High resolution profiling of neuronal lineages by functional characterisation and sequencing of barcoded RNA

谱系追踪是生物学研究的基本方面，研究分裂细胞如何产生许多不同类型的成熟细胞。这对于理解生物组织的发育和功能是至关重要的。它对于理解疾病过程也至关重要，因为许多疾病涉及细胞的异常产生或特定细胞类型的损失。谱系追踪提供的信息解释了每种不同类型的细胞在哪里出生，这些细胞如何移动到它们的最终位置，以及它们如何相互作用以产生一个功能组织。例如，在神经系统中，科学家们使用谱系追踪来了解分裂的“祖”细胞如何产生成人大脑中发现的许多不同类型的神经细胞。我们也刚刚开始了解祖细胞对其产生的神经细胞有多大的影响。例如，最近的研究表明，甚至一个神经细胞与其他神经细胞形成特定连接的方式，也可能与它诞生的祖细胞有关。尽管它对理解生物过程很重要，但科学家们在确定哪些细胞来自分裂的祖细胞方面的工具有限。最具结论性的方法是用一串遗传物质制成的独特标签来标记同一谱系的成员，这些遗传物质中的核苷酸“字母”序列是独一无二的。这被称为“DNA条形码”，因为它使用独特的标签，类似于条形码用于识别超市中不同产品的方式。然而，一系列问题限制了该方法的应用。首先，恢复DNA条形码的效率非常低。其次，DNA条形码是在固定组织上进行的，这意味着不可能在细胞活着的时候研究细胞的重要特性。为了解决这个问题，我们开发了一种新的方法，它与一种叫做RNA的不同类型的遗传物质一起工作。在我们的初步实验中，我们设计了一种RNA条形码方法，在每个细胞中产生许多相同条形码的副本，并表明这将条形码序列的读取成功率提高到几乎100%。此外，由于RNA条形码可以在活细胞中进行，这种方法为将我们的方法与研究活神经细胞的技术相结合提供了令人兴奋的新机会。例如，我们可以将我们的方法与记录神经细胞电活动和详细三维形状的技术结合起来。也许最重要的是，我们的方法允许我们使用强大的新遗传方法来测量我们研究的每个细胞中数千种不同基因的水平。这提供了一种非常丰富的方法来对神经细胞的确切类型进行分类，并提供了对祖细胞如何影响其产生的细胞的非常具体方面的见解。在这个项目中，我们将通过评估其在谱系追踪小鼠皮质细胞和来自多能干细胞的人类皮质细胞方面的效用来研究我们的新方法的潜力。证明我们的方法在这些不同的系统中有效，将表明它是一种重要的方法，可以为多个科学领域做出新的贡献。我们还将使用我们的方法来解决重要的生物学问题，包括哪些基因可能导致相关的神经细胞彼此之间更相似。最后，我们建议制作我们的条形码工具的新版本，这将进一步增加应用范围，我们有具体的计划，关于如何使我们的工具可用于科学界。因此，这项工作将产生双重影响，它将促进我们对大脑的生物学理解，同时也为许多领域的科学家提供新技术，包括干细胞生物学和癌症生物学。

Lineage tracing is the fundamental aspect of biological research that examines how dividing cells can give rise to many different types of mature cell. This is crucial for understanding how biological tissue develops and functions. It is also crucial for understanding disease processes, because many diseases involve the abnormal production of cells or the loss of a particular cell type. Lineage tracing provides the information that explains where each of the different cell types are born, how these cells move to their final position and how they interact with one another to produce a functional piece of tissue. In the nervous system for example, scientists use lineage tracing to understand how dividing 'progenitor' cells give rise to the many different types of nerve cells found in the adult brain. We are also just beginning to understand how much influence a progenitor can have upon the nerve cells that it produces. For example, recent work has suggested that even the way that a nerve cell forms its particular connections with other nerve cells can be linked to the progenitor from which it was born.Despite its importance for understanding biological processes, scientists have had limited tools for working out which cells come from a dividing progenitor cell. The most conclusive method has involved labelling members of the same lineage with a unique tag made from a string of genetic material in which the sequence of nucleotide 'letters' are unique. This has been referred to as 'DNA barcoding', as it uses unique labels, similar to the way that barcodes are used to identify different products in the supermarket. However, a series of issues have limited the application of this method. First, recovering the DNA barcode has proved to be very inefficient. Second, DNA barcoding has been performed on fixed tissue, which has meant that it has not been possible to study important properties of the cells whilst they are alive.To address this problem we have developed a new approach that works with a different type of genetic material, called RNA. In our preliminary experiments we have designed an RNA barcoding approach that produces many copies of the same barcode in each cell and have shown that this increases the success of reading the barcode sequence to practically 100%. Furthermore, since RNA barcoding can be performed in live cells, this approach offers exciting new opportunities to combine our method with techniques for studying live nerve cells. For example, we can combine our method with techniques for recording the electrical activity and detailed three-dimensional shape of the nerve cell. Perhaps most importantly, our approach allows us to use powerful new genetic methods to measure the levels of thousands of different genes in each cell that we study. This provides a very rich way of classifying the exact type of nerve cell, as well as offering an insight into how a progenitor cell can influence very specific aspects of the cells it produces.In this project we will investigate the potential of our new approach by assessing its utility for lineage tracing mouse cortical cells and also human cortical cells derived from pluripotent stem cells. Demonstrating that our method works in such different systems will show that it is an important method and can make new contributions to multiple areas of science. We will also use our method to address important biological questions, including what genes might cause related nerve cells to be more similar to one another. Finally, we are proposing to make new versions of our barcoding tool that will further increase the range of applications and we have specific plans about how we can make our tools available to the scientific community. The work will therefore have dual impact in the sense that it will advance our biological understanding of the brain, whilst also providing new technologies for scientists in many fields, including stem cell biology and cancer biology.