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

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.

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.

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.