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

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Although COVID has had a significant impact on our work (through furlough of key experimental workers during phases of national lockdown, and through delays in returning to work caused by national and local COVID measures), we have made significant and important progress. Our key research question is to understand how a set of progenitor cells (neural crest cells - a key paradigm for understanding how progenitor and stem cells work) become assigned to specific cell-types. In particular, we wished to test the feasibility and validity of our novel Cyclical Fate Restriction (CFR) paradigm. To date we have shown that the progenitor cells show combinations of gene expression at early differentiation stages that are surprising under the old models, but more consistent with our CFR model. Importantly, we have developed a theoretical framework for the CFR idea, which shows some very interesting predictions of gene expression in cells, which we think can be related directly to observations of neural crest cell development. In addition, recent publications from studies in mouse, which focus on describing the process of differentiation, appear compatible with our view, suggesting that the principles we have identified may be widely applicable.
Exploitation Route It is too early to say, but we have noted that a published model of neuron development in the spinal cord shares features of the model we have studied in a neural crest context, so we hope that some of our insights may well apply more widely. At the Stem Cell Fate Choice workshop that we organised, there was considerable discussion around our model and how this related to those of others in the field.
Sectors Healthcare

Other

URL https://www.bath.ac.uk/announcements/developing-cells-likely-can-change-their-mind-about-their-destiny/
 
Title SevaVigg/NanostringDanioNCCscAnalysis: Analysis of Nanostring and Taqman data of developing neural crest single cell RNA profiling in Danio rerio 
Description Analysis process for data in Subkhankulova et al., 2023, Nature Comms 
Type Of Material Data analysis technique 
Year Produced 2023 
Provided To Others? Yes  
Impact Contributed to publication 
URL https://zenodo.org/records/7585731
 
Title SevaVigg/NanostringDanioNCCscAnalysis: Analysis of Nanostring and Taqman data of developing neural crest single cell RNA profiling in Danio rerio 
Description Includes datasets for study published as Subkhankulova et al., 2023, Nature Comms. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Supported acceptance of publication 
URL https://zenodo.org/records/7585731
 
Title Zebrafish pigment cells develop directly from persistent highly multipotent progenitors 
Description Nanostring expression profile of zebrafish neural crest single cell dataset as published initially as a preprint: Nikaido et al., 2021 https://www.biorxiv.org/content/10.1101/2021.06.17.448805v1 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Has led so far to preprint and now published in Nature Comms. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE185592
 
Description Andrea Rocco 
Organisation University of Surrey
Country United Kingdom 
Sector Academic/University 
PI Contribution Rocco is our mathematical modelling partner, co-funded by BBSRC; we are generating data that he uses for modelling
Collaborator Contribution Mathematical modelling of Gene Regulatory Network is underway
Impact Greenhill et al, 2011 PLoS Genetics Vibert et al, submitted Multidisciplinary - Maths and Genetics
Start Year 2010
 
Description Natascia Tiso 
Organisation University of Padova
Country Italy 
Sector Academic/University 
PI Contribution We have investigated baz1 mutants
Collaborator Contribution COntributed studies of delta, jagged gene expression to help validate a model
Impact Delfino-Machín, M., Nikaido, M., Zhang, D., Law, E.W.P., Colanesi, S., Tiso, N., Blader, P. and Kelsh, R.N. An imbalance of Sox10-dependent activation and Notch-dependent repression of ngn1 underlies the neurogenic phenotype in a novel sox10 mutant allele. In prep.
Start Year 2010
 
Description Vsevelod Makeev 
Organisation Vavilod Institute of General Genetics
Country Russian Federation 
Sector Academic/University 
PI Contribution We have produced and performed a preliminary analysis of single cell transcriptomic data, which resulted in an unexpected but exciting finding
Collaborator Contribution Dr Makeev's team of bioinformaticians are exploring this data, to assess the validity of our initial interpretation
Impact Subkhankulova et al, in prep.
Start Year 2016
 
Title NanostringDanioNCCscAnalysis 
Description This repository contains software and two datasets of developing neural crest single cell expression profiling of Danio rerio. Expression profiling was performed in the lab of Prof. Robert Kelsh (Univ. of Bath, https://researchportal.bath.ac.uk/en/persons/robert-kelsh) with the help of the Nanostring nCounter (R) for the inhouse panel of 45 genes (main portion of files in the SourceData folder) and with the TaqMan assay (tables in the Taqman subfolder of the SourceData folder) The code in the R directory conducts the analysis and plots Figures for the manuscript accepted to Nature Communications. Title: Zebrafish pigment cells develop directly from highly multipotent progenitors. Authors: Tatiana Subkhankulova[1], Karen Camargo Sosa[1], Leonid A. Uroshlev[2], Masataka Nikaido[1,a], Noah Shriever[1], Artem S. Kasianov[2,3,4], Xueyan Yang[1,b], Frederico S. L. M. Rodrigues[1], Thomas J. Carney[1,c], Gemma Bavister[1], Hartmut Schwetlick[5], Jonathan H.P. Dawes[5], Andrea Rocco[6,7], Vsevolod Makeev[2,3,8] and Robert N. Kelsh[*,1] 
Type Of Technology Software 
Year Produced 2023 
Open Source License? Yes  
Impact Too early to say 
URL https://www.nature.com/articles/s41467-023-36876-4
 
Description A Keynote talk at 11th Curie-Paris Sud International Graduate Course on "Development and Cancer" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Keynote lecture, 11th Curie-Paris Sud International Graduate Course on "Development and Cancer", Paris-Sud/online, Nov 2021 to audience of PGR, undergarduate students and academics. Meeting was in mixed format, and due to emergence of COVID Omicron variant I was at last minute unable to attend in person, instead giving my seminar online; unfortunately, this limited the interactions possible.
Year(s) Of Engagement Activity 2021
URL https://training.institut-curie.org/courses/development-cancer-7
 
