Epithelial Sheet Dynamics during Primitive Streak Formation as Active Matter

An important goal of the study of development of higher animals including humans is understanding gastrulation. Gastrulation is a critical stage in early embryonic development where the main body plan of the embryo is laid down and the main body axes emerge. It involves large-scale, long-range cell movements during which cells of the three tissue layers, the ectoderm, the mesoderm and endoderm take up their correct positions in the embryo. The endoderm is located innermost in the embryo and adult, lining the digestive tract and associated glands. It is surrounded by the mesoderm that will give rise to the muscles and the skeleton, which is in turn covered by the outmost layer, the ectoderm, which will form the epidermis and the nervous system. Defects in cell movements during gastrulation result in severe cases in death and in less severe cases form the basis of many birth defects.

The cellular processes and chemical signalling underlying gastrulation in higher vertebrates (such as humans) are experimentally studied in so-called model systems, especially chick and mouse embryos. The chick embryo has the advantage that development takes place outside the mother and is therefore easily experimentally accessible. It 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. During very early stages development the chick embryo consists of two concentric disks of tissue sitting on top of the yolk; the inner, one cell layer thick, ring will form the embryo proper. Cells in a sickle shaped domain on one side of this epiblast disc will differentiate into the mesoderm and endoderm. During gastrulation this sickle shaped domain of mesendoderm cells deforms into a stripe of tissue extending from one edge of the embryo through the central midline; the structure is known as the primitive streak. The central cells of the primitive streak then move inwards and away from this site of ingression to form the inner mesodermal and endodermal layers of the embryo.
In this project we will study gastrulation in the chick embryo using two complementary approaches. First, we use experiments to follow the mechanical and chemical cell-to-cell signalling in the developing embryo at a cell-level detail. In order to do so 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 (50,000-200,000) in a special chick strain in which the cell membranes of all cells are marked with a green fluorescent protein. We study how different cell behaviours such as division, shape changes and motion are coordinated to generate these tissues and which chemical and mechanical cell-cell signalling mechanisms control them. Second, we build a computational model based on active, interacting cells using concepts from the physics of collective motion and use it to understand cell flow both at the local and the full embryo scale.
Our study of the interplay between cell-cell signalling, cell differentiation, proliferation and migration is not only important to the community of researchers whose interest is focused on embryogenesis but will also be of great importance to scientists whose research is centred on processes such as wound healing, tissue repair and regeneration. Furthermore, in order to progress with the proposed research we will develop several new mathematical and computational techniques which are expected to be of great value for further mathematical investigation of other biological and biomedical/engineering problems.

This study focuses on characterising the signals and cellular mechanisms that drive gastrulation in the chick embryo, closely combining experiments and physical modelling. Using advanced Light Sheet Microscopy imaging we have shown that the large-scale tissue flows that drive streak formation result from complex spatiotemporal patterns of shape changes, ingressions and intercalation of mesendoderm cells. While appearing stochastic at the cell-level, these events result in highly robust behaviour at the tissue level. Our experiments strongly suggest that myosin-dependent mechanical cell interactions play critical roles in the spatial coordination of these behaviours. We will now further characterise these cellular interactions experimentally and use concepts and modelling methods from soft active matter physics to understand emergence of the global behaviour. Specifically we will further develop computational methods to detect and quantify the direction and frequency of cell intercalations during streak formation. Using a combination of laser cutting, local and global mechanical perturbation experiments we will investigate whether these intercalations are tension dependent resulting in elastic propagation. We will address the role of tension-dependent MyosinII cable assembly in elastic propagation and begin addressing the signalling pathways that control it. Characterizing the spatiotemporal patterns and mode of mesendoderm cell ingression will allow us to assess their role in tension generation and address whether secreted factors are involved in the integration of these mechanical processes. These experimental results will feed into soft active matter models at the cell and at the continuum scale, that will allow us to understand how individual disordered cell behaviours lead to highly organised behaviour at the tissue level. Embryo-scale simulation will allow us to explore parameter regions not readily accessible to the experiment and guide experimental design.

The research proposed here investigates the mechanisms governing gastrulation, a central process in the development of all higher animals. Findings made here will greatly increase our understanding of how cell-cell signalling directs cellular events, like differentiation, proliferation and migration. This is important for understanding development and the origin and cause of many congenital defects. Gastrulation is core material in many Life Sciences and Medical textbooks. Key research findings made here could become textbook material and therefore affect students of the medical and life sciences.
Soft and active matter are recent directions within the physical sciences. They are specifically set up to deal with out of equilibrium, living systems and the patterns they form. As such, the research proposed here forms part of a concerted effort to construct a general theory of living things, in this case the collective properties of cells behaving as a tissue. Development, with its germ layers, patterning and segmentation, and geometrical constraints, is one of the most promising areas for this approach. Research findings from this proposal are expected to find their way into teaching materials for students in the new, interdisciplinary field of biological physics / quantitative biology.
The key processes of gastrulation such as directed collective migration, 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 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 correctly 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 5-10 year term, the research proposed here will undoubtedly have practical applications in these increasingly important areas of medicine and healthcare, affecting researchers and practitioners in both the academic and the commercial sector.
An important aspect of the proposed research is that it will strengthen links between the Biology and Physics communities. This closer integration will be beneficial to both: Biology will benefit from the depth of modelling experience, and wealth of analytical and numerical techniques developed in the statistical mechanics community, and Physics will benefit from opening up to a new community and gaining impact on a rapidly developing area. A vital aspect here is the development of a productive interdisciplinary culture. Though this is accepted reality, current undergraduate and postgraduate training remains largely monodisciplinary. Important interdisciplinary training will be provided to the PDRAs involved as well as associated PhD and master students. For example, Dr Manli Chuai was trained as a medical doctor but is now also trained in methods of advanced LSFM and large scale data analysis. Hence, an important added benefit of our proposed research will be to develop careers within an interdisciplinary culture.
The Life Sciences sector has an important economic impact in Dundee, contributing around 16% of the city's GDP. A range of activities and organisations in the city connect scientists with the public. In recognition of the economic and social impact of these interactions, the College of Life Sciences won the BBSRC "Excellence with Impact" Award in 2011.
Finally, this research generates exquisite experimental and simulated images. These have been and will be part of exhibitions at the local, national and international level.