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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Srinivas S (2017) A Tale of Division and Polarization in the Mammalian Embryo. in Developmental cell

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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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Stower MJ (2015) Bi-modal strategy of gastrulation in reptiles. in Developmental dynamics : an official publication of the American Association of Anatomists

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Stower MJ (2018) The Head's Tale: Anterior-Posterior Axis Formation in the Mouse Embryo. in Current topics in developmental biology

 
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.
Exploitation Route The approaches we have developed for imaging mouse embryos by light sheet microscopy can be used by others to image their samples.
Sectors Education,Healthcare,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.
First Year Of Impact 2017
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Description Gastrulation in reptiles: Characterisation of turtle and chameleon embryos
Amount £6,000 (GBP)
Funding ID IE121500 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 06/2013 
End 06/2014
 
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
 
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 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 Private 
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
 
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