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Unravelling blood stem cell formation at single cell resolution

Principal Supervisor: Dr Rui MonteiroLink opens in a new window, Institute of Cancer and Genomic Sciences, University of Birmingham

Co-supervisor: Dr Sascha Ott, University of Warwick

PhD project title: Unravelling blood stem cell formation at single cell resolution

University of Registration: University of Birmingham


Project outline:

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 pre-requisite 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recently shown that the Notch ligand Dll4 is required for the expression of the critical HE genes, runx13 and gata2b (unpublished). In this project, we aim to study the role of the Notch ligand Dll4 in the formation of HE and subsequent production of HSCs. To achieve this, we will:

  1. Generate transgenic lines to drive photoconvertible fluorescent proteins or Cre recombinase specifically in arterial endothelial or HE cells using tissue specific enhancers previously identified in the lab5,6. The Cre driver lines will be crossed to an effector line containing a fluorescent reporter for lineage tracing in vivo.
  2. Perform lineage tracing using this transgenic system to identify the haematopoietic cell populations that derive from dll4+ (arterial) or gata2b+ (HE) cells. Characterization of these haematopoietic cells will be accomplished by single cell transcriptional profiling of the labelled cells.
  3. Understand the molecular cascade elicited by dll4 in arterial and HE cells. To achieve this, we will use a Runx1-citrine transgenic line3 that allows separation of arterial and HE cells to compare gene expression between wildtype and dll4 loss of function by scRNAseq and identify potential regulatory mechanisms.

Methods

The student will train in vertebrate embryology and micromanipulation/microinjections of embryos, 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.

These experiments should establish the molecular cascade downstream of dll4 that specifies HE, whether the arterial fate is a pre-requisite for the formation of HSCs and how this fate could be manipulated to generate HSCs in vitro.

Other potential projects on this theme are also available in the lab.


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).

BBSRC Strategic Research Priority: Understanding the rules of life Stem Cells

Techniques that will be undertaken during the project:

Genome editing (CRISPR/Cas9 technology) and transgenesis

Single cell transcriptional profiling (scRNAseq)

Analysis of high throughput Next Generation Sequencing data

Microinjection and embryo manipulation

Confocal microscopy

Molecular biology

 

Contact: Dr Rui MonteiroLink opens in a new window