Investigating Vegfa transcriptional regulation by co-repressors ETV6 and ETO2 in haematopoietic stem cell development

Lead Research Organisation: University of Oxford
Department Name: Weatherall Inst of Molecular Medicine


Tissue stem cells are multipotent cells that have the unique capacity to generate all cell types of a specific organ. For this reason, they are extensively studied for their regenerative potential in clinical settings: researchers are aiming at producing stem cells "in a dish" from more immature, pluripotent precursor cells, such as those present in the early embryo. To achieve this ambitious goal, one will have to be able to reproduce in vitro the developmental cues normally integrated by these early precursors as they differentiate into specialised stem cells in vivo in the embryo.

The blood (or haematopoietic) system is amongst the best studied tissues and haematopoietic stem cells (HSCs) often serve as a paradigm in stem cell biology. So far, however, no one has successfully been able to generate HSCs in vitro. A full dissection of the regulatory mechanisms underlying HSC development in the embryo is therefore necessary to be able to develop the culture conditions that will sustain in vitro HSC production.

One key molecule in blood development is the growth factor VEGFA. VEGFA is not only necessary for blood vessel formation but also for HSC specification during embryonic development. We have recently described specific stages that require this growth factor for development of HSCs. Specifically, we have shown that distinct inputs from molecules (called transcriptional regulators) that control VEGFA level and spatio-temporal expression lead to the distinct activities of VEGFA in (i) formation of the vessel where the first HSCs emerge and (ii) production of HSCs themselves.

We now propose to further investigate how expression of VEGFA is controlled in the embryo through characterisation of the nature and function of the transcriptional regulators directly involved in this process. We believe that a complex interplay between these molecules is responsible for the exquisite timely expression of VEGFA, and we will aim at dissecting their mechanisms of action. In the longer term, we will use this information, together with findings from other scientists in the field, to define the critical regulatory signals that will help make HSCs in vitro.

This research will further our understanding of fundamental biological processes and benefit researchers working on stem cell development, regulation of gene expression and VEGFA signaling. Ultimately, it will contribute to the improvement of human health. Establishment of protocols for production of HSCs will benefit patients with blood disorders such as leukaemia who require stem cell transplantation.

Technical Summary

Stem cell specification relies on numerous intrinsic and extrinsinc signalling and transcriptional inputs. We have recently demonstrated that, during embryonic development, expression of the growth factor VEGFA in the somites is specifically required in haematopoiesis for formation of the dorsal aorta (site of emergence of the first adult haematopoietic stem cells (HSCs) in the embryo) and specification of these HSCs.

The main objective of this study is to identify the key direct regulators of Vegfa somitic expression as well as their mechanisms of action in the developmental stages leading to HSC specification. To achieve this, we will use in vivo Xenopus models and high-throughput technologies (ChIP- and RNA-sequencing) to describe the genomic targets of two upstream, indirect regulators of VEGFA expression in the somites, i.e. co-repressors ETV6 and ETO2. We will then assess the function of selected target genes on Vegfa expression through development of appropriate cellular and genetic assays that will include loss- and gain-of-function analyses, ChIP and mechanistic studies. Based on our previous phenotypic and molecular analyses, we expect to describe fundamental molecular mechanisms involved in transcriptional repression and alternative splicing of Vegfa. This work will be extended to mouse embryos in an attempt to characterise Vegfa regulation and function in HSC specification in higher vertebrates. This will inform further research aiming at developing protocols for in vitro production of HSCs.

Planned Impact

Our study aims at identifying some of the molecular pathways leading to haematopoietic stem cell (HSC) development. This research will therefore immediately benefit scientists attempting to produce HSCs in vitro from pluripotent stem cells. Indeed, so far, no one has successfully generated HSCs without prior genetic manipulation of the cells. We believe that successful derivation of HSCs will only be achieved when we fully understand the signalling and transcriptional cues that are successively required during embryonic development to instruct the precursors of HSCs.

