Molecular control of fate decisions: reconstructing neural, neural crest and placode cell lineages

The human body originates from a single cell, the fertilised egg, which over the first few weeks develops into an embryo with recognisable features like the head, eyes, brain and limbs. How is such complexity generated from identical cells and how does this work reliably almost every single time? How do cells that share a common history segregate and become diversify? Complete cell lineage trees are only available for simple organisms like worms, whose body is composed of less than 1000 cells. Although it is now possible to follow many cells in vertebrates during early development, and hence to follow their lineage, it is challenging to correlate their behaviour in vivo with their molecular makeup to assess how specific genes influence cell fate choices. Thus, many of the fundamental principles that control cell fate decisions in vivo remain poorly understood. Here we combine newly available technology to uncover the molecular mechanisms that govern cell lineage decisions in the vertebrate nervous system.
During early development, cells that form the central and peripheral nervous system (CNS and PNS) arise from common progenitors. In the first step to generate CNS and PNS these progenitors are subdivided into neural plate (CNS) and neural crest and placode cells (PNS). While some of the signals that induce these fates have been established, the mechanisms that integrate the signals and implement fate decisions are largely unknown. Here we will use the chick to establish the hierarchy of how multipotent progenitors become committed to specific CNS and PNS cell types and link this to cell behaviour in vivo.
First, we will explore the molecular differences of individual cells as progenitors become neural, neural crest, placode and epidermal cells using a novel technology (single cell RNAsequencing). Using computational tools, we can then assess how many different cell types are present as the different lineages emerge and group cells into different classes. This will provide a catalogue of cell types and the genes that characterise them, and thus allow us to predict the mechanisms that rule fate decisions.
Second, we will use the single cell sequencing data to reconstruct a lineage tree using sophisticated, newly developed bioinformatics tools. This approach organises cells in 'pseudo-time', predicting the order and mode in which cell fate decisions are made. This will allow us not only to construct a tree, but also to predict genes that occupy special positions around branch points of the tree. These may simply be markers for a specific lineage, factors that control fate choice or both. We will verify their expression in the embryo using newly developed methods that detect expression at single cell resolution. This will allow us to correlate the transcriptional profile of individual cells with their location in the embryo.
Third, we will use our existing data to identify regions in the genome that control the expression of genes surrounding branch points. We will use these regions to drive fluorescent proteins in the same cells that express these genes. This will enable us to follow these cells in the living embryo by time lapse imaging and thus observe cell fate decisions as they happen. Because we use a genetic label of individual cells we can then correlate the molecular makeup of cells with their position and behaviour.
Finally, we will assess whether genes expressed around branch points play an active role in controlling cell fate decisions by manipulating their expression.
Together, these experiments will provide deep understanding of the molecular nature of nervous system progenitors, reconstruct the decisions that lead to the segregation of neural, neural crest and placode cells and relate this to cell behaviour in vivo. Thus, the power of the project lies in the combination of molecular and live imaging techniques at the level of single cells in an in vivo system.

This project will characterise progenitors for the neural plate, neural crest, placode and epidermal cells and unravel the molecular cascade leading to their segregation. It will establish the molecular heterogeneity of these progenitors, how their transcriptional profile changes as they become committed and how gene expression in progenitors relates to cell behaviour in vivo. The power of this project lies in the combination of state-of-the-art molecular and in vivo approaches to reconstruct the lineage tree for central and peripheral nervous system progenitors.

Specifically, we will:

- Use single cell RNA to determine the transcriptome of progenitor cells and of neural, neural crest, placode and epidermal precursors. This will classify cells into different groups and determine how these change over time
- Use bioinformatics algorithms to reconstruct fate decision trees by organising cells in pseudo-time. This will generate cell trajectories from progenitor to definitive neural, neural crest, placode and epidermal cells
- Use this tree to define genes expressed around branch point that may represent fate markers or even fate determinants
- Verify the expression of these genes using hybridisation chain reaction in situ detection at single cell resolution
- Use our existing data that identified enhancers for progenitor, neural, neural crest and placode genes to identify enhancers for genes surrounding branch points
- Drive fluorescent proteins using these enhancers for live imaging to follow cells with a specific transcriptional profile, monitor their behaviour and assess their ultimate fate
- Assess whether genes expressed around branch points play a role in fate decisions.

The proposed project is multidisciplinary combining biology, imaging, molecular and computational approaches and addresses the fundamental question of how cells acquire their unique fate to build functional organs during development. It cuts across several BBSRC priority areas e.g. data driven biology, replacement, refinement and reduction in research using animals, systems approaches to biosciences and technology development in biosciences, as well as in the long term healthy ageing.

There are various academic beneficiaries as the project addresses a basic biology question of how multipotent ectodermal progenitors generate precursors for the central and peripheral nervous system. These include researchers in the field of neuroscience, developmental, stem cell and systems biology, regenerative medicine, and tissue engineering. In addition, the project will benefit clinical research involved for example in developing cell replacement strategies or in re-activation of endogenous stem cells. We have summarised above how these different communities will benefit from our research.

Our data will be published in scientific journals, at conferences and through teaching and outreach events, with all genomics, imaging and experimental data being made publicly available. Therefore, these benefits will occur during the course of the project or shortly thereafter. In the long term, the project will contribute to strategies to manipulate cell fate in vitro and in vivo by identifying crucial genes involved as well as by elucidating general principles. In turn, this will be beneficial to regenerative medicine and clinical approaches.

The project also has benefits beyond academia, although they may take longer to bear fruit. In particular, these will include
- Training of highly skilled researchers in interdisciplinary research; this training will not only equip the PDRAs with skills for a career in science, but also with many transferable skills such as organisation, critical thinking, problem solving, modelling complex scenarios, cross-disciplinary interactions and many more. This will therefore contribute to strengthening the UK economy by providing highly skilled personnel for the academic or private sector
- Enhancing the international reputation of UK science will increase international collaborations including associated funding.
- Enthusing young people to take up a career in science; our outreach activities specifically target young individuals as future talents (school pupils in short lab projects, school visits through the German scientist association, KCL Science Gallery targeting the local young population, Crick chats for the general public). This will support the UKs ambition for strong science underpinning growth of the economy, entrepreneurial activities and industrial development. The focus on computational approaches will contribute to alleviating current shortage in bioinformatics skills by attracting new talent.
- Benefits for animal and human health through impact on regeneration and repair. There are many potential applications including the identification of crucial factors to re-programme cells or generate patient-specific cells, identifying mechanisms to activate repair with endogenous stem cells, grow and manipulate cell fates in vitro, using such cells to develop new drugs or treatments and many more.