Description Applied and Interdisciplinary Mathematics Seminar Series, Department of Mathematical Sciences, University of Bath, presented by Jon Dawes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Seminar given by Jon Dawes to mixed academic audience, inc. PGR and PDRAs, as part of the main departmental applied and interdisciplinary mathematics (AIMS) seminar series which takes place every week during teaching semesters. The overall purpose of the seminar series is to disseminate recent research results and enhance the training experience of PhD students and post-docs. There were audience questions during the seminar and a follow-up question by email. There was positive impact on the audience, inspiring them to start or continue interdisciplinary work, as evidenced by the comment, from a mathematician, received by email: "It was great to see your work just now - I really liked it! I was pleasantly surprised that I could just about follow the biology".
Year(s) Of Engagement Activity 2022
URL https://www.bath.ac.uk/corporate-information/applied-and-interdisciplinary-mathematics-seminars-cent...
 
Description Invited Talk - Ringberg Symposium: Advances in cell and developmental genetics, pattern formation and evolution, Ringberg, Germany, Oct 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A focused meeting organised by Nobel Laureate, with selected audience of well-known developmental biologists from around the world. Ideas received with interest, based upon personal feedback afterwards.
Year(s) Of Engagement Activity 2022
 
Description Invited seminar - Gur group, Weizmann Institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact PDRA invited to speak about the project to the Gur Group. Ideas presented were greeted with interest, based on personal feedback
Year(s) Of Engagement Activity 2022
 
Description Invited seminar - Keynote talk at 12th Curie-Paris Sud International Graduate Course on "Development and Cancer", Nov 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I presented the opening lecture on this course, and remained for the duration. Informal feedback from participants and other lecturers showed significant interest in the new model we've developed
Year(s) Of Engagement Activity 2022
URL https://training.institut-curie.org/courses/development-cancer-9
 
Description Invited talk - Gordon Research Conference 'Neural Crest and Placodes', Italy Feb 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation in session on stem cells, to very focused audience within the field. Talk very well-received, with multiple members of audience reporting how they liked the model we are proposing. One audience member commented 'Most stimulating talk of meeting; I was writing notes'!
Year(s) Of Engagement Activity 2023
URL https://www.grc.org/neural-crest-and-cranial-placodes-conference/2023/
 
Description Invited talk - Joint Research Day, Bath, UK, Jan 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact An invited talk to departmental audience, presenting new ideas. Direct oral feedback indicated growing understanding of new model and impact on audience.
Year(s) Of Engagement Activity 2022
 
Description Invited talk at Neural Crest and Placodes Gordon Research Conference 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact My seminar triggered much discussion, and directly led to plans for collaboration with scientists from Sweden and Switzerland. We are currently working on grant applications to fund our proposed work.
Year(s) Of Engagement Activity 2023
URL https://www.grc.org/neural-crest-and-cranial-placodes-conference/2023/
 
Description Invited talk at Research Day, Department of Life Sciences, University of Bath 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Oral presentation (PDRA). Cyclical Fate Restriction, a new model for neural crest cell fate specification. Research Day, Department of Life Sciences. University of Bath. UK. February 2023. c 100 people attended; interest in ideas presented, based upon oral feedback.
Year(s) Of Engagement Activity 2022
 
Description Myron Gordon Award Plenary Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was awarded the Myron Gordon Award by the International Federation of Pigment Cell Societies, and gave an award talk at the International Pigment Cell Conference
Year(s) Of Engagement Activity 2023
URL https://www.ipccbilbao2023.org/en/awards
 
Description Poster presentation at BSDB/BSCB Spring Meeting, Warwick 2022, presented by Saeed Farjami 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Poster presentation
Year(s) Of Engagement Activity 2022
URL https://registrations.hg3conferences.co.uk/hg3/frontend/reg/thome.csp?pageID=75504&eventID=208&trace...
 
Description Seelcted talk at 17th International Zebrafish Conference, Montreal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Oral presentation at 17th International Zebrafish Conference. Looking again at neural crest cell ate restriction: low-level co-expression of fate-specification factors reveals retained broad multipotency of migrating neural crest cells in vivo. Large international conference, with audience c 200+. Growing interest in ideas presented, base don oral feedback from other participants.
Year(s) Of Engagement Activity 2022
URL https://www.izfs.org/education/past-meetings/17izfc
 
Description Selected Talk - 24th European Society for Pigment Cell Research Conference, online, Nov 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A selected abstract, but online format made evaluation of impact difficult. Nevertheless, postive feedback from one individual indicates some effect on growing awareness of our work.
Year(s) Of Engagement Activity 2022
URL http://www.espcr.org/index32.php
 
Description Selected talk at British Applied Mathematics Colloquium Apr 2022, presented by Saeed Farjami 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk to be presented at this conference.
Year(s) Of Engagement Activity 2022
URL https://bamc2022.lboro.ac.uk/
 
Description Selected talk at IOP Conference Physics Meets Biology 2021, presented by Saeed Farjami 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk presented as part of online conference. Audience c 15, generated considerable interest in our approach to understanding stem cell biology
Year(s) Of Engagement Activity 2021
URL http://pmb2021.iopconfs.org/Home
 
Description Stem Cell Fate Choice: Mechanisms and Modeling Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Jon Dawes and I organised a workshop with 8 invited speakers (from UK (5), USA, Sweden/Austria, Spain), and an audience of c 50. This focused meeting generated a lot of interest, with active engagement from the audience and speakers
Year(s) Of Engagement Activity 2023
URL https://www.bath.ac.uk/events/stem-cell-fate-choice-mechanisms-and-modelling/