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

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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.