CRUK Career Development Fellow

Email: as889@cam.ac.uk

Research themes:

Regulation of Haematopoiesis in Homeostasis and Disease

Description of research

Blood stem cells need to both perpetuate (self-renew) themselves and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells, and how it accommodates the transition from a stem cell to a mature blood cell. Clarifying how HSCs differentiate into diverse cell types is important for understanding how this process is subverted in the generation of blood pathologies. The aim of my group is to bridge this knowledge gap by providing a method in a relevant model organism (zebrafish, Danio rerio) that will allow us to dissect the role of novel blood genes and to determine their hierarchical position in regulatory networks that underlie haematopoiesis.

Our current program is divided into two research themes:

1. Functional genomics
2. Single cell transcriptomics

1. Functional genomics

In the last few years, DNA sequencing technologies have been developed that allow the identification of every genetic change in a given cancer sample, promising a new harvest of cancer genes. However, many of the identified genes were have not previously been implicated in blood formation and there is a real need to investigate the biology and potential therapeutic aspects of these genes. This will be achieved by pursuing two main objectives:

First, a high-throughput screen in zebrafish to dissect the functional role of novel cancer genes, implicated in myelodysplasia and myeloproliferative neoplasms, in haematopoiesis. This effort will define the function of novel cancer genes in blood cell formation by a morpholino and CRISPR/Cas knock-down approach in zebrafish.

This early objective will be followed by a far more ambitious long-term one. This objective will be based on in-depth functional characterisation of a subset of genes.

2. Single cell transcriptomics

Traditionally, different blood cells are distinguished by virtue of the fact that they express unique combinations of cell surface markers. Our knowledge of the different haematopoietic cell types is a direct result of the development of reagents to distinguish these various cells by their cell surface markers, followed by functional, transplant-based tracking of their activities. An inherent problem with this approach is that the presence of specific cell surface markers doesn’t directly reflect the transcriptional state of a cell. In addition, the variable loss or gain of marker expression occurs according to the activation/proliferative state of the cell. Although the transcriptomes of populations of HSC and progenitor cells have previously been assessed on microarrays and more recently using RNA-Seq of bulk cells, individual cells can exhibit substantial differences in gene expression with important functional consequences.

A population of seemingly homogenous haematopoietic cells captured at any one time may include many distinct or intermediate cellular states. This has been nicely illustrated by recent studies showing that the early diversification into cells with distinct lineage bias within the HSC compartment may exist and that individual HSCs lead to different reconstitution patterns (e.g. a balanced production of myeloid and lymphoid cells or deficiency in lymphoid potential). Considering only the average properties, by e.g. bulk transcriptomics analysis, masks subpopulations of cells. Therefore, a study of blood development with single-cell resolution would allow us to define cellular level heterogeneities that presage distinct differentiation decisions.

We will apply an integrative strategy, combining genetic perturbation with computational sequence and network analysis methods, to reconstruct the regulatory networks that maintain the dynamic balance between different blood cell types. This will be achieved by pursuing two main aims:

1) We will create a comprehensive atlas of single cell gene expression in adult zebrafish blood cells and computationally reconstruct the blood lineage tree. We will order cells according to their most likely developmental chronology and identify genes and gene regulatory networks that define distinct cell types.

2) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo. To achieve this we will generate a number of loss-of-function and transgenic zebrafish lines.

This programme sets out to determine the transcriptomes of thousands of single haematopoietic cells obtained from zebrafish kidney marrow. Data-driven classification of cell types will provide high-resolution transcriptional maps of both main and intermediate cellular states during differentiation and help us (re)define the haematopoietic lineage branching map in zebrafish in vivo. This will be crucial in understanding cell fate-determining events and to what extent differentiation outcome is determined by a limited set of TFs versus distinct gene sub-networks. Finally, the research proposed here will generate a plethora of biologically plausible candidate genes for further in-depth biological explorations, as a part of future studies.

Keywords: Haematopoiesis; Zebrafish; Functional genomics; Single Cell Transcriptomics

Clinical conditions: Haematopoietic malignancies

Methodologies: Single Cell RNA-Seq; Microinjections; In situ hybridisation; CRISPR/Cas knock-down; Live imaging

 

Key Publications:

Macaulay IC, Svensson V, Labalette C, Ferreira L, Hamey F, Voet T, Teichmann SA, Cvejic
 A (2016) Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells. Cell Rep, 2016 Feb 2;14(4):966-77.

