Characterising Epithelial Cell Movements during Anterior Patterning

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

During embryonic development, the head-tail axis is properly oriented by the migration of a special group of cells called the anterior visceral endoderm (AVE). If these cells fail to migrate, the embryo develops abnormally and generally aborts. The AVE moves within a sheet of cells called the Visceral Endoderm (VE). Work from my group indicates that AVE migration is regulated by the surrounding VE cells changing their shapes in a coordinated manner. Our work has implicated a specific molecular pathway called the Planar Cell Polarity (PCP) pathway in AVE migration.

Using a novel imaging technology that provides images of living samples at extremely high quality, this project will characterise in detail the movement of all the cells of the VE, to provide the contexts in which to understand AVE migration. It will also characterise the dynamics of the molecular motors that drive the cell shape changes that regulate AVE migration. To better understand the influence of PCP signaling on AVE migration, we will disrupt it in various way, to determine what effect this has on AVE migration. We will examine various mutants that are known to show a failure of AVE migration to see if they also show a perturbation of PCP molecules. Finally, we will examine mutants of major PCP genes for AVE migration defects.

Sheets of cells like the VE are generically called epithelia and play an important part in many developmental processes. In adults, epithelia give rise to the majority of cancers, and the spreading of such tumors is the major cause of mortality in patients. PCP signaling is important in many contexts, such as during the formation of the heart and spinal cord. Disruption of PCP can lead to congenital heart defects or condition such as spina bifida, where portions of the spinal cord are exposed through the skin on the back of the body. A better understanding of both epithelia and PCP signaling will contribute to the development of treatments for pathological conditions arising from their perturbation.

Technical Summary

The anterior visceral endoderm (AVE) is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Using high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy we have shown that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct and dynamic patterns of F-actin and myosin IIA localisation.

Little is known about how surrounding cells in the VE respond to or influence AVE migration and the significance of the dynamic localisation patterns of Dvl-2, F-actin and myosin IIA. This project aims to use light sheet microscopy and automated image segmentation and analysis algorithms to perform a comprehensive characterisation of VE cell behaviour in wild type embryos and various mutants with AVE migration defects. Using fluorescent reporters of F-actin and myosin IIA, we will determine the dynamics of localisation of these molecular motors. By mislocalisaing Dvl-2 in the VE, we will test hypotheses relating to the significance of its specific localisation pattern.

Planned Impact

The results of this research will be conveyed to other researchers through the publication of findings in peer-reviewed journals, by reporting unpublished work at conferences and through personal communication with other scientists.

Image data that has been converted into vector models will be made available on a publicly accessible web site. Though meant primarily for other scientist, such data will also be readily available to the general public.

Though the results will primarily be disseminated through scientific journals, attempts will be made to inform the media of results prior to publication, so that the general public is more likely to be made aware of the results.

The results of this project will be communicated primarily by the PI, but also by the postdoc working on the project. Manuscripts will be written by the two together. Websites making the data publicly accessible will be plain but functional. Such simple web sites can be created relatively easily with commercially available programs.

Publications

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Leonavicius K (2020) Spatial protein analysis in developing tissues: a sampling-based image processing approach. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Stower MJ (2014) Heading forwards: anterior visceral endoderm migration in patterning the mouse embryo. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Cheewaruangroj N (2019) Peristaltic Elastic Instability in an Inflated Cylindrical Channel. in Physical review letters

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Leonavicius K (2018) Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have discovered how the way a cell divides might influence the fate of the daughter cells. Our ongoing research is also clarifying how cells migrate during the formation of the embryo.

In terms of techniques, we have developed ways of imaging mouse embryos using light sheet microscopy, a new, advanced type of microscope.

Updated March 2020

The techniques and initial insights enabled by this award have enabled us to further characterise coordinated movement between differnt embryonic tissues and has enabled us to study cell movements int he pluripotent epiblast, the tissue that gives the majority fo the fetus. We have discovered that there coordinated movements within the peiblast, and that they are aligned to the future head-tail axis, suggesting the movements may be responsible for setting up this axis.
Exploitation Route The approaches we have developed for imaging mouse embryos by light sheet microscopy can be used by others to image their samples.

Updated March 2020
As a larger part of this project, we have developed various computational approaches to handle the lightsheet image data. These computational approaches can be used by others. We are it he process of preparing a manuscript reporting these results, and wil lbe able to deposit the associated code on public repositories such as GitHub.

We have also initiated a collaborations with a physicist (Dr Antonio Scialdone) interested in modelling cellular interactions in the early embryo. We have provided them our image data for them to use as a starting point for their analyses.

