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The role of dll4 in Haematopoietic Stem Cell emergence

Primary Supervisor: Dr Rui Monteiro, Institute of Cancer and Genomic Sciences

Secondary supervisor: Dr Sascha Ott, University of Warwick

PhD project title: The role of dll4 in Haematopoietic Stem Cell emergence

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 recently 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recently shown that the Notch ligand Dll4 is required for the expression of the critical HE genes, runx13 and gata2b (unpublished).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 Cre recombinase specifically in arterial endothelial (dll4-Cre) and HE cells (gata2b-Cre). These 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 epigenetic and 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; kdrl-mCherry transgenic line that allows separation of arterial and HE cells to compare gene expression between wildtype and dll4 loss of function by RNAseq.

These experiments should establish the molecular cascade downstream of dll4 that specifies HE and whether the arterial fate is a pre-requisite for the formation of HSCs. If that is the case, efforts to produce HSCs in vitro may benefit from identifying the optimal conditions to generate aortic arterial endothelium as a necessary first step.

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).

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
  • Epigenetic analysis of chromatin states (ChIP-seq, ATACseq)
  • Single cell transcriptional and epigenetic profiling (scRNAseq, scATACseq)
  • Analysis of high throughput Next Generation Sequencing data
  • Microinjection and embryo manipulation
  • Confocal microscopy
  • Molecular biology

Contact: Dr Rui Monteiro, University of Birmingham