Construction of a novel Digital Scanning Lightsheet Microscope and its application in measuring 3D cell behaviour and movement in embryos

Understanding embryonic development is one of the big challenges of Life Science Research The first stage in the embryonic development of all higher organisms is the formation of the zygote from the fusion of a sperm with an egg cell, which is typically followed by a series of rapid cell divisions in which many cells are generated. Due to asymmetries present in the cytoplasm of the egg, or external asymmetries provided by the mother, different cells initiate distinct gene expression programs, which allow the cells to proliferate, undergo programmed cell death, differentiate and cell move. For proper development to occur these processes have to be coordinated precisely in 3 dimensional space and time, which is achieved by extensive cell-cell communication. Cell-cell communication can involve signaling through direct cell-cell contacts (short-range) or through secretion of signaling molecules that can diffuse in the space in between the cells and travel a relatively long distance. There are only a limited number of forms and shapes that can be generated by these processes and in many cases cells will need to move from the place where they produced to the site where they are required. This is particularly important in the process of gastrulation and during the formation and wiring of the nervous system. Gastrulation is a critical stage in embryogenesis where the main body plan of the embryo is laid down and the axes of symmetry emerge. It involves large-scale long-range cell movements during which cells of the three germlayers (ectoderm, mesoderm, and endoderm) take up the correct topological positions in the embryo. The endoderm is located innermost in the embryo and adult and endoderm cells form the lining of the digestive tract and associated glands (liver pancreas etc). The endoderm is surrounded by the mesoderm that will give rise to the muscles and the skeleton. The mesoderm is covered by the outmost layer, the ectoderm that will form the epidermis and the nervous system. Improper cell movements during gastrulation results in severe cases in abortive development and in less severe cases form the basis of many congenital defects in animals and humans. The signaling mechanisms and the cellular processes underlying gastrulation have been studied in a variety of experimental model systems. The study of gastrulation in higher vertebrates such as amniotes (birds reptiles and mammals) has focused on the development of the chick and mouse embryo. The chick embryo has the advantage that development takes place outside the mother and is therefore easily accessible to experimental manipulation. The chick embryo is flat and translucent which makes observation of cell movements during gastrulation possible. To understand complex processes such as gastrulation it is essential to be able to follow the movements of all cells in the embryo. This requires very powerful microscopic techniques and one of the aims of this research is to build and develop a microscope with which this will be possible. This will require close collaboration between physicists, computer scientist and life scientists and we have assembled such a consortium. Once the instrument is build we will use it to map out cell division and movement during early chick development and generate a blueprint of this process. In a second phase we will start to investigate the signaling systems that control these movements by experimental perturbation, we will up and down regulate critical signaling molecules and study their quantitative effects on early development. From this we will build up a picture of the most critical processes that control gastrulation which will help in understanding many congenital defects and diseases in later life and knowledge obtained in these studies will be essential to be able to prevent and cure some of these cases in the future.

We aim to obtain a complete description of the cell division and movement patterns that occur during gastrulation in the chick embryo, a model system for amniote development. This requires the implementation and development of new optical methods that allow the recording of the behavior of tens of thousands of cells in three dimensional space. We will build and develop a Digital Scanning Lightsheet Microcope (DSLM) that is based on optical sectioning of the specimen under investigation with a thin light sheet and detection of fluorescence at right angles through high speed, high resolution, digital cameras This methods combines minimal light exposure, with fast confocal microscopy of large specimens at high resolution and an optimal signal to noise ratio and is especially suited for in-vivo imaging of cell behaviours in complex three samples such as embryos. It will be integrated with photo-activation and laser cutting options to allow in-vivo labeling and manipulation of specific cells in specimens under investigation. This will be the first such instrument in the UK and can be applied to many different questions. We will use two new transgenic GFP expressing transgenic chick strains that express membrane targeted and photoactivatable GFP to obtain a description of cell division and movement of all cells in the chick embryo from the time of egg laying to the 4-5 somite stage. These data are expected to give new insights in the mechanisms that underlie early amniote development. It will show how much and where cells intercalate to from the hypoblast, where and when cells undergo an epithelial to mesenchymal transition and by which mechanisms the cells move. In subsequent experiments this information will be used to quantitate and analyze the consequences of the perturbation of cell-cell signaling on gastrulation. We will especially investigate how the FGF, PDG and Wnt signaling pathways control division, cell polarisation and movement.