Genetic and imaging studies of eye morphogenesis in development and disease

Lead Research Organisation: University College London
Department Name: Cell and Developmental Biology

Abstract

Eye colobomas encompass a group of common eye defects affecting patients of all ages, but especially young children. These pathologies are a common cause of visual problems, can cause retinal detachment and cataracts, and often induce blindness in affected patients. Colobomas are generally hereditary diseases with a highly variable clinical presentation, being diagnosed by the detection of a notch, gap, hole or fissure in any of the structures of the eye, including the cornea, retina, optic nerve or even the eyelid. The defects in the ocular structures associated with the colobomas result from a failure in the embryonic formation of the eye. During embryogenesis, the forming eye and optic nerve undergo dramatic shape changes that lead to the closure of a fissure present on one side of the eye (the choroid fissure), and eventually to the formation of the eye globe. If choroid fissure closure is disrupted, an ocular coloboma develops. Recently, some of the congenital defects responsible for certain forms of coloboma have been identified. These defects consist of mistakes or ?mutations? in certain genes that normally make proteins important during eye formation. Despite these recent advances, we know virtually nothing about how cells behave during closure of the choroid fissure and how they are affected in coloboma conditions. Understanding these issues is fundamental to understand coloboma pathologies and to find ways of treating them. For this purpose, we will use the zebrafish, a small striped fish that has been used for years as a model to study embryonic development and that recently has been successfully used to model other human diseases. Highly developed imaging techniques allow visualisation of the developing eye in the living zebrafish embryo. By using zebrafish that exhibit colobomas similar to those in humans, we will compare the process of choroid fissure closure between these coloboma disease models and healthy conditions. In addition, we will perform a number of genetic screens to search for other genes affected in coloboma. These analyses will provide us with a list of candidate genes responsible for human ocular pathologies, will lead us to identify and generate new models for human eye diseases and will allow us to gain further insight into normal eye development and into the causes of hereditary ocular malformations.

Technical Summary

Ocular colobomas constitute a family of ocular pathogeneses that are caused by non-closure of the choroid fissure, an event that normally occurs during morphogenesis of the eye. These abnormalities are the most common hereditary ocular malformations, causing as much as 10% of childhood blindness. However, their genetic bases remain elusive. Identification and functional characterisation of genes responsible for hereditary coloboma is not easy to do in humans as the low number of affected families and wide phenotypic and locus heterogeneity, make it difficult to identify disease loci. Moreover, a thorough understanding of the cellular events leading to choroid fissure closure during morphogenesis of the eye is essential to understand the causes of these hereditary ocular malformations. In this project, we aim to gain insight into the cellular events leading to optic cup morphogenesis and choroid fissure closure and to identify molecules involved in this process. Preliminary results have prompted us to identify two well-known signalling pathways, the Wnt and the Eph/ephrin pathways, as strong candidates for participating in choroid fissure formation and closure and we will pursue the functional analysis of these pathways. Other molecules important for this process have recently been identified by ourselves and others but, with the exception of Ncadherin, all are signalling molecules and their target transcription factors. The final downstream effectors of optic cup morphogenesis activated by these signals are still largely unknown. To identify genes with roles in choroid fissure morphogenesis and closure, we will make use of both forward and reverse genetic approaches in zebrafish. We will interfere with the function of genes known to be important for optic fissure closure and will use microarray expression profiling of cells from the resulting colobomatous eyes to identify genes misregulated when colobomas occur. As a complementary forward genetic approach, we will characterise existing zebrafish mutants with coloboma phenotypes and will participate in a genetic screen to identify mutations affecting eye morphogenesis. In parallel with these approaches, we will make use of highly advanced imaging techniques to elucidate the cellular mechanisms underlying these morphogenetic processes in living fish. These analyses will identify candidate genes responsible for human ocular pathologies and will lead us to identify and generate new models for human eye hereditary diseases. Analysis of the cell biology underlying the identified genetic defects will allow us to gain further insight into the normal eye morphogenesis of the eye and the aetiologies of hereditary ocular malformations.

