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

人体起源于一个单细胞，受精卵，在最初的几周内发育成一个胚胎，具有可识别的特征，如头部，眼睛，大脑和四肢。这种复杂性是如何从相同的细胞中产生的，以及它是如何几乎每次都可靠地工作的？具有共同历史的细胞是如何分离并变得多样化的？完整的细胞谱系树仅适用于蠕虫等简单生物，其身体由不到1000个细胞组成。虽然现在可以在脊椎动物的早期发育过程中跟踪许多细胞，从而跟踪它们的谱系，但将它们的体内行为与它们的分子组成相关联以评估特定基因如何影响细胞命运选择是具有挑战性的。因此，许多在体内控制细胞命运决定的基本原理仍然知之甚少。在这里，我们结合联合收割机新的可用技术，揭示在脊椎动物神经系统中控制细胞谱系决定的分子机制。在早期发育过程中，形成中枢和外周神经系统（CNS和PNS）的细胞来自共同的祖细胞。在产生CNS和PNS的第一步中，这些祖细胞被细分为神经板（CNS）和神经嵴和基板细胞（PNS）。虽然诱导这些命运的一些信号已经确定，但整合信号和实施命运决定的机制在很大程度上是未知的。在这里，我们将使用小鸡来建立多能祖细胞如何致力于特定的CNS和PNS细胞类型的层次结构，并将其与体内细胞行为联系起来。首先，我们将使用一种新技术（单细胞RNA测序）探索祖细胞成为神经细胞、神经嵴细胞、基板细胞和表皮细胞时个体细胞的分子差异。使用计算工具，我们可以评估有多少不同的细胞类型存在，因为不同的谱系出现，并将细胞分为不同的类别。这将提供一个细胞类型和基因的目录，从而使我们能够预测的机制，规则的命运决定。第二，我们将使用单细胞测序数据重建谱系树使用先进的，新开发的生物信息学工具。这种方法在“伪时间”中组织细胞，预测细胞命运决定的顺序和模式。这将使我们不仅能够构建一个树，而且还可以预测占据树的分支点周围的特殊位置的基因。这些可能只是特定谱系的标记，控制命运选择的因素或两者兼而有之。我们将使用新开发的检测单细胞分辨率表达的方法来验证它们在胚胎中的表达。这将使我们能够将单个细胞的转录谱与它们在胚胎中的位置相关联。第三，我们将利用现有的数据来识别基因组中控制分支点周围基因表达的区域。我们将使用这些区域来驱动表达这些基因的相同细胞中的荧光蛋白。这将使我们能够通过延时成像跟踪活胚胎中的这些细胞，从而观察细胞命运的决定。因为我们使用单个细胞的遗传标记，我们可以将细胞的分子组成与它们的位置和行为联系起来。最后，我们将评估在分支点周围表达的基因是否通过操纵它们的表达在控制细胞命运决定中发挥积极作用。总之，这些实验将提供对神经系统祖细胞分子性质的深入理解，重建导致神经、神经嵴和基板细胞分离的决定，并将其与体内细胞行为联系起来。因此，该项目的力量在于在体内系统中的单细胞水平上结合分子和活体成像技术。

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.