Abstract
Successful embryogenesis requires the formation of the correct cell types in appropriate positions. In order to make the right decisions, cells communicate using a conserved repertoire of signalling factors and transduction pathways. The ERK signalling pathway is one of these important pathways that is used repeatedly during development in animals. However, we still do not understand how ERK signalling is involved in such diverse functions. It has been proposed that the characteristics of the spatio-temporal dynamics themselves encode the diversity of effects observed and a variety of kinetics of ERK activity is now observed in many cell types or in response to different stimuli. Thus, although the study of the dynamics of ERK activity during development is essential to further our understanding of the function of this pathway and its impact on cellular behaviour, quantitative studies of ERK activity at cellular resolution in living embryos are only beginning to emerge and our understanding remains limited to a few unrelated elements.The study of the ERK pathway in vertebrates is complicated by the duplication of their genomes and the presence of multiple paralogs. Thus, during my PhD, I studied the ERK pathway in ascidians, small marine invertebrates that are members of the tunicates, the sister group of vertebrates. These animals possess unique genes for each component of the ERK pathway and use this pathway to control many induction events during their embryogenesis. Embryos of the ascidian Phallusia mammillata have two other favourable properties: they are small and fully transparent, thus easy to image, and they develop rapidly with invariant cell lineages, so that each cell can be named and found at the same position, providing a rigorous framework for analyses at cellular resolution. In addition, prior to the start of my PhD, our team developed a unique ability to identify/segment/track cells during Phallusia development.My thesis project therefore combined quantitative in vivo imaging, computer science, theoretical modelling and experimental perturbation to quantitatively and systematically study the spatio-temporal dynamics of the ERK signalling pathway during Phallusia embryogenesis. I first focused on the development of experimental methodologies allowing the manual in vivo quantification of ERK activity at the 64-cell stage using the ERK-KTR biosensor. As a proof of principle, we used this tool as part of a paper review to test the hypothesis that contact surfaces between transmitter and receiver cells provide the quantitative information that determines the outcome of inductions in ascidians. I then participated in the development of a theoretical framework and algorithmic methods to automatically identify and name each cell in reconstructed Phallusia embryos. By connecting the embryos into a common repository, this work establishes stereotyped ascidian embryos as a numerical framework of choice for building and integrating functional 4D atlases with cellular resolution. Finally, I focused on the development and validation of the methodology to automatically measure ERK activity for each cell of living Phallusia embryos every 2 minutes. This work will allow us to identify new ERK-dependent inductions and to study the variability of ERK signalling between embryos in relation to the variability of cell contacts in order to test the generality of the surface-contact dependent induction model in ascidians.