Publications

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Bazin-Lopez N (2015) Watching eyes take shape. in Current opinion in genetics & development

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Cavodeassi F (2012) Brain regionalization: of signaling centers and boundaries. in Developmental neurobiology

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Hernández-Bejarano M (2015) Opposing Shh and Fgf signals initiate nasotemporal patterning of the zebrafish retina. in Development (Cambridge, England)

 
Description PhD training
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description MRC Project Grant
Amount £518,324 (GBP)
Funding ID G0900994 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2010 
End 08/2013
 
Title Novel transgenic lines expressing GFP and other proteins 
Description Our work involves generation and characterisation of transgenic lines expressing GFP and other reporters in specific cell types. These lines (too many to list here) are made available to the community and are widely used. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact By generating and characterising new transgenic lines, we are providing the research community with new tools for the study of developmental processes. 
 
Title Zebrafish models for analysis of cell movement defects 
Description Our work characterises zebrafish lines carrying mutations that affect developmental processes and as such, we create models for human congenital conditions. These mutants are freely available to the research community and so there are many outside researchers who are using the same lines as us. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact Our studies in zebrafish lines that are models for human congenital diseases provides valuable information to better understand the role of the affected genes in the induction and/or progression of those pathologies in humans. 
 
Title Zebrafish models for human congenital conditions 
Description Our work characterises zebrafish lines carrying mutations that affect developmental processes and as such, we create models for human congenital conditions. With the CRISPR/Cas-Mediated Genome Engineering we have generated several new mutants. These mutants are freely available to the research community and so there are many outside researchers who are using the same lines as us. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact Our studies in zebrafish lines that are models for human congenital diseases provides valuable information to better understand the role of the affected genes in the induction and/or progression of those pathologies in humans. 
 
Description Bill Harris: Retinoic Acid signaling in eye morphogenesis 
Organisation University of Cambridge
Department Department of Physiology, Development and Neuroscience
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have been able to identify that retinoic acid receptor signaling regulates choroid fissure closure through independent mechanisms in the ventral optic cup and periocular mesenchyme.
Collaborator Contribution Prof. William Harris works on eye patterning, development and differentiation in Zebrafish embryos and had started a project on retinoic acid (RA) target during eye morphogenesis. We have been able to identify several RA targets genes potentially involved in the coloboma phenothype induced by RA and this has led to one publication so far (21555593 Lupo et al. 2011).
Impact So far, one publication has resulted from our collaboration (21555593 Lupo et al. 2011) .
Start Year 2007
 
Description Brian Link 
Organisation Medical College of Wisconsin
Country United States of America 
Sector Academic/University 
PI Contribution We have studied the retinal phenotype of fish lacking Lmx1b function. We are looking at the role of POM in eye morphogenesis, from a cellular point of view while Link is generating useful transgenic lines. We are both interested in the role of YAP1 gene in RPE genesis and coloboma. While we focused more on the role of YAP1 in coloboma formation Link is complementing our analysis looking at the role of YAP1 in RPE specification.
Collaborator Contribution Our collaborators performed experiments and provided lines of zebrafish that helped us to resolve the role of Lmx genes and POM cells in eye development.
Impact So far, one publication has resulted from this collaboration (19500562). While: Yap and Taz regulate retinal pigment epithelial cell fate; Joel B. Miesfeld, Gaia Gestri, Brian S. Clark*, Michael A. Flinn, Richard J. Poole, Jason R. Bader, Joseph C. Besharse, Stephen W. Wilson, and Brian A. Link. Development. 2015 Sep 1;142(17):3021-32. doi: 10.1242/dev.119008.
Start Year 2007
 
Description Michael Brand: Dynamic coupling of eye morphogenesis and patterning 
Organisation Technical University of Dresden
Department Biotechnology Center
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution We have contributed with experiments and discussions, to the understanding of how the processes of morphogenesis and allocation of retinal identity are coordinated during maturation of the optic cup. The work deriving from our interaction with Dr. Picker has recently been published in a high profile Journal.
Collaborator Contribution Our collaborator, Dr. Picker, is an expert in eye patterning and morphogenesis. Our interaction with him has resulted in a publication in a high profile Journal that contributes to our understanding of how the processes of morphogenesis and allocation of retinal identity are coordinated during maturation of the optic cup.
Impact So far, one publication has resulted from our collaboration (19823566).
Start Year 2007
 
