Symmetry breaking and axial patterning in aggregates of mouse Embryonic Stem cells

Organisms develop from a single fertilized egg by increasing the number of cells through cell division, making those cells different from each other and, most importantly, organizing them in space to give rise to tissues and organs. This organization requires the emergence of systems of spatial coordinates that guide the arrangement of the different cells. A well accepted view of the process contends that there are gradients of special proteins, called morphogens, that can instruct cells what to do in a concentration dependent manner. This means that in a developing group of cells, there is always some pattern of instructions that cells read and that acts as a template for the process. An alternative view is that there is no such template and cells self organize from an initial situation in which all cells are equivalent. Understanding this second possibility has been difficult for lack of an adequate experimental system. Recently we have used mouse Embryonic Stem cells to create a system that recapitulates the events that take place in the early mouse embryo. This system is robust and reproducible and, together with classical genetic analysis, provides a versatile experimental tool to study processes of pattern formation. Here we propose to use this system to explore the mechanisms that pattern the early mouse embryo. Specifically we focus on a protein called Nodal that genetic analysis has shown to be crucial for the early patterning of the mouse embryo.

One of the challenges of modern biology is to integrate large amounts of data, particularly from gene expression, into coherent frameworks that account for specific processes e.g the development of an organ like the heart, or a tissue, like the skin. In this process the acquisition of quantitative data about the system and its integration into predictive models is a most important part of the research. In this project we propose to do exactly this by focusing on Nodal and following preliminary results that suggest that it acts as the key element in the process of pattern formation in the aggregates as it does in the embryo, though we do not understand the mechanism of the process that mediates the patterning. In the proposed experiments we shall engineer versions of Nodal and associated proteins that will allow us to follow the patterning process live, extract quantitative data about it and combine it with classical genetic analysis in a useful and fruitful manner. The experimental system will be our patterned aggregates that will allow us to bypass the embryo and explore the role that mechanical forces play in the pattern forming process and how it interferes with the better understood biochemical events.

Pattern formation is a central biological problem which requires an understanding of how biochemical process interact with mechanical forces and boundary conditions during the emergence of tissues and organs. Traditional approaches to answer this question have relied on genetic analysis, a process whereby the isolation of mutations that interfere with the normal process leads to the identification of genes and proteins associated with it and creates inferences about the molecular underpinning of specific events. However, there are limitations to this approach, significantly the problems that arise from assigning a protein a function in a complex process based on a mutant phenotype. A complementary approach has its roots in engineering. It involves rebuilding a specific biological system from its component parts, deduce the minimal number of elements required for this process and use Genetics to perturb the process and thus learn about the contribution of each of the parts. It is this approach, very much in the spirit of Synthetic Biology, that we propose to use here in the context of the symmetry breaking events that lead to antero-posterior axis determination in mammalian embryos. The experiments are grounded on a novel experimental system that we have developed using three dimensional aggregates of Embryonic Stem cells. We have shown that under specific culture conditions, this system recapitulates the early patterning events of the embryo and allows for controlled perturbations of the process; spatiotemporal control of the signals and the response as well as, importantly, the ability to test the role that mechanics plays in the patterning process. We propose to use this experimental system to test it whether Nodal is involved in a Reaction-Diffusion system during the establishment of the anteroposterior axis and how mechanical and chemical systems interact in this system and how transcription react to these interactions.

In this proposal we build on results that we have obtained with a novel three dimensional culture system that we have established for mouse Embryonic Stem cells, to study pattern formation in ensembles of mammalian embryonic stem cells. Understanding the mechanisms underlying pattern formation is an important challenge in modern biology which has acquired particular significance with the emergence of organoid biology. Identifying and understanding those mechanisms will lead to a rational engineering of organs and tissues in the future. Unfortunately there are challenges in these studies: experiments in whole organisms are difficult and, even in the instances in which it is possible, the experiments are difficult. This is compounded by the emerging need to understand the interactions between mechanical forces and biochemical events which, anecdotal evidence suggests, is an important element of pattern formation. It is here that our 3D system and the proposed experiments will be of use. We have already shown that this experimental system recapitulates many of the events in the embryo and we have also shown that it is reproducible. The proposed experiments will consolidate these observations and will also provide evidence of their value to address the challenging interactions between mechanics and chemistry which are essential to developmental proesses but very difficult to analyse experimentally. The assembled team has the expertise and knowledge to do so.

For these reasons, the work falls within several priority areas of the BBSRC. Notably, the potential that it has for tissue engineering means that the project falls within the remit of Biosciences for health and, within this realm, it will importantly impact the areas of synthetic biology and systems biosciences.

Two significant aspect of the proposed work are the implementation of quantitative cell biology, computational biology and associated data analysis, and its integration with synthetic biology. The collaboration between a strong experimental group and a theoretical group is bound to give rise to significant results and there is added value in the proposed participation of PF Lenne and of the ongoing collaboration between AMA and Matthias Lutolf (see letters of support). The notion of an iteration of theory and experiments that is at the core at the project is a trademark of modern biological research, one that the participating labs have much at the core of their endeavors and one that is the essence of the project. It is possible that the work will give rise to patents, particularly in the interaction between mechanical and chemical signals; these will be handled by the University in conjunction with the BBSRC.

At a different level, the creation in culture system that mimics the events in the embryo provides further support for the implementation and value of systems that promote the 3Rs (Replacement, Reduction and Refinement).

Very significantly, the structure of the project will have an impact on the professional development of the named postdocs who will develop a common language, an important skill in todays world, and guide them towards independence.

As the work will mainly be carried out in University departments, there will be ample opportunity for the researchers to get involved with summer visitors and undergraduate students. The AMA lab for example has a very active summer program for interns and in particular has been partnering with the Nuffield Sixth form college internship program which is very successful. The emphasis on quantitative and analytical projects provides a great opportunity to guide the development of the next generation of scientists by highlighting to them the importance of these skills, and will provide them with a new vision of Biology.