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

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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.