We have also received a Wellcome public engagement award to collaborate with a choreographer, who is currently embedded in our group. She has taken inspiration from our lightsheet movies in developing a contemporary dance piece.
Sectors Education,Healthcare,Culture, Heritage, Museums and Collections,Pharmaceuticals and Medical Biotechnology

 
Description The culture and imaging approaches developed have enabled us to capture higher resolution time lapse image volumes of developing embryos than was previously possible, both with standard confocal, as well as light sheet microscopy. The knowledge generated as a result of this project have led to a better understanding of early vertebrate development (mammalian and reptile) at pre-implantation and post-implantation stages. Two of the main manuscripts published as a result of this study (Watanabe et al 2014 and Stower et al. 2015) have been cited 36 and 10 time respectively as of March 2018. Updated March 2020: We have also received a Wellcome public engagement award to collaborate with a choreographer, who is currently embedded in our group. She has taken inspiration from our lightsheet movies in developing a contemporary dance piece. The findings arising from this project have also led to the establishment of a new collaborative project with Dr Jeyan Thiyagalingam (who was a collaborator on this grant) developing new approaches to track cell movements.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Description Human Fertilisation & Embryology Authority, Scientific & Clinical Advance Advisory Committee
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact Provide advice on advances in science and clinical practice which are relevant to the Authority's work in the are of Human Fertilisation and Embryology
URL https://www.hfea.gov.uk/about-us/our-authority-committees-and-panels/scientific-and-clinical-advance...
 
Description Gastrulation in reptiles: Characterisation of turtle and chameleon embryos
Amount £6,000 (GBP)
Funding ID IE121500 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 06/2014
 
Description Gastrulation in reptiles: Characterisation of turtle and chameleon embryos
Amount £6,000 (GBP)
Funding ID IE121500 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 06/2014
 
Description Mechanisms regulating the timing of developmental events in the early mouse embryo
Amount £791,589 (GBP)
Funding ID MR/T028637/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 08/2023
 
Description Molecular mechanisms of cell fate decisions in gastrulation and early organogenesis (WT 220379/Z/20/Z)
Amount £4,000,000 (GBP)
Funding ID 220379/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2020 
End 11/2025
 
Description UK Human Developmental Biology Initiative
Amount £6,148,973 (GBP)
Funding ID 215116/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2024
 
Description Wellcome Trust Senior Investigator Award
Amount £1,760,000 (GBP)
Funding ID 103788/Z/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 01/2020
 
Description Wellcome Trust Strategic Award
Amount £2,400,000 (GBP)
Funding ID 105031/C/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 10/2019
 
Description Wellcome Trust Technology Development Award
Amount £1,500,000 (GBP)
Funding ID 108438/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 10/2020
 
Title Spatial protein analysis in developing tissues: a sampling-based image processing approach 
Description Computational pipeline for estimating protein levels from fluorescent image data. The approach incorporates features to relate expression levels with information about spatial location within the sample. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Publication of the research tool 
URL https://process.innovation.ox.ac.uk/software/p/13299a/silentmark-academic/1
 
Description Antonio Scialdone - single cell approaches to understanding early mammalian development 
Organisation Helmholtz Zentrum München
Country Germany 
Sector Academic/University 
PI Contribution Expertise in early mammalian embryology
Collaborator Contribution Expertise in computational approaches to analysing developmental processes
Impact Preprints on BiorXiv Multidisciplinary, developmental and computational biology
Start Year 2017
 
Description Collaboration with condensed matter physicist Dr John Biggins 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Pre-implantation embryology and cell fate specification
Collaborator Contribution Quantitative analysis of cellular parameters during early lineage specification
Impact publication: Watanabe et al 2014 multidisciplinary: biology and physics
Start Year 2010
 
Description Ruth Baker 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental work designed to understand how cells migrate int he context of epithelia in the embryo
Collaborator Contribution Mathematical modelling of cell movements in epithelia
Impact Multidisciplinary collaboration between experimental biologists (my group) and mathematical biologists (Prof. Ruth Bakers group)
Start Year 2015
 
Description Tracking cell movements. Dr Jeyan Thiyagalingam 
Organisation Science and Technologies Facilities Council (STFC)
Country United Kingdom 
Sector Public 
PI Contribution We have provided image data that has been precessed by a machine learning algorithm to increase signal/noise.
Collaborator Contribution Our partners have developed tracking algorithms that they are adapting to track the movement of cells in the crowded epithelia of the mouse embryo.
Impact Software for tracking cells
Start Year 2019
 
Description Tristan Rodriguez 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution imaging expertise
Collaborator Contribution molecular embryology expertise
Impact publications: Trichas et al. 2011, Stuckey et al. 2011, Trichas et al 2012
Start Year 2006
 
Description Wolf Reik single cell sequencing 
Organisation Babraham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in early mouse embryogenesis
Collaborator Contribution Expertise single cell sequencing approaches to understand the molecular basis of generation of cellular diversity.
Impact Multi-disciplinary collaboration between developmental biologists, molecular biologists, bioinformatic specialist and mathematical biologists.
Start Year 2014
 
Title 'SilentMark' software for the automated quantification of fluorescent signal in biological material 
Description Karolis Leonavicius (a BBSRC funded DPhil student in my group) created software that uses a novel sampling based approach for the quantification of fluorescence signal in different compartments of the cell. Used on microscopy images of immnuno-fluorescence stained samples, this allows one to quantify the amount of specific proteins in various cellular compartments. Importantly, the software also allows one to relate the fluorescence intensity to the spatial landmarks with the sample, which is particularly important in developmental biology applications. 
Type Of Technology Software 
Year Produced 2016 
Impact Thie software has been used by us in our research and by a collaborator in an unrelated project. We are in the process of publishing this software so that the wider community has access to it. 
 