Cvejic A. (2015) Mechanisms of fate decision and lineage commitment during haematopoiesis. Immunol Cell Biol. 2015 Nov 3.

Hung LL, Bielczyk-Maczyńska E, Ferreira L, Fleischmann T, Weis F, Fernández-Pevida A, Harvey SA, Wali N, Warren AJ, Barroso I, Stemple DL, Cvejic A. (2015) The ribosome biogenesis protein Nol9 is essential for definitive hematopoiesis and pancreas morphogenesis in zebrafish. PLoS Genet, 1;11(12):e1005677.

Chen L, Kostadima M, Martens JH, Canu G, Garcia SP, Turro E, Downes K, Macaulay IC, Bielczyk-Maczynska E, Coe S, Farrow S, Poudel P, Burden F, Jansen SB, Astle WJ, Attwood A, Bariana T, de Bono B, Breschi A, Chambers JC; BRIDGE Consortium, Choudry FA, Clarke L, Coupland P, van der Ent M, Erber WN, Jansen JH, Favier R, Fenech ME, Foad N, Freson K, van Geet C, Gomez K, Guigo R, Hampshire D, Kelly AM, Kerstens HH, Kooner JS, Laffan M, Lentaigne C, Labalette C, Martin T, Meacham S, Mumford A, Nürnberg S, Palumbo E, van der Reijden BA, Richardson D, Sammut SJ, Slodkowicz G, Tamuri AU, Vasquez L, Voss K, Watt S, Westbury S, Flicek P, Loos R, Goldman N, Bertone P, Read RJ, Richardson S, Cvejic A, Soranzo N, Ouwehand WH, Stunnenberg HG, Frontini M, Rendon A. (2014) Transcriptional diversity during lineage commitment of human blood progenitors. Science, 345(6204):1251033.

Bielczyk-Maczyńska E, Serbanovic-Canic J, Ferreira L, Soranzo N, Stemple D, Ouwehand WH, Cvejic A (2014). A loss of function screen of identified genome-wide association study loci reveals new genes controlling haematopoiesis. PLOS Genet, 10;10(7):e1004450.

Cvejic A (2014). From genome-wide association study hits to new insights into experimental hematology. Exp Hematol, 42(8):630-6.

Cvejic A*♯, Haer-Wigman L*, Stephens JC*, Kostadima M, Smethurst PA, Frontini M, Sipos B, Akker Evd, Bertone P, Bielczyk E, Farrow S, Fehrmann RSN, Gray A, Haas M, Haver VG, Jordan G, Karjalainen J, Kerstens HHD, Kiddle G, Loyd-Jones H, Needs M, Poole J, Soussan A, Rendon A, Rieneck K, Sambrook JG, Schepers H, Siljer HHW, Swinkels D, Tamuri AU, Verweij N, Watkins NA, Westra HJ, Stemple D, Franke L, Soranzo N, Stunnenberg HG, Goldman N, Harst Pvd, C Schoot Evd, Ouwehand WH♯, Albers C♯ (2013). The red blood cell GWAS gene SMIM1 underlies the Vel blood group and is a novel regulator of red blood cell formation. Nature Genetics, 45(5):542-5.

Gieger C*, Radhakrishnan A*, Cvejic A*, Tang W*, Porcu E*, Pistis G*, Serbanovic-Canic J*, [150 other contributing authors], Sanna S, Hicks AA, Rendon A, Ferreira MA, Ouwehand WH, Soranzo N (2011). New gene functions in megakaryopoiesis and platelet formation. Nature, 480(7376): 201-8.

Serbanovic-Canic J*, Cvejic A*, Soranzo N, Stemple DL, Ouwehand WH, Freson K (2011). Silencing of RhoA nucleotide exchange factor, ARHGEF3 reveals its unexpected role in iron uptake. Blood, 118(18): 4967-76.

Albers CA*, Cvejic A*, Favier R, Bouwmans E, Alessi MC, Jordan G, Kiddle G, Kostadima, R Read, Sipos B, Smethurst P, Stephens J, Voss K, Nurden A, Rendon AM, Nurden P and Ouwehand WH (2011). Exome sequencing identifies NBEAL2 as the causative gene for Gray Platelet Syndrome. Nature Genetics, 43(8): 735-7.

(*joint first authors, ♯corresponding authors)