The molecular control of glial progenitor proliferation in Drosophila and mammals

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

Abstract

Devastating nervous system injury (e.g. spinal cord injury, brain damage), neurodegenerative diseases of the ageing brain (e.g. Alzheimer's disease) and demyelinating diseases (e.g. multiple sclerosis) cannot be cured and future therapy requires understanding of the underlying biology. The key therapeutic approach to repair central nervous system (CNS) damage and disease is the transplantation of stem cells or glial progenitors to the site of injury. For instance, transplantation of stem cells or glial progenitor cells in paraplegic mice repairs the broken axons and restores normal movement. However, the current lack of knowledge of how the transplanted cells behave prevents a guarantee of repair, and prevents control over undesirable outcomes such as cancer (i.e. gliomas). Thus a molecular understanding of neural stem cell and glial progenitor proliferation is urgently required. There are glial progenitors in the adult human CNS, which upon injury or disease divide in what is known as the glial-repair response (GRR), leading to a spontaneous brief recovery. Although the GRR does not result in functional repair, it reveals an intrinsic tendency of the nervous system to repair itself. If we knew what the underlying genes are and how they work, we could manipulate them to induce repair. A golden opportunity to discovering the gene network controlling glial progenitor cell division and CNS repair is provided by the GRR. Working out gene networks important for human development and disease is frequently done using the fruit-fly Drosophila because most gene networks are evolutionarily conserved. Drosophila research enables powerful genetic approaches to investigating gene function, it has high cellular resolution, it is technically sophisticated, cheap, quick, it can be done in whole and in living animals, and it does not raise ethical concerns. We have discovered a GRR in Drosophila and we have a detailed working model of the underlying molecular genetic mechanism: a gene network involving a tight relationship between the genes Notch, Prospero/Prox1, Eiger/ TNF and Dorsal/NFkB. The aim of this proposal is to work out the molecular genetic mechanism underlying the control of glial progenitor division and the GRR in the Drosophila and mammalian CNSs. To meet this aim, the following experimental objectives will be addressed: (1) To test our working model on the involvement of the candidate genes in the control of proliferation of quiescent glial precursors in Drosophila. (2) To translate our findings to the mammalian CNS, by testing the functions of the mammalian homologues in the context of glial progenitors and the GRR of the mouse spinal cord. (3) To use Drosophila to test and identify further genes involved in the GRR and glial proliferation, which can then be extrapolated to mammalian glial proliferation and GRR. This project is a collaboration between a Drosophila and a mammalian expert to use the powerful genetics of Drosophila to advance mammalian glial progenitor research. Research into repair of the damaged or diseased CNS typically relies on mammalian animal models, requiring a severity of damage to animals ranging from sacrifice at different stages of development to inflicting physical damage (e.g. breaking the spinal cord). While Drosophila research does not raise ethical concerns, basic research using fruit-flies requires an active involvement of Drosophilists to promote the effective translation to mammalian gene discovery. Here, we will use Drosophila to propel mammalian research while in this way replacing and reducing the use of mice. Our Drosophila paradigm is simple and will become available to the wider research community for further research into the GRR and drug testing for therapeutic purposes using fruit-flies. This proposal responds to the call for the '3Rs: replacing protected animals with invertebrate models' and the strategic priority of 'Ageing and lifelong wellbeing' research.

