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Dissecting the role of novel haematopoietic regulators in Haematopoietic Stem Cells

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

Secondary supervisor: Dr Roland Arnold, Institute of Cancer and Genomic Sciences

PhD project title: Dissecting the role of novel haematopoietic regulators in Haematopoietic Stem Cells

University of Registration: University of Birmingham 

Project outline:

Background

Blood is made up of different cell types with different functions, such as carrying oxygen (red blood cells), help with wound healing (platelets) and fighting infections (white blood cells). All these cells arise from Haematopoietic stem cells (HSCs) that live in our bone marrow. They are generated during embryonic development, arising 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 uses the zebrafish model to investigate the formation of HSCs in the early embryo1-6.

We have recently demonstrated that haematopoietic gene expression in the HE requires input from the transcription factor Gata2a, followed by the Notch signalling pathway. This pathway can rescue embryonic haematopoiesis in the absence of Gata2a4. In this project, we will use a combination of classical developmental biology, transgenesis and gene editing, together with (single cell) transcriptional profiling and computational tools to identify novel haematopoietic factors that are targeted both by Gata2a and Notch signalling in the HE and study how they shape haematopoiesis in embryonic development and in homeostasis in the adult.

Zebrafish is very amenable to genetic modifications including generation of gene mutations and transgenic reporters for live imaging of cellular behaviour or isolation of specific cellular lineages for downstream molecular analyses. This is a collaboration with a bioinformatics lab (Arnold Lab) and ample opportunity for training will be provided. A better understanding of the factors that generate and maintain a healthy haematopoietic system in a whole organism is critical both to design new approaches to generating stem cells in vitro but also to help design effective interventions when haematopoiesis is perturbed in diseases such as leukaemias.

References:

  1. 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).
  2. Ciau-Uitz, A., Monteiro, R., Kirmizitas, A. & Patient, R. Developmental hematopoiesis: ontogeny, genetic programming and conservation. Exp Hematol 42, 669-683, doi:10.1016/j.exphem.2014.06.001 (2014).
  3. Dobrzycki, T., Krecsmarik, M., Bonkhofer, F., Patient, R. & Monteiro, R. An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation. Biol Open 7, doi:10.1242/bio.031096 (2018).
  4. 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).
  5. Monteiro, R. et al. Transforming Growth Factor beta Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells. Dev Cell 38, 358-370, doi:10.1016/j.devcel.2016.06.024 (2016).
  6. Monteiro, R., Pouget, C. & Patient, R. The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1gamma. EMBO J 30, 1093-1103, doi:10.1038/emboj.2011.34 (2011).

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

  • Transcriptional profiling (bulk and single cell RNAseq)

  • Analysis of high throughput Next Generation Sequencing data (RNAseq, ATACseq)

  • Microinjection and embryo manipulation

  • Confocal microscopy

  • Molecular biology

Contact: Dr Rui Monteiro, University of Birmingham