Rethinking the neural crest - a novel dynamic hypothesis of neural crest fate restriction

All cell types in the body 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 (e.g. neurons, blood or skin cells). A major problem in developmental biology is to understand how these flexible precursors make a specific choice of cell-type to adopt. The scale of the problem is illustrated by the fact that for one key exemplar, neural crest stem cells, there is still uncertainty about how the process works even after four decades of research - do fully multipotent cells 'jump' straight to a specific chosen fate, or do they go through a series of steps in which their options become more and more limited, until eventually they choose a single cell-type? These two models - Direct Fate Restriction (DFR) and Progressive Fate Restriction (PFR) - have each received support from different studies, but are conflicting. Although PFR is now the textbook view of neural crest development, a prominent paper studying mouse neural crest recently concluded firmly with a DFR interpretation.

As a result of work done on an ongoing BBSRC grant studying neural crest stem cells in zebrafish embryos, we are proposing a revolutionary new view, which we believe reconciles these conflicts. We have been looking at the formation of pigment cell-types from the neural crest, as a model of neural crest development in general. Specifically, we have been looking at melanocytes (black pigment cells, well-known for their roles in skin and hair colour in humans, and giving rise to melanoma), and iridophores, a shiny silver cell-type that is prominent in most fishes. We see evidence for only some very broadly multipotent precursors, leading us to propose our novel Cyclical Fate Restriction model. We think that neural crest precursors are variable because they are highly dynamic, constantly changing. This view is consistent with, and reconciles, the conflicting data and interpretations in the field. Increasingly, stem cell biology is being explored using a mathematical modelling approach which has often given key insights into how they function. Surprisingly, perhaps, almost all this work has focused on 'binary choices', and so has ignored the possibilities of a DFR-type process. Even for PFR, modelling has not explored how binary choices might be interlinked to generate multiple diverse derivatives.

In this project, we will test and explore our new Cyclical Fate Restriction model, using experimental studies and mathematical modelling to gain insight into how the process might work. A key experiment is to use a complementary technique to look at gene activity in thousands of neural crest cells, looking comprehensively at their cell-profiles so as to study the range of identifiable precursor states in the neural crest. We will then use a sensitive technique to look at such cell-types directly in the embryo. In parallel we will explore the mathematical basis for the three models, developing current models to describe PFR, and applying novel theoretical insights to stem cell biology to investigate the plausibility of both DFR and our novel Cyclical model. We will integrate the two approaches, experimentally investigating biological features relevant to the models, including direct assessment of the direction of change of progenitor cells, and quantitative investigation of the key fate specification signals in the neural crest.

Together, these studies will test a revolutionary view of neural crest stem cell biology. 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 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.

Despite decades of modern research, a fundamental question remains unresolved: how does a fully multipotent neural crest (stem) cell generate each of the diverse derivatives? Since the end of the last century the neural crest field has been divided into two intellectual camps, one favouring a Direct Fate Restriction mechanism, the other a Progressive Fate Restriction mechanism. Based on an unexpected finding in our analysis of single cell expression profiles, we propose a new, Cyclical Fate Restriction hypothesis, which resolves the conflict and reconciles the two viewpoints. We suggest that intermediate progenitors are more multipotent than previously thought, but also highly dynamic in vivo. Hence, highly multipotent progenitor states exist in a quasi-stable state, but cycling through a 'cloud' of transient cell states biased towards different fates. As a result, snapshot examination of marker expression reveals heterogeneity, while lineage labeling may show high diversity of fates within large cell clones. Our focus in this project is on testing this revolutionary view of neural crest development by using an interdisciplinary approach. Mathematical modelling has been influential in understanding stem cell development, but theory currently neglects Direct and Cyclical Fate Restriction models. Using diverse experimental and modelling approaches, we will develop a comprehensive picture of neural crest cell heterogeneity in vivo, establish a detailed mathematical framework based on Dynamic Systems Theory for interpreting this heterogeneity in the light of all models, and quantitate key fate specification signals experienced by neural crest cells. We will also re-examine the key experimental data underpinning the Progressive Fate Restriction model. Together, these studies will test our revolutionary new hypothesis of this key stem cell, with implications for stem cell biology and its applications reaching well-beyond the basic biology studied here.

This research will contribute directly to the BBSRC's priority areas, including the strategic priority areas of Data driven biology, Systems approaches to the biosciences 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 will contribute to Healthy ageing across the lifecourse priority. We note also our continued international collaboration with Dr V. Makeev (Vavilov Institute of General Genetics, Moscow), extending our Royal Society-funded collaboration (ends Feb 2019), so that we also contribute to International Partnerships.

Academic impact
Due to its fundamental nature, the major direct benefits to human health or to the UK economy are longer term. In the shorter term this research will be important to develop new techniques for systems biology of vertebrates, by building in silico developmental models to understand a highly medically-relevant process, fate choice in multipotent stem cells. Our work's broader importance lies principally in its interdisciplinary nature, exploring new mathematical modelling approaches in stem cell development. Thus, the most immediate impact will be via transfer of knowledge to other researchers. The most direct beneficiaries will be academic researchers in development, stem cell biology, pigment cell biology, mathematical biology, biological physics and systems biology.

Economic and societal impact
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 of stem cell biology, both in general and in pigment cell development, through methodological advances in modelling of fate specification processes and 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.

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 and other neurocristopathies. Our contribution will be indirect, by showing the value of the interdisciplinary approach we are pioneering, and also direct, towards understanding healthy pigment cell function and their stem cell origins (important, for example, since melanoma development is often viewed, in part, as a dedifferentiation of melanocytes to a more proliferative stem cell-like state). 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, so that patients will also be beneficiaries in the longer term.

Within the public sector, and for the public themselves, our work will contribute to the public understanding of science. Pigment cell biology is so 'visual', and thus of interest to organisations such as the Bath Royal Literary and Scientific Institution. At Surrey we will engage with secondary schools and colleges to promote the progression of students on to higher education. 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 is of considerable interest to the public.

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