Technical Summary

We aim to work out the molecular mechanism underlying the control of glial cell proliferation in the central nervous system (CNS) of Drosophila and mice. The hypothesis is that the glial repair response (GRR) is evolutionarily conserved and Drosophila can be exploited to understand mammalian NG2 oligodendrocyte progenitor proliferation and the mammalian GRR. With preliminary findings from Drosophila we have established a working model: quiescent glial precursors are normally on the brink to divide due to a positive feed-back loop between Notch (activator of cell division) and Prospero/Prox1 (repressor of cell division). This balance is broken and pushed towards cell division by Eiger/TNFa released upon injury, which activates Dorsal/NFkB, the expression of which depends on Prospero/Prox1. The experimental objectives are: (1) To consolidate our Drosophila model using: genetics, immunohystochemistry and confocal microscopy; an experimental paradigm of the GRR developed in AH's lab; and automatic counting of glial cells using software developed in AH's lab. (2) To translate our Drosophila findings to the mammalian CNS, by testing the functions of the mammalian homologues in NG2 glial progenitors and the GRR of the mouse spinal cord. This will be done by: purifying NG2 cells from the spinal cord of normal mice, followed by immunohystochemistry and microscopy; carrying out transfections for gain of function and siRNA analyses in isolated cells and in situ in the spinal cord, and one transection experiment as a GRR proof of principle. (3) To use Drosophila to test and identify further genes involved in the GRR and glial proliferation, using genetics, which can then be extrapolated to mammalian NG2 cells. We already know that at least two genes involved in the control of NG2 proliferation - EGFR and FGFR - also regulate glial proliferation in Drosophila. The output will be a molecular platform for the control of glial proliferation and the GRR in Drosophila and mammals.

Publications

10 25 50
 
Description We have discovered a gene network that controls the glial regenerative response to central nervous system injury, using the fruit-fly as a model organism.It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NF_B. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment.

Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness.

After working out this gene network in the fruit-fly, we tested it in the mouse. We found that the molecular mechanism is essentially evolutionarily conserved from flies to mammals. It regulates the balanced between glial progenitor proliferation and differentiation. We identified a key gene - Prox1 - that promotes oligodendrocyte differentiation. This is a very important finding in the context of how to manipulate glial progenitors and stem cells to promote CNS regeneration and repair, eg after spinal cord injury.

Our findings fall within the area of regenerative biology.
Exploitation Route As indicated above, in the longer term, our findings will have medical relevance in the context of repair and regeneration of the central nervous system after damage (e.g. spinal cord injury, stroke, demyelinating diseases). In the long term, the discovered gene network may lead to finding therapeutic solutions to promote repair after central nervous system damage (e.g. spinal cord injury). For instance, it might be possible to control glial progenitors or stem cells by manipulating the discovered gene network, or use drugs targeting these genes.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Regeneration_and_Glia.html
 
Description BBSRC Committee A Core Member
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description BBSRC Pool of Experts
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 ISIS
Amount £4,450 (GBP)
Funding ID BB/K02146X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2013 
End 04/2013
 
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 Glia 
Description software programme to count the number of glial cells in Drosophila in vivo 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact sharing tools 
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. 
 
Title Regeneration and repair in the living Drosophila larval CNS 
Description CNS injury in Drosophila living larvae to investigate regeneration and repair 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact this model was presented at a conference for the first time in 2014 
 
Description Glial proliferation, differentiation, repair 
Organisation University of Birmingham
Department College of Medical and Dental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This was a BBSRC grant with Dr Alicia Hidalgo (School of BIosciences, University of Birmingham) as the Principal Applicant, and Prof Ann Logan (IBR, Medical School, University of Birmingham) as Co-applicant. The project involved investigating the genetic bases of glial cell proliferation and differentiation in the fruit-fly Drosophila, and in the mouse. Dr Hidalgo supervised all the Drosophila work, Prof Logan supervised all the rodent work. The appointed post-doctoral researcher, Dr Kentaro Kato, carried out all the experimental work.
Collaborator Contribution Prof Ann Logan's team trained the post-doctoral researcher Dr Kentaro Kato to carry out all the experiments in mice, and provided advice.
Impact • Kato, Forero and Hidalgo (2011) The glial regenerative response to CNS injury is enabled by Pros-Notch ad Pros-NFkB feed-back. PLoS Biology 9, e1001133 • Forero, Kato and Hidalgo (2012) Automatic cell counting in vivo in the larval nervous system of Drosophila. J Microscopy. 2012 May;246(2):202-12. doi: 10.1111/j.1365-2818.2012.03608 • Kato, K., Hidalgo, A. An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila.(2013) J. Vis. Exp. (73), e50306, doi:10.3791/50306. http://www.jove.com/video/50306/an-injury-paradigm-to-investigate-central-nervous-system-repair
Start Year 2010
 
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 British Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Engagement with the general public

Public understanding of science and research
Year(s) Of Engagement Activity 2014
 
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, 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