Description Nicky Ragge: Retinal coloboma and TFAP2 function 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have been able to develop fish models of human conditions affecting eye development.
Collaborator Contribution Dr Nicky Ragge works with human patients carrying mutations in genes that we are functionally characterising in fish. We have been able to develop fish models of the human conditions and this has led to one publication so far (Gestri et al. 2009).
Impact So far, one publication has resulted from our collaboration (19685247 Gestri et al. 2009) .
Start Year 2007
 
Description Outreach - A Visit to Local Primary Schools (portugese speakers at local primary schools) 
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 Native Portuguese speakers Ana and Renato organised an exciting Portuguese science session at St. Mary's Roman Catholic Primary School in London as part of the Native Scientist program. During the session the students leant about behaviour and brain function, explored genetic concepts such as inherited traits and examined embryos and investigated how long different species take to develop. Last but not least, the students had a chance to extract DNA from strawberries!

To learn more about Native Scientist visit: www.nativescientist.com
Year(s) Of Engagement Activity 2015,2016
URL http://www.nativescientist.com
 
Description Outreach - A-level placement - Individual (5 day work experience) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact This work experience is very popular and heavily oversubscribed - we usually get about 8 application per place. The feed-back is always very positive, often students write back to us after they have successfully secured a university place, often at Oxbridge or other Russell Group Universities. Students cite their work experience as having been very stimulating and helpful to list on their UCAS application and at a subsequent interview.

Every year we host between several Biology A-Level students for a 5-day work experience once or twice a year, in recent years generally 8 students per work experience. The placement week is designed to give students an insight into the daily life in the lab by carrying out an independent research project under supervision.

Participating students work in groups of two, for example in 2016 they worked on three projects that cover the research interests of our lab: eye development, CNS asymmetry, and our neuroanatomy atlas. For their project students used standard Molecular Biology methods (e.g. TUNEL staining to detect apoptotic cells, Wholemount In Situ Hybridisation and fluorescent immuno-histochemistry to distinguish normal and altered RNA or protein distribution in mutant and normal sibling fish (mutation affecting the eye); PCR to genotype fish embryos). Students also examined transgenic fish and fish from heterozygous mutant line crosses FISH (fluorescent In Situ Hybridisation) using confocal microscopy to analyse altered neuroantomy in mutant larval fish. We all have fun; the students are extremely enthusiastic and a pleasure to interact with and supervise, and the students also seem to enjoy the challenges and work extremely well together. Students present their results and place their work in the greater context of research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
URL http://www.ucl.ac.uk/zebrafish-group/outreach/
 
Description Outreach - Visits from Local Schools 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact We have open days for the labs involving school kids from around London. These happen 2 or 3 times a year and are massively oversubscribed, about 10-20 students attend per day visit, about 30-50 students per year.

We have many requests each year from schools and try to accommodate them as best as we can.

The last visit was on 22 November 2016: 12 students plus 1 teacher from Haringey Sixth Form Centre spent an afternoon in our lab.

We have had very positive feedback from students, some indicating that the visits have encouraged them to take science at University
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
URL http://www.ucl.ac.uk/zebrafish-group/outreach/
 
Description Research Conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We have presented our data to colleagues through many seminars and presentations at Research Conferences.

Our discussions and interaction with other researchers help us to move forward in our research, and to identify future collaborations.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description Website outreach 
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 Our website (www.ucl.ac.uk/zebrafish-group/ www.ucl.ac.uk/zebrafish-group/outreach/) contains descriptions of our research projects and publications written at a level accessible to a well-educated public audience.

Several researchers interested in establishing collaborations with us contacted us after seeing our web page. It is also a very good advertising medium to get potential students and postdoctoral researchers interested in our lab. We also host visits and work-shadowing by school kids at least twice a year. We run the openlab programme that invites any UCL students for lab visits. We have presented to design students and have hosted art students.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012