the world to have generated these reagents and we have already distributed them to several other labs world-wide. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Collaborations, publications, knowledge, networking 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Projects.html
 
Title Generation of Toll-6 and Toll-7 reagents 
Description We generated antibodies to Toll-7, and constructs for both Toll06 and Toll-7 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Citations 
 
Title Nerve cord injury paradigm in Drosophila 
Description During the course of my MRC-CEG, and funded by an EU Marie Curie Post-doctoral fellowship and subsequently (ie 2010) by a BBSRC Project Grant, we established an injury paradigm in the Drosophila larva that enables us to investigate the genetic and molecular basis underlying central nervous system regeneraiton. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact This work is not published yet, the manuscript is currently under review, but the method and reagents will become available after publication. 
 
Description DNTs 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution My team and I did bionformatics, all molecular biology and functional in vivo analysis of DNTs.
Collaborator Contribution Kenji Mizuguchi did the structural analysis of DNTs for our project, based on an informal collaboartion. This resulted in him being an author in our paper Zhu et al in PLOS Biology.
Impact 19018662
 
Description Longitudinal glia 
Organisation Autonomous University of Madrid
Country Spain 
Sector Academic/University 
PI Contribution Most of the project was carried out in my lab; this was a microscopy and functional analysis of Prospero function in glial division.
Collaborator Contribution Joni Benito-Sipos joined my lab for one summer and he did experiments with us that resulted in a joint publication Laura Torroja was his supervisor in Spain).
Impact 18634579
Start Year 2006
 
Description Receptors 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a collaboration with Prof NIkc Gay, Department of Biochemistry, University of Cambridge. My research team provides the neuroscience context, and genetics, microscopy and molecular biology approaches. My collaborator provides the biochemistry expertise.
Collaborator Contribution We have recently submitted a joint research paper for publication.
Impact The outputs currently are a submitted paper, and a project grant awarded by The Wellcome Trust. This grant is multi-disciplinary, involving molecular biology, genetics, biochemistry and imaging.
Start Year 2011
 
Title Automatic Fly Sorter 
Description An image processing based automatic method to select transformant flies bearing a GFP marker in the eye. This is useful for the Drosophila community of researchers. 
IP Reference Insufficient Information 
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact none yet.
 
Title DeadEasy Caspase 
Description software to count the number of apoptotic cells in the Drosophila embryo in vivo 
Type Of Technology Software 
Year Produced 2009 
Open Source License? Yes  
Impact sharing with the scientific community; research advance; publications 
 
Title DeadEasy Caspase Larvae 
Description adaptation of DeadEasy Caspase to work in larvae, in 3D stacks of confocal images taken from the whole larval CNS 
Type Of Technology Software 
Year Produced 2011 
Open Source License? Yes  
Impact sharing with scientific community and research advance 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title DeadEasy Larval Glia 
Description to count the number of glial cells in the Drosophila larva in 3D in vivo 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact sharing and scientific advance, publications 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title DeadEasy MitoGlia 
Description to count the number of glial cells or dividing cells in Drosophila embryos in 3D in vivo 
Type Of Technology Software 
Year Produced 2010 
Open Source License? Yes  
Impact sharing and scientific advance 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title DeadEasy Neurons 
Description to count the number of HB9 or other sparesely distributed neurons in the Drosophila embryonic CNS in 3D in vivo 
Type Of Technology Software 
Year Produced 2010 
Open Source License? Yes  
Impact sharing with scientific community 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title DeadEasy Synapse 
Description to count the volume occupancy of active zones or other synaptic components 
Type Of Technology Software 
Year Produced 2013 
Open Source License? Yes  
Impact sharing and scientific advance 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title FlyTracker 
Description to track crawling larvae and walking flies 
Type Of Technology Software 
Year Produced 2013 
Open Source License? Yes  
Impact sharing and scientific advance 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Description Community Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Communicating scientific discovery to the public using fruit-flies

outrach
Year(s) Of Engagement Activity 2013
 
Description Community Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Communicating science to the general public

outreach
Year(s) Of Engagement Activity 2014
 
Description Lab web-site 
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 Soon after starting this project, I created a lab web-site where information on this project, and other lab projects, can be found. www.biosciences.bham.ac.uk/labs/hidalgo

making our work known to the public
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Member of the organising committee for NeuroFly, Belgium 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I was a member of the scientific and organising committee for the NeuroFly meeting held in Leuven, Belgium

running the meeting, networking
Year(s) Of Engagement Activity 2006
 
Description Member of the organising committee for NeuroFly, Manchester 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I am a member of the organising committee for the biannual meeting NeuroFly, which was held in Manchester in 2010.

networking
Year(s) Of Engagement Activity 2010
 
Description Organiser of the BSDB-SEBD meeting, Seville, Spain 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I was co-organiser with Dr Robert Kelsh of the British component of the joint meeting between the British and Spanish societies for developmental bology (BSDB and SEDB respectively) which was held in Seville, Spain.

running the meeting, networking.
Year(s) Of Engagement Activity 2008
 
Description Video on this project in scivee.tv 
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 We made a video pubcast on this MRC funded project, which is available online at www.scivee.tv/node/8389 , where it has been in the most recommended and amongst the most viewed since its launch in November 2008. It has received so far (21-12-2009) 16537 viewings. Its DOI: 10.401618389.01

making our work known to the general public
Year(s) Of Engagement Activity 2008