As an example, our recent research in this field has highlighted very specific activities of VEGFA's isoforms in HSC formation. So far, only VEGFA medium isoform is routinely used in culture media to try and support HSC formation from pluripotent cells. Our findings will prompt scientists to test whether addition of the other VEGFA isoforms in culture media results in instruction of the HSC programme.

Our current proposal focuses on the upstream regulators of VEGFA. Understanding how VEGFA is turned on and off and the interplay between the activities of ETV6 and ETO2 in this regulation will confer another level of knowledge of the fine tuning of expression of this key signalling molecule. We envisage that, beyond the scientists directly working in this research field, our work will benefit the commercial sector and companies willing to develop small molecules mimicking the activities of VEGFA's regulators.

Finally, in the longer term, our research will benefit patients with blood disorders such as leukaemia and myelodysplastic syndromes who rely on allogeneic bone marrow transplantation for disease-free survival. Being able to reliably produce disease-free HSCs in a dish from patient-specific iPS cells would eradicate rejection and graft-versus-host disease and improve human health.


10 25 50
Description After developing new reagents essential for the research, we have identified positive and negative gene regulatory networks that control expression of one of the key cytokines controlling blood stem cell development during embryogenesis, VEGFA.
Exploitation Route We anticipate that a better understanding of the pathways leading to production of blood stem cells will guide protocols supporting production of such cells in vitro for regenerative medicine purposes. Moreover, we have identified similarities between the regulatory network supporting Vegfa expression during blood stem cell development and the transcriptional control of Vegfa expression in cancer cell lines. In the longer term, our findings may therefore inform tumorigenic processes.
Sectors Healthcare

Title Anti-Xenopus antibodies 
Description Generation of antibodies against Xenopus proteins that are not commercially available. 
Type Of Material Antibody 
Year Produced 2019 
Provided To Others? Yes  
Impact This tool will allow molecular studies that were not possible until now, or only in an indirect manner. 
Title Expression vectors 
Description Generated a series of truncated expression vectors to study the function of a newly identified potential regulator of blood development 
Type Of Material Biological samples 
Year Produced 2021 
Provided To Others? No  
Impact Will allow detailed analysis of the protein domain involved in the activity of a regulator of blood development 
Title Transcriptional mechanisms leading to expression of the growth factor VEGFA during vascular and blood stem cell development 
Description The identification of some of the transcription factors regulating expression of VEGFA in vivo through high throughput genomic sequencing (ChIP- and RNA-seq) has led to the description of positive and negative gene regulatory networks upstream of VEGFA that are controlled by the ETS factor ETV6. This will open up a field of mechanistic investigations not only in developmental hematopoiesis, but also in tumorigenesis where VEGFA expression, critical in metastatic processes, is regulated by some of the regulators we have identified in our screen. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact No impact yet. 
Title Whole genome sequencing databases (somites) 
Description RNA- and ChIP-seq databases from somitic cells. This has helped build the gene regulatory networks activating expression of VEGFA in the somites, a critical regulator of vascular and hematopoietic stem cell (HSC) development during embryogenesis. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact No impact yet. 
Description Transcriptional control of VEGFA 
Organisation University of Oxford
Department Radcliffe Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental and intellectual. My team brings technical expertise that complements that of the partner team. Together, we have a broader, more comprehensive approach.
Collaborator Contribution Experimental and intellectual. The partner team brings their expertise of a model organism.
Impact One publication in Nature Communications (2019) One publication in Seminars in Cell and Developmental Biology (2022)
Start Year 2015
Description VEGF signalling from the somites. 
Organisation University of Oxford
Department Weatherall Institute of Molecular Medicine (WIMM)
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual and experimental.
Collaborator Contribution Intellectual and experimental.
Impact None yet.
Start Year 2014
Description Women in Leadership programme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact 12 women post-doctoral scientists attended a leadership programme designed to equip them with tools required to develop their leadership skills.
Year(s) Of Engagement Activity 2021