MOLECULAR CONTROL OF HAEMOGENIC ENDOTHELIUM FATE

During embryonic life, the blood system is established in successive waves from specialized endothelial subsets. The generation of blood cells from these endothelial subsets is carefully timed and coordinated to provide the growing embryo with specific blood cell types that are needed at each successive stage of development. The molecular mechanism that regulates when these subsets of specialized endothelial give rise to blood cells is still poorly understood.

In the present project, we will aim to determine the underlying molecular mechanism that controls how these endothelium subsets are temporally maintained or how they are instructed by external stimuli to generate blood cells. In this context, we will explore the interaction between two transcriptional regulators, one controlling the endothelial programme and one controlling the blood programme. We hypothesise that the interaction between these two regulators of gene expression is critical to control the timing of blood cell generation during embryonic development. To address these questions, we will use an experimental system based on mouse embryonic stem cells that mimics normal development in vitro and in which we can easily manipulate gene expression and generate enough cells to examine many different features. Using fluorescent reporter proteins, we will follow the expression of these two transcriptional regulators and we will also determine how these two regulators interact with each other. Our work will answer a number of fundamental questions on how blood cell emergence is controlled at the molecular level during embryonic development.

During embryonic development, all haematopoietic cells are generated from transient haemogenic endothelium populations. Understanding the molecular control of haemogenic endothelium and its transition to blood is of fundamental importance since this population of cells is central to the establishment of both embryonic and adult haematopoietic systems. We recently showed that the transcription factors SOX7 and RUNX1 are found co-expressed in haemogenic endothelium at all sites of haematopoietic emergence during embryonic development. Additionally, we showed that in haemogenic endothelium, SOX7 binds to RUNX1 and hinders its interaction with CBFbeta which is an essential co-factor of RUNX1 for stabilization and DNA binding. Together, this prevents the transcriptional activation of haematopoietic targets by RUNX1, a critical event for haematopoietic specification. Here, we hypothesise that the interaction between SOX7 and RUNX1 establishes a metastable haemogenic state in which the endothelial to haematopoietic transition is poised. We postulate that this poised state, released by external cues and by the destabilization of the SOX7-RUNX1 complex, allows the coordinate spatial and temporal generation of blood progenitors during embryonic development. Here we propose to investigate the molecular network that regulates this transient maintenance of haemogenic endothelium state and how changes in this molecular network lead to haematopoietic commitment. The successful completion of this proposal will provide critical information and knowledge to understand further how blood cells are generated during embryonic development. The data generated in this project will also provide novel experimental cues on how to maintain or manipulate haemogenic endothelium populations in vitro to produce haematopoietic stem and progenitors cells.

Who will benefit from this research?
Our work will have an impact not only on our immediate research field but also far beyond. If successful, our work will benefit a number of diverse research fields. This includes:
1. Developmental biology, because the relevance of transcription factors for the regulation of metastable state is not well understood and shared by many developmental processes.
2. Stem cell research, because the molecular details of early haematopoietic specification and haematopoietic stem cell generation are still not fully understood.
3. Transcriptional regulation, because the molecular details and the dynamics of how different transcription factors participate in complexes to activate or inhibit gene expression in development is not well understood.
4. Haematology, because our work will provide fundamental basis in understanding how the blood system is established during development.

How will they benefit from this research?
1. We will make our system-wide data sets and novel experimental tools publicly available.
2. We will generate data that will be highly relevant to scientists studying other developmental/differentiation pathways both in academia and industry.
3. One significant potential outcome of our work is the identification of how haemogenic endothelium is specified and maintained. Such knowledge will immediately be applied to our human ESC cultures and could be used to drive the production of haematopoietic stem cells. This methodology will be of significant commercial benefit. We will make our expertise available to members of the industry and academia who wish to explore this possibility.
4. Our work will enhance the skills base in the UK. Future advances in biology and medicine will depend on building a skills base consisting of researchers who will be capable of thinking both in cellular and molecular terms as well as in system-wide terms, and researchers working on this grant will be exposed to the forefront of research in this field.
5. Along the same lines, we will attract students into this area. The University of Manchester have a number of studentship schemes.