Understanding haematopoietic stem cell development through global single-cell gene expression analysis

Background

The first blood cells in mammals appear in the yolk sac, however haematopoietic stem cells (HSCs) which give rise to the adult haematopoietic system appear inside the embryo body within the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, Cell, 1996). HSCs gradually mature in the AGM region before colonizing the foetal liver and subsequently bone marrow. However, genetic mechanisms underlying HSC development in mammals are poorly understood partly due to in utero development. We therefore established a culture system which replicates HSC development in the AGM region (Taoudi et al., Cell Stem Cell, 2008; Rybtsov et al., Stem Cell Reports, 2014). Using this system, we characterized the hierarchy of HSC precursors by cell surface markers. However, these markers are shared with other blood progenitors, which hampers precise identification of developing HSCs and genes which drive HSC development.

Recent advancement in single-cell transcriptome analysis have transformed our understanding of cell types and lineages. It permits exploration of heterogeneity of cell populations and identification of novel cell sub-types which otherwise remain unrecognized. Using highly parallel barcoding of individual cells and computational tools specifically developed for single-cell data, it is now possible to build detailed developmental/ differentiation trees and identify key candidate genes underpinning HSC development (Macosko et al., Cell, 2015; Mohammed et al., Cell Reports, 2017).

Aims

In this interdisciplinary project, we aim to explore molecular mechanisms underlying embryonic HSC development. The student will explore the transcriptional heterogeneity of the developing haematopoietic system in mouse embryo using system-wide single-cell transcriptome analysis. In addition to wild type, we will study gene expression changes in a key mouse mutant line where HSC development is blocked. This will allow us to dissect the entire gene network supporting HSC development. Single-cell gene expression analysis will be performed using cell barcoding in a microfluidics device. Using bioinformatics methods the student will reconstruct the developmental tree of the developing haematopoietic system and will identify molecular signatures specific to clades of the developing HSC lineage. Candidate genes identified in the molecular signature that could potentially be involved in HSC development will be validated using functional in vitro and in vivo approaches and their expression in the embryo will be characterized using confocal microscopy. Our data-driven approach using cutting-edge single cell methodology in mouse is expected to be highly informative for identifying homologous cellular differentiation pathways in human and will have substantial predictive power for analysing mechanisms underlying human HSC development. This project will fill a crucial gap in our mechanistic understanding of HSC development, which could in future inform improved strategies for manipulating HSCs ex vivo.

Training outcomes

This is a collaborative interdisciplinary project addressing important biological questions at the system-wide level, between the groups of Prof. A. Medvinsky, an expert in embryonic development of mouse and human HSCs and groups of Dr. Chandra, experts in single cell analysis including computational analysis of single-cell data. To enhance the outcome of the project we have established a collaboration with Prof. Bertie Gottgens in Cambridge, a leading expert in the analysis of blood transcriptional networks.

The student will work in a highly collaborative environment and will become proficient in advanced methods of early analysis of embryonic HSC development (embryo manipulations, fluorescence activated cell sorting and analysis, HSC culture, qRT-PCR, confocal microscopy) as well as single-cell analysis and computational biology methods. Importantly, UoE has been awarded a £650K MRC Discovery Award to implement Drop-Seq-like approaches to single cell transcriptomics. This creates an excellent opportunity for the student to become an expert in this rapidly evolving cutting-edge technology. The project will allow the student to obtain important insights into the analysis of complex biological systems and to acquire important interdisciplinary skills in systems biology approaches, which should have a highly positive impact on their future career in science.

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location:

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note, you must apply to one of the projects and you are encouraged to contact the primary supervisor prior to making your application. Additional information on the application process if available from the link above.

For more information about Precision Medicine visit:

http://www.ed.ac.uk/usher/precision-medicine