Investigation of the mechanics of gastrulation in the chick embryo using new transgenic chicken lines

An important goal of the study of development of higher animals including humans is to understand how hundreds of thousands to millions of cells assemble into tissues that make organs and structures that make up an organism and which mechanisms control and integrate these behaviours. This coordination of cell behaviours is especially important at a critical early stage in embryonic development, gastrulation. During gastrulation the main body plan of the embryo is laid down and the main body axes emerge. Gastrulation involves large-scale, long-range cell movements and tissue deformations during which cells of the three body layers, the ectoderm, the mesoderm and endoderm take up their correct positions in the embryo. The endoderm is located inner most in the embryo forming the lining of the digestive tract and associated glands. It is surrounded by a middle layer the mesoderm that will make the muscles and the skeleton. The mesoderm is surrounded by the outmost layer, the ectoderm, which will form the epidermis (skin) and the brain and nervous system. Defects in the coordination of cell movements during gastrulation results in death of the embryo or in less severe cases in many major birth defects, such as the various forms of spina bifida and diverse heart defects.
We study the mechanism of gastrulation in the chick embryo, that has the advantage that development takes place in the egg outside the mother animal and is therefore easily accessible to experimental observation and manipulation. The embryo is also flat and translucent which helps observation of cell movement during gastrulation. Gastrulation in chick embryos greatly resembles gastrulation in humans, which means that findings can be extrapolated to human development.
We study how different cell behaviours such as cell division, shape changes, differentiation and motion are coordinated to generate these three embryonic tissues through specific chemical and mechanical cell-cell signalling mechanisms. Since these processes are highly dynamic we use time lapse microscopy to visualise these behaviours of the around 500.000 cells that make up the embryo of this stage of development. To do this we have developed and built a novel type of microscope, a light-sheet fluorescence microscope, that allows us to see almost all the cells in the embryo of a special chicken line in which the membranes of cells are marked with a green fluorescent protein. We have also developed extensive computational methods to extract quantitative information about relevant cell and tissue behaviours that drive gastrulation under normal and experimentally perturbed conditions. In this project we specifically test the hypotheses that forces produced by cell behaviours such as cell growth and movement are instrumental in the coordination of the complex cell behaviours of many cells in the developing embryo. Furthermore we expect that these forces feedback on the control of gene expression controlling the location and quantity of the various different types of cells in the embryo. To test this hypothesis we will generate new chicken lines that allow us to monitor and perturb the activity of critical elements of the actin-myosin cytoskeleton of cells that is responsible for generating these forces and correlating these with distinct cell and tissue behaviours using the above described live imaging and data analysis methodology.

This study aims to analyse and model the mechanisms that quantitatively coordinate key epiblast cell behaviours that drive primitive streak formation in the chick embryo. Previous work has shown that tissue flows are driven by myosin dependent directed cell intercalations in conjunction with apical contraction and ingression of mesendoderm cells. Now we will investigate the mechanism by which forces generated by these complex cell behaviours can organise the spatio-temporal coordination of cell divisions, ingressions and intercalations of hundreds of thousands of epiblast cells to drive the embryo wide tissue flows. Specifically, we will investigate whether mechano-sensitive myosin accumulation and activation organises the observed large scale orientation of cell intercalations and characterise the mechano-transduction pathways involved. We will investigate how cell behaviour generated tension drives the balance of cell divisions and ingressions through the streak and out with the streak to achieve cellular homeostasis of the embryonic region during streak formation. We will initiate an analysis of how mechanical feedback is involved in the control of gene expression controlling cell behaviours and differentiation. This work will be based on our recent advances in light sheet microscopy, quantitative large scale data analysis, and the local and large scale biological, chemical and physical manipulation of cells and tissues. The work will also rely heavily on the generation of novel knockin chick lines where critical components of the actin myosin cytoskeleton are endogenously labelled and knocked out using a highly innovative avian transgenesis and gene editing platform. This will establish the chick as a power full amniote model system that combines easy of live culture and experimental accessibility of embryos with extensive and cost effective genetic manipulation.

The research proposed here investigates the mechanisms governing gastrulation, a central process in the development of all higher animals. It makes use of state of the art long term lightsheet microscopy, advanced large scale automated image processing, data analysis and mathematical modelling in combination with novel transgenesis and gene editing techniques to study gastrulation in the chick embryo. Our work focusses on elucidating the mechano-chemical mechanisms that coordinate individual cell behaviours such as division, ingression and intercalation of a large population of hundreds of thousands of differentiating cells to generate and organise complex tissues at the organism scale. Findings made here will greatly increase our understanding of the development, the origin and causes of many congenital defects associated with early development, e.g. spina bifida, partial twinning, heart and vasculature defects and should be of interest to researchers working on these problems.
This research will advance the establishment of the chick embryo as a key model system to study amniote development, by a combination of experimental accessibility of the embryo, which allows detailed analysis of dynamic processes at the cell and tissue level over extended periods of time, with the ability to genetically tag and precisely manipulate individual key components of the gene regulatory, signalling and executing systems involved, hitherto only available in invertebrates and lower vertebrates such as amphibians and fish. Furthermore, the availability of another genetically tractable relatively cost effective model system besides mice is a pre-requisite for a comparative approach necessary to distinguish generic characteristic from species specific adaptations of the mechanism that drive gastrulation in amniotes. This should be of interest to many researchers working at different aspects of amniote development and development in general.
Key processes of gastrulation such as directed collective migration, tissue deformation, ingression and EMT are also central to other biological processes using similar cellular mechanisms like wound healing, tissue repair and regeneration. Failure to properly control these is key to the development of autoimmune diseases and metastasis of cancer cells. Therefore findings made here will be directly relevant to researchers in these areas.
Understanding these developmental processes is also essential for the rational use of embryonic stem cells in regenerative medicine. It is by no means clear how embryonic stem cells migrate to the right positions and organise themselves to repair defects in-situ. Clearly, successful manipulation of stem cells will require understanding of directed cell migration, cell-cell interactions and interactions between behaviour and signalling. Therefore, in the longer term, the research proposed is expected to have practical applications in these increasingly important areas of medicine and healthcare, affecting researchers and practitioners in both the academic and the commercial sector. Life science research activities in Dundee are already responsible for 16% of the Tayside economy.
The project will train a number of young researchers (PDRAs and PhD students) in a unique combination of live imaging, computational data analysis, modelling and advanced transgenesis and gene editing methods.
Insights and materials (movies of development) acquired in this research are already extensively used in lectures for undergraduate and postgraduate students by us and colleagues. Gastrulation is core material in many Life Sciences and Medical textbooks and key research findings made here will become textbook material for medical and life sciences.
This research generates exquisite experimental and computational images that have and will be part of exhibitions at the local, national and international level and will stimulate further interactions with branches of the creative industries.