The encoding and interpretation of FGF signals in mammalian cell fate choice

Lead Research Organisation: University of Cambridge
Department Name: Genetics

Abstract

During the development of an embryo, cells become different from each other and do so in a manner that is highly regulated in time and in space. This process of diversification is regulated by the coordinated activity of transcription factors inside cells, which determine what the cells become, and by signals emitted and received by cells that allow them to communicate their states with each other. The workings of transcription factors as the masters of cell identity are increasingly well understood: they bind to the DNA and can activate or repress the expression of cohorts of genes that will endow cells with specific functions and identities. Transcription factors act in a combinatorial manner, i.e. some gene expression programs are the result of overlaying the activities of several individual transcription factors. Signals are received by specific receptors and passed on to their targets via a signal transduction pathway in the cell and are used to share information about their state. How signals interact with the activity of transcription factors, remains open to discussion. It could be that an effector of a signalling pathway acts like a transcription factor i.e. that it contributes to some sort of code that defines a unique identity for every cell. Alternatively the effector might have a more permissive role e.g. by opening up a molecular landscape (chromatin structure) for the action of the transcription factors, or as a factor that enhances or weakens the activity of those transcription factors at the level of single cells. Our proposal aims to distinguish amidst these possibilities by focusing on the FGF/MAPK signalling pathway, in two experimental systems that lend themselves to quantitative single cell analysis: the preimplantation mouse embryo and its correlate, the ES cells.

FGF is an extracellular signalling molecule which triggers a signalling pathway that activates a protein kinase, MAPK. The activity of MAPK is known to influence the behaviour of cells, in particular their differentiation into more specialized cell types. FGF/MAPK activity is widespread in developing embryos, and although many different cells receive this activity, the response is different in different cells and at different times. Our project is aimed at understanding the basis for this observation. Signalling through MAPK is very common in normal and pathological conditions and we are therefore certain that our findings will have an impact beyond our experimental system.

To gain a better understanding of the modes of action of FGF/MAPK signalling, we will focus on the earliest cell fate decisions in a mammalian embryo. Before the embryo implants in the uterus, a group of naïve cells, the Inner Cell Mass (ICM), becomes subdivided into two different populations: the Primitive Endoderm (PrE), that will give rise to extraembryonic tissue, and the naïve pluripotent Epiblast (Epi) that will give rise to the embryo. The decision to become PrE depends on a small number of transcription factors and FGF/MAPK signalling. We can reproduce these events in culture using engineered Embryonic Stem (ES) cells in which we can induce the fate decision event by activating a transcription factor and modulate the response by manipulating FGF/MAPK signalling. By focusing on single cells we can learn details of the decision which cannot be gauged from coarser approaches.

An essential part of the problem is to develop reporters for MAPK signalling in single cells and link signalling activity to the transcriptional inputs and outputs of the process. This combination will allow us to obtain quantitative, dynamic data of the process, and inform novel mathematical models of cell decision making.

Technical Summary

Signalling through FGF and MAPK plays a significant role in development, being iteratively used in many different cell fate decisions. While it is well understood how FGF activates MAPK, very little is known about the actual molecular mechanisms through which activated MAPK affects cell fate choice and promotes the execution of different gene expression programs in the context of lineage decisions. The specification of embryonic and extraembryonic cell types during pre-implantation development of the mouse embryo is an ideal test case to address this question. In the inner cell mass of the 32-cell stage embryo, FGF/MAPK signalling together with GATA transcription factors specifies the extraembryonic primitive endoderm at the expense of the embryonic epiblast. In the absence of GATA factors FGF/MAPK signalling promotes the transition of the epiblast from primed to naïve pluripotency. We can recreate these events in culture using ES cell lines carrying doxycycline-inducible GATA transgenes. We will integrate into these cell lines fluorescent reporters for FGF/MAPK signalling and perform live imaging to record signaling dynamics, i.e. the amplitude and/or duration of the signal, with high resolution at the level of single cells. By cell tracking and subsequent immunostainig for differentiation markers we will correlate signalling with lineage decisions. This will be done both in ES cells and embryos. Finally, we will test the hypothesis that lineage-specific transcription factors guide direct effects of MAPK on the transcriptional machinery and the chromatin to effect these lineage decisions. To achieve this, we will make use of our inducible GATA factors and measure the activity state of the transcriptional machinery in the presence and absence of these factors and for different MAPK signalling levels.

