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Unravelling blood stem cell formation at single cell resolution


   Institute of Cancer and Genomic Sciences

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  Dr R Monteiro, Dr S Ott  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

About the Project

Background

Haematopoietic stem cells (HSCs) are generated during embryonic development and are responsible for the production and maintenance of all the blood lineages throughout adult life. They arise from the haemogenic endothelium (HE), a specialized subset of endothelial cells located in the floor of the main embryonic artery, the dorsal aorta. One of the bottlenecks in the production of HSCs in vitro for substitution therapies is to determine the right conditions that mimic the embryo microenvironment and induce a HE-like intermediate that can differentiate into HSCs. The Monteiro lab is interested in understanding how endothelial and blood stem cells grow and differentiate during embryonic development and how lineage fate decisions lead to the formation of HSCs from the HE. A major player in determining the arterial and haemogenic endothelial fates is the Notch signalling pathway1. For example, the ratio between the levels of Notch ligands Dll4 and Jag1 in endothelial cells is thought to define the arterial vs haemogenic fate: Jag1 determines the haematopoietic fate whereas Dll4 drives the arterial fate2. However, we and others have uncovered evidence suggesting that establishing the (aortic) arterial cell fate is a prerequisite for the formation of HE3,4.

Objectives

We have recently shown that the Notch ligand Dll4 is required for the expression of the critical HE genes, runx13 and gata2b5. In this project, we aim to study the role of the Notch ligand Dll4 in the formation of HE and the subsequent production of HSCs. First, the student will use an existing transgenic line2 to isolate arterial and haemogenic cells to compare gene expression between wildtype and dll4 loss of function by scRNAseq. They will then generate transgenic lines to drive Cre recombinase in specific endothelial and haemogenic populations to perform lineage tracing and identify the haematopoietic cell populations that derive from arterial or haemogenic cells. Characterization of these haematopoietic cells will be accomplished by single-cell transcriptional profiling of the labelled cells.

Methods

The student will train in vertebrate embryology and micromanipulation/microinjections of embryos, and use well-established Tol transgenesis and CRISPR/Cas9 genome editing tools and confocal microscopy. The project requires the acquisition and analysis of single-cell transcriptomics data which the student will develop in close collaboration with the co-supervisor.

This project combines classical developmental biology techniques with lineage tracing, genome editing and cutting-edge single-cell transcriptomics to understand the fundamental biology regulating the formation of HSCs. This will aid ongoing efforts to produce HSCs in vitro.

Person Specification

Applicants should have a strong interest in developmental biology, haematopoiesis or transcriptomics. They should hold or realistically expect to obtain at least an Upper Second Class Honours Degree in Genetics, Biological Sciences or related subjects. Detailed instructions for applicants, academic requirements and eligibility criteria can be found in the University of Birmingham and University of Warwick websites:

https://warwick.ac.uk/mibtp/

https://www.birmingham.ac.uk/research/activity/mibtp/index.aspx

and here: https://warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/application/

How to apply:

Applicants are encouraged to contact Dr Rui Monteiro directly ([Email Address Removed]) to discuss the project before applying. This project can be found here: https://warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phd_opportunities/stemcells2023/cellresolution

The University of Birmingham are waiving International Fees, however, international students will still have to pay for their own visas and a healthcare surcharge (approximately £2500).


References

1 Butko, E., Pouget, C. & Traver, D. Complex regulation of HSC emergence by the Notch signaling pathway. Dev Biol 409, 129-138, doi:10.1016/j.ydbio.2015.11.008 (2016).
2 Gama-Norton, L. et al. Notch signal strength controls cell fate in the haemogenic endothelium. Nat Commun 6, 8510, doi:10.1038/ncomms9510 (2015).
3 Bonkhofer, F. et al. Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium. Nat Commun 10, 3577, doi:10.1038/s41467-019-11423-2 (2019).
4 Uenishi, G. I. et al. NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells. Nat Commun 9, 1828, doi:10.1038/s41467-018-04134-7 (2018).
5 Dobrzycki, T. et al. Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis. Commun Biol 3, 71, doi:10.1038/s42003-020-0798-3 (2020).
6 Mahony, C. B. et al. Lineage Skewing and Genome Instability Underlie Marrow Failure in a Zebrafish Model of GATA2 Deficiency. SSRN, doi:http://dx.doi.org/10.2139/ssrn.4088882 (2022).

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