Title Virtual Microscopy 
Description Software for exploring and interacting with multidimensional image volumes in virtual reality. The user can view up to four channels of data, rendered in up to six different modes. The user can also section the volume data in any arbitrary plane, in addition to zooming in/out, walking around the object, turing the object around in space etc. 
Type Of Technology Software 
Year Produced 2018 
Impact We use this software in my group to explore our image volumes and arrive at insights about the spatial distribution of proteins, or of cells within tissues. We have also found that this software is very useful in outreach activities, bringing alive our research to the lay public. We have used it in approximately 6 outreach activities, in schools, and science festivals. 
 
Description 'Dynamic Origins': Dance collaboration with Anan Atoyama 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Embryo development is a dynamic and essential time that starts our lives - something we have all undergone in our mothers' wombs but generally do not think about. An embryo starts out as a single cell which divides to form many cells that together are shaped ultimately into the new-born baby. Our research focuses on understanding how cell movement are coordinated to mould embryonic form.

A major aspect of our work relies on using microscopes to image cell movements in the embryo and computer programs to visualise these data, so that we can understand how they lead to the emergence of form. However even with these modern tools, we have difficulty sometimes truly assimilating the complicated three-dimensional changes occurring over time during development.

AToU is a dance company with a strong emphasis on creating dance piece to visualise invisible and unknown aspects of human society. Our project brings researchers and AToU artists together to visualise and more importantly, experience processes that are not easily seen, through the medium of dance. As scientists, we are very interested in AToU's work to connect concepts of embryonic body formation to our physical body movement, a unique opportunity for us to understand and inform our research through our own body, while at the same time, sharing our fascination for development with the wider public.

This project will:
1. foster deep interaction between our research group and artists, to inspire the creation of performance art based on our science by Anan Atoyama of AToU.
2. conduct science and dance workshops for students at The Cheney School in which students will co-create dance pieces with AToU and scientists.
3. organise a dance performance by the students to an audience at Cheney School.
4. produce an original music score inspired by our science and Anan's work, to enhance engagement with the Cheney School students and contribute to AToU's development of performance pieces.

Through our activities, we hope to inspire changes in attitude towards movements, art and science in the students, scientists and the artists.
Year(s) Of Engagement Activity 2019,2020,2021
 
Description Cheny School Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Participation in a Science Festival organised by Cheny Secondary School. In collaboration with Prof Mathilda Mommersteeg form my department and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) we had a table at the festival where we described out work on imaging embryonic morphogenesis over a period of approximately 5 hours. The co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant.

Acting on experience gained from our previous activity (Oxford Science Festival 2016), we were able to refine our activities for this event, as well as trial two additional activities. A student in my group created an 'World' in the computer game 'Minecraft' depicting different stages of heart development, as well as a maze through a developing heart. This attracted much interest from the secondary school students who visited our table. We also had on display cultured mouse caridomyocytes that visitors could watch beating using a microscope. In addition to these, as with our last event we had: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development.
Year(s) Of Engagement Activity 2017
 
Description Oxford Science Festival 2016 
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 Participation in the Oxford Science Festival. In collaboration with two colleagues from my department (Profs. Jo Begbie and Mathilda Mommersteeg) and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) my group had a table over two full days (Saturday and Sunday) in which we described out work on studying embryonic morphogenesis. The co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant.

Items used to facilitate engagement included: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development; and moulding clay that visitors could use to make models of embryos.

Participation in this resulted in an invitation to another science festival at a local secondary school.
Year(s) Of Engagement Activity 2016
 
Description Oxford Science Festival 2017 
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 For the second consecutive year, in collaboration with two colleagues from my department (Profs. Jo Begbie and Mathilda Mommersteeg) and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) my group had a table over two full days (Saturday and Sunday) in which we described out work on studying embryonic morphogenesis. As before, the co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant. Items used to facilitate engagement included: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development; and moulding clay that visitors could use to make models of embryos. This year, we had a new display item, a 'World of Minecraft' rendering of the inside of a mouse heart. This attracted much interest from a segment of the public in the age range of 8-18 year old that we have had only limited success engaging with in the past.
Year(s) Of Engagement Activity 2017