Planned Impact

The proposed study is a basic research project on the role that signalling and transcription factors play in cell fate decisions in development and on the molecular mechanisms whereby they implement their function. Very significantly we shall compare differentiation events in cell culture and the embryo and therefore test to what extent the experiments in ES cell culture reflect the situation in vivo. This is not commonly done, but will be increasingly important, as the differentiation of ES cells in culture becomes an important avenue for the generation of tissues for medical purposes and pathological models.
The project aims at a very precise set of important questions about FGF/MAPK signalling which is a very widespread event in normal and pathological conditions. The deepened understanding of the dynamic regulation and modes of action of this signalling pathway will be of interest to scientists that work to interfere with dysregulated signalling in pathological conditions. It is very likely that our research will reveal new, more quantitative, targets for drug development.
The quantitative investigations of the interactions between signalling and transcriptional regulators that we propose in this project will lead to a more rational understanding of the molecular rules that underlie the differentiation of pluripotent cells along different lineages. Given the importance and expectations that lie on Embryonic Stem cells in the emerging field of Regenerative Medicine and the central role that MAPK signalling plays in the biology of these cells, these findings of our research have the potential to influence the use of these cells for the purposes of direct differentiation. In this context, our research will be of interest to clinicians with an active interest in the potential of stem cells for regenerative purposes.
The research programme will, have an impact on the professional development of the Researcher co-investigator who has provided the background to the project and will carry out most of the experimental work. Liaising with collaborators and organizing the project will allow him to develop essential skills in preparation to his career as an independent researcher.

Another significant contribution resides in the novel interdisciplinary and quantitative nature of the approach that we are taking that combines classical genetic and cell biological studies with quantitative cell biology, advanced microscopy and image analysis techniques. This combination, we believe, highlights the path to the future in biological research. Biology has been very qualitative, this project will be part of a movement that ushers an era in which it is quantitative.

As the work will mainly be carried out in a University department, there will be ample opportunity for the researchers to get involved with summer visitors and undergraduate students. The AMA lab for example has participated for 5 years in the Nuffield Sixth form college programme which is very successful. Furthermore, part II projects are regularly being carried out in the participating labs, and we expect that the research proposed here will lead to several of those. Through this, the project will provide a great opportunity to guide the development of the next generation of scientists. Our quantitative and analytical approaches will highlight to them the importance of these skills, and will provide them with a new vision of Biology.

Publications

10 25 50
 
Description A manuscript has been written and published describing FGF/MAPK signalling during mouse development, using the reporters built with this award (PMID: 29964027). The line is being used now in several labs.

Importantly, making use of our in vitro embryonic organoid system we have started to explore the role of FGF/ERK signalling in early gastrulation stages and the early stages of differentiation. This is being done with reporters as well as by, as proposed at the level of single cell gene expression. The grant has also enabled the development of our gastruloid system. Over the last 12 months we have been able to use this system to get some insights into the role of FGF in early fate decisions.
Exploitation Route The embryonic organoid system, to which this award has contribuited and which we and others are starting to use routinely is likely to be a useful and successful replacement (in the context of the 3Rs) for mouse embryos.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://bbsrc.ukri.org/news/health/2018/181011-pr-artificial-embryos-may-reduce-the-need-for-animals-in-research/
 
Description The discovery of the gastruloid system has led to the filing of two patent applications (1815438.5 and 1815439.3) which are being used in interactions with a variety of pharmaceutical companies. These commercial interactions are being done together with Cambridge Enterprise.
First Year Of Impact 2018
Sector Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Cultural,Societal,Economic

 
Description Isaac Newton Trust Research Grant
Amount £23,386 (GBP)
Funding ID 16.24(b) 
Organisation University of Cambridge 
Department Isaac Newton Trust
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 07/2017
 
