A systems biology approach to neural crest development: The role of noise in fate choice from bipotent precursors.

All the body's different cell types derive from stem or other precursor cells. These precursors are multipotent, having the flexibility to develop into any one of many types of working cells (such as neurons, blood or skin cells). A major problem in developmental biology is to understand how these precursors maintain flexibility and are thus able to generate very different cell types, while at the same time, once the choice of cell-type to adopt has been initiated, they then develop into stable cells of that type. So far, trying to dissect the genetic and non-genetic components involved in this process, known as differentiation, has proven difficult. Despite many advances in the field, the mechanism allowing the fine balance between flexibility of the multipotent stem cell and stability of the differentiated state remains mysterious. In this project we adopt a Systems Biology approach to investigate this issue. Systems Biology approaches rely on the combination of mathematical modelling techniques and experiments, to make progress towards our understanding of the system under study. Within this framework, we plan to collect a variety of experimental data capable of informing detailed dynamical models, which will be used to make predictions to be tested experimentally, and then iteratively refined. In particular we hypothesize that, counter-intuitively, an important factor helping to create alternative fates in the stem cell is 'noise' - random fluctuations in biological processes. Noise originates in many aspects of the biology of gene expression, and of other cellular activities, and accounts for much of the variability that we see in all biological systems. While we imagine that the architecture of genetic components has evolved so as to minimize any negative impact of noise, and make biological systems robust despite its presence, recent theory suggests the unexpected hypothesis that noise is an important factor that is actually required to help drive fate choice. In the context of the cell differentiation process, we will investigate a system of two important pigment cells, black melanocytes and shiny iridophores, descending from a common progenitor cell, in zebrafish. The zebrafish is a very useful model system, because the embryo is transparent and readily allows a visual inspection by using microscope techniques, and because we can readily alter gene activity and see what effects this has on the pigment cells. We will use genetics to discern the key gene interactions underlying development of this pigment cell progenitor. Then we will make detailed measurements using state-of-the-art techniques of the different activities of the relevant genes at different time-points during differentiation. At the same time we will combine this information with a mathematical model of the gene interactions. The experimental studies and the modelling will be developed in parallel and with each informed by the results of the other, so as to reconstruct the gene regulatory network responsible for pigment cell choice from the progenitor. We will also measure the amount of noise affecting the components of this network, and from this information we will be able to develop a deeper understanding of the mechanisms leading to the choice of different fates and the stable differentiation into these two cell types. In particular, we will be able to assess for the first time in the living embryo, the degree to which noise in the system helps or hinders cell differentiation. Understanding these processes has implications well beyond the basic biology we are studying here. In particular, it is important in a medical context, in that this process of stem cells choosing between different cell-types, and the process of stabilisation of these cell-types, is of fundamental importance to understanding the healthy body and how it goes wrong in ageing and in disease. It thus will shed light on the mechanisms underlying congenital diseases and cancer.

Noise originates in many aspects of the biology of gene expression and accounts for much of the variability that we see in all biological systems. The robustness of such systems, with discrete cell-types stably differentiated, indicates that the architecture of genetic components has evolved to minimize the impact of noise. Counter-intuitively, however, theory predicts that cells may also use dynamical noise to help drive fate specification and commitment processes i.e. understanding noise may be vital to understanding these cellular processes. Thus we propose a study to account for the effects of noise on the Waddington landscape of embryonic development. We will employ an iterative cycle of experimental genetics and quantitative mathematical modelling within a systems biology framework to begin to test this hypothesis in vivo. Our model system, zebrafish pigment cell differentiation, is ideal since this process is genetically well-characterised, easily manipulable, and shares many characteristics with mammals. We will investigate two pigment cell-types, black melanocytes and shiny iridophores, descending from a common progenitor cell, building directly on our recent breakthroughs in modelling the gene regulatory network (GRN) of melanocyte differentiation and in identifying key genetic mechanisms active in the common progenitor. We will identify the topology of the GRN of the common pigment cell progenitor. We will construct in an iterative manner both deterministic and stochastic models of this GRN, using key experimental data obtained in vivo to aid parameter fitting. Mathematical analysis will then allow us to assess the effects of intrinsic and extrinsic noise. This new approach will be widely applicable to understanding fate choice in other stem cell and developmental systems. The detailed understanding we will generate has important implications for lifelong health as the destabilisation of differentiation is strongly linked to aging and cancer.

This research will contribute directly to the BBSRC's priority areas, including in the short term forming a pioneering in vivo exemplar of the BBSRC's priority areas Systems approach to biological research and Technology development for bioscience. In the medium to long-term, potential healthcare benefits (including improved diagnosis/personalised treatment) resulting from better understanding of basic biological processes are likely to contribute to Aging research and Economic and social impact. Finally, by developing quantitative models of differentiation, we expect to contribute to the priority of 3Rs in research using animals.

Due to its fundamental nature, this research is unlikely in the short term to have major direct benefits to human health or to the UK economy. However, it will be important for developing new techniques for systems biology of vertebrates, for developing in silico models of a medically-important cell-type, the melanocyte, and for understanding a highly medically-relevant process, fate choice in multipotent stem cells. The broader importance of our research lies principally in its interdisciplinary nature, exploring capabilities and limitations of in silico modelling in development. Thus, the most immediate impact will be via transfer of knowledge to other researchers. The most direct beneficiaries will be academic researchers in the zebrafish development and genetics, pigment cell biology, mathematical biology, biological physics and systems biology fields. Researchers in the commercial private sector, including research charities (e.g. CRUK) and the pharmaceuticals/regenerative medicine communities (e.g. Pfizer) will benefit from better understanding gene function in pigment cell development, phenotypic information, much expanded gene regulatory networks (GRNs), methodological advances regarding GRN development and testing and the use of dynamical systems and stochastic processes in development, as well as through secondary use of our data. This will have impact far beyond the immediate biological significance of our research. By reaching these groups of academic and biotechnology researchers, we will influence the quality of life of the UK public, by providing basic research informing our understanding of ageing and disease, and allowing safe and effective use of stem cells.

Policy-makers, including National Centre for 3Rs Research, will benefit in the longer term from developing improved methods for modelling in vivo GRNs; as these models become more sophisticated and quantitative, this will in time help to reduce the numbers of animals used in research.

In the commercial private sector, the data and models generated will be important to the pharmaceutical industry and research charities working on pigmentation disorders and melanoma. Our contribution will be indirect, by showing the value of the interdisciplinary approach we are pioneering, and also direct, towards understanding healthy melanocyte function. This research is vital to our better understanding of abnormal function and to the development of therapies against diseases such as melanoma and Waardenburg syndrome.

Within the public sector, and for the public themselves, our work will contribute to the public understanding of science, especially since pigment cell biology is so 'visual', and thus of interest to organisations such as the Bath Royal Literary and Scientific Institution. Our work could be used to explain the concepts of systems and mathematical biology, and differentiation in health and disease. Because of the relevance to melanoma, this topic could be of considerable interest to the public.

This project will have high impact on PDRA Training, in its combination and integration of innovative techniques in experimental in vivo biology and mathematical modelling. As such, the two PDRAs will obtain a superb training in this increasingly attractive area, making them highly employable in academe or in industry.