Morphogenesis: the role of the vertebrate planar-cell-polarity (PCP)-related pathway in the regulation of cell behaviour

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

Abstract

Morphogenesis is a term that describes the process by which tissues, organs and embryos acquire their specific shapes and forms. For morphogenetic events to occur properly requires the orchestrated and coordinated movements and re-arrangements of many cells and so these cells must have mechanisms to communicate with each other. When morphogenetic processes go wrong, the consequences can be disastrous for the developing embryo. For instance, in humans, defects in the development of the neural tube (which forms our central nervous system) arises in 1 in 1000 live births and causes debilitating conditions like Spina bifida. Recent work using mice and other model organisms has revealed that the genetic pathways required for neural tube development are implicated in other major morphogenetic processes such as gastrulation, where simple embryonic structures undergo complex spatial rearrangements to give rise to the different layers (muscle, intestine, skin and so on) of the body. Furthermore, these pathways are evolutionarily conserved between species, including fish, frogs, mice and humans. This evolutionary conservation provides a solution to the ethical and practical limitations of studying morphogenetic movements in mammalian systems, where development occurs inside the mother making it very difficult to observe the cell and tissue movements occurring. However, Zebrafish (Danio rerio) embryos develop from externally fertilised eggs and are transparent, making them well suited for observations of cell behaviours in living embryos. Through the use of sophisticated imaging techniques, we can compare morphogenetic events in between normal (what are called wild-type) embryos and embryos in which activity of various genes is disrupted. In this project, we will focus on a group of genes that regulate the patterning of tissues in animals as diverse as flies and humans. We will work with zebrafish embryos and study the behaviours of cells at very early developmental stages. Our work will help understand how certain genes help to orchestrate complex morphogenetic processes and will elucidate the genetic cascades fundamental to understanding some of the most common and severe birth defects in humans.

Technical Summary

For morphogenesis to occur properly requires the orchestrated and coordinated re-arrangements of large populations of cells and when morphogenetic processes go wrong, the consequences can be disastrous for the developing embryo. Morphogenetic defects including neural tube and craniofacial abnormalities are relatively common in humans and so understanding the aetiology of these defects is an important goal for current research in this field. Emerging data implicates a signalling pathway related to the fly planar cell polarity (PCP) pathway in the regulation of many morphogenetic processes in vertebrates. Among the phenotypic defects attributed to altered activity of genes functioning in this pathway are disrupted neural tube closure, altered cochlear hair cell organisation, disturbed gastrulation movements, failure in neuronal migration, cyclopia and craniofacial abnormalities. A key effector of PCP signalling in flies is Flamingo (Fmi), a seven pass transmembrane protein that regulates the sub-cellular localisation of other components of the pathway thereby contributing to cell polarisation. In vertebrates, it is less clear how Fmi and other PCP pathway genes exert their effects upon cell morphology and on cell and tissue movements. In this project, we will take a multi-facetted approach to elucidating how the PCP-related pathway, and in particular Fmi, regulates morphogenetic events during gastrulation in the zebrafish embryo.

Our aims are:

1) Does Fmi regulate cell morphology and process outgrowth? Using advanced 4D imaging approaches in both gain and loss of function assays, we will determine how Fmi regulates cell migration, cell shape and formation, length, number and directionality of cell processes.
2) Is Fmi processed to form a novel seven-pass membrane receptor? We will explore the possibility that full length Fmi is cleaved, most likely removing all extracellular cadherin repeats and potentially generating a novel Fmi receptor.
3) What are the binding partners of Fmi? The intracellular domain of Fmi is predicted to interact with other proteins ? we will use a proteomic approach to identify these binding partners.
4) What genes are affected by two mutations that enhance the severity of morphogenesis phenotypes? We will phenotypically characterise and clone two mutations that have been isolated in a screen for enhancers of a gastrulation morphogenesis phenotype.
5) Do proteins functioning in the PCP-related pathway show polarised sub-cellular localisation? In flies, polarised localisation of PCP pathway components provides a beautiful read-out for activation of the pathway. Can we see similar localisation of PCP-related pathway components in vertebrates?

Publications

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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 Dr Alfonso Martinez Arias 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of zebrafish PCP mutant constructs in Drosophila
Collaborator Contribution Analysis of zebrafish PCP mutant constructs in Drosophila
Impact The work has been published in 19091770.
Start Year 2006
 
Description Dr Carl-Philipp Heisenberg 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Cell Biology and Genetics
Country Germany 
Sector Academic/University 
PI Contribution Screen of genetic modifiers for the planar cell polarity (PCP) pathway, and imaging analysis of cell behaviour
Collaborator Contribution Screen of genetic modifiers for the planar cell polarity (PCP) pathway, and imaging analysis of cell behavioursignificant intellectual input into your research
Impact There are two publications from this collaboration; 17130287 and 18701549.
 
Description Dr Carl-Philipp Heisenberg 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Cell Biology and Genetics
Country Germany 
Sector Academic/University 
PI Contribution Screen of genetic modifiers for the planar cell polarity (PCP) pathway, and imaging analysis of cell behaviour
Collaborator Contribution Screen of genetic modifiers for the planar cell polarity (PCP) pathway, and imaging analysis of cell behavioursignificant intellectual input into your research
Impact There are two publications from this collaboration; 17130287 and 18701549.
 
Description Dr Hitoshi Okamoto 
Organisation RIKEN
Department RIKEN Brain Science Institute
Country Japan 
Sector Public 
PI Contribution Provided us with flamingo mutant fish
Collaborator Contribution Provided us with flamingo mutant fish
Impact The work has been published in 19091770.
 
Description Dr Miguel Concha 
Organisation Pontifical Catholic University of Chile
Department Institute of Biomedical Sciences
Country Chile 
Sector Academic/University 
PI Contribution analysis of PCP genes in the regulation of morphogenesis
Collaborator Contribution significant intellectual input into our research
Impact The work has been published in 20843857.
 
Description Dr Yasuyuki Fujita 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Molecular interaction analysis of PCP gene products.
Collaborator Contribution Molecular interaction analysis of PCP gene products
Impact The work has been published in 19091770 and20026643.
Start Year 2008
 
Description Prof. Phil Beales 
Organisation University College London
Department Institute of Child Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Functions of PCP genes in ciliogenesis and laterality.
Collaborator Contribution training of staff and significant intellectual input into your research
Impact The work has been published in 20643117.
 
Description Hosting A-Level school class from London June 2019 
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 Biology A-Level students spend half a day with us in the research lab. They were shown live zebrafish (for example, transgenic 'glow-in-the-dark' embryos) as well simple experiments, the fish facility etc.
Year(s) Of Engagement Activity 2019
 
Description Meeting presentation - EMBO workshop 2005 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Dr Tada has presented our work through oral presentation at the workshop

This work has been published in Development
Year(s) Of Engagement Activity 2006
 
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 2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017
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 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016
URL http://www.ucl.ac.uk/zebrafish-group/outreach/
 
Description The Native Scientist - engagement with disadvantaged pupils in local, inner city schools - school visit/demonstrations 2019 
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 Researchers from my group went to visit a school in underprivileged area of London. We showed them live zebrafish and simple experiments as well as talking about the options for a career in science.
Year(s) Of Engagement Activity 2019
 
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