Title ES cell lines and mice 
Description We have developed a culture system which allows us to use mouse Embryonic Stem Cells (ESC) to generate embryo like structures in vitro that we call gastruloids. We have shown that this system mimics the embryo and that it can be used to study developmental events in great detail which is not possible with embryos. The system fits well the requirement for 3Rs and promises to be a most important tool for the study of mammalian development. One of the advantages of this system is that it bypasses the embryo and that allows the generation and assay of many cell lines. We have built ESC lines bearing reporters for FGF/ERK signalling which, used in this system, are allowing us to explore the role of FGF signalling in peri-implantation development and differentiation. These cell lines are being converted into mice. Recently we have developed a second generation of gastruloids from Epiblast Stem Cells which depend completely on FGF for their generation. 
Type Of Material Cell line 
Year Produced 2016 
Provided To Others? Yes  
Impact This is an important achievement as we have shown that gastruloids faithfuilly recapitulate the events in the mouse embryo between the initiation of gastrulation and the start of organogenesis. The system has the potential to substitute mouse embryos for the study of developmental biology and is starting to be adopted by labs around the world. 
URL https://en.wikipedia.org/wiki/Gastruloid
 
Description Construction and establishment of a reporter mouse line for FGF signallinhg. 
Organisation Memorial Sloan Kettering Cancer Center
Department Developmental Biology Program
Country United States 
Sector Hospitals 
PI Contribution My research team provided the sensors and, through Christian Schroeter, helped characterize the reporter and provided the basic work for the collaboration.
Collaborator Contribution Kat Hadjantonakis laboratory at Sloan Kettering has provided the mouse transgenic facilities to transform ES cell lines bearing reporters that sense FGF/ERK signalling and transforming them into mice. They have also performed the initial characterization of the mice by looking at the activity of the reporter in conditions of gain and loss of FGF signalling.
Impact There is a joint manuscript about to be submitted for publication describing the mouse and making it available to the community.
Start Year 2015
 
Description FGF signalling dynamics in ES cells 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Physiology
Country Germany 
Sector Public 
PI Contribution The PI in MPI Dortmund was the postdoc initially associated with the grant. We are keeping some of his projects going as a collaboration that was initiated with this grant
Collaborator Contribution In MPI Dortmund, there are sophisticated quantitative methods which allow the measurement of FGF/ERK signalling
Impact Christian Schroeter provided elements and results for a manuscript, about to be submitted, characterizing the reporter as well as a mouse line.
Start Year 2016
 
Description Physical analysis of symmetry breaking 
Organisation Aix-Marseille University
Department Institute of Developmental Biology of Marseille Luminy (IBDM)
Country France 
Sector Academic/University 
PI Contribution We have provided the experimental system and results
Collaborator Contribution Lenne spent a sabbatical in my lab where he learnt the experimental system. He is now back in Marseille and we continue the collaboration we initiated during his stay here. He is making significant contributions to the analysis and modelling of the experimental data
Impact For the moment, only joint grant applications
Start Year 2017
 
Description Role of extracellular environment in symmetry breaking 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Department Laboratory of Stem Cell Bioengineering
Country Switzerland 
Sector Academic/University 
PI Contribution We have provided the basic experimental system
Collaborator Contribution They are providing their engineering abilities and materials.
Impact The collaboration is interdisciplinary as it involves building bridges between Engineering, Physics and Biology. It also is leading to some patents and the possibility of a start up.
Start Year 2015
 
Title Human polarized three dimensional cellular aggregates 
Description Aggregates of defined numbers of human ES cells which, under defined culture conditions undergo gastrulation like movements and generate a mammalian body plan 
IP Reference GB1815439.3 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact None yet beyond discussions following some commercial interest
 
Title Polarized three dimensional cellular aggregates 
Description Aggregates of defined numbers of mouse ES cells which, under defined culture conditions undergo gastrulation like movements and generate a mammalian body plan. 
IP Reference GB1815438.5 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact There is interest from some pharmaceutical and stem cell companies