ANALYSIS OF MECHANISMS OF EARLY HAEMATOPOIETIC STEM CELL DEVELOPMENT CONTROLLED BY FLK1/SCL REGULATORY NETWORK

The blood system in the animal is maintained through daily generation of billions of different types of erythrocytes, granulocytes and lymphocytes. At the foundation of the blood system lie blood stem cells called haematopoietic stem cells (HSC). Thus, all blood cells in the animal are generated from HSCs. This is the cell type which is responsible for the long-term reconstitution of the blood system of patients who receive bone marrow transplants. Since the number of bone marrow donors is limited, scientists and clinicians are seeking for alternative routes for generation of HSCs in the laboratory conditions. Embryonic stem (ES) cells hold a great promise for regenerative medicine, including production of blood. However, the generation of HSCs from ES cells pose a serious challenge, mainly because many essential details of how the embryo generates HSCs are missing.
To a large extent, the progress has been slow since no culture system which would allow us to replicate development of HSCs was available until recently. This research proposal is mainly based on a novel powerful and analytical culture system developed in our laboratory. It allowed us to identify specific stages of development of HSCs in the embryo, at a time when these stages are concealed from scrutiny by traditional methods. We will use this culture system to investigate two genes, called Flk1 and Scl, that are, major regulators of early HSC development. These genes are expressed in various populations of cells of the developing embryo which are potentially involved in development of HSCs. In addition, these genes have a potential to regulate each other?s activity.
We will study how Flk1 and Scl genes regulate stages of HSC development by deleting these genes in our culture system using modern recombination technology. Results of such gene ablation will always be verified by a long-term transplantation assay, which is the gold-standard approach for detection of the presence and numbers of HSCs.
Additionally, we will carry out more comprehensive analysis of genes expressed at different stages of HSC development and determine how this genetic regulatory network is modified after deletion of Flk1 and Scl. To this end, we will use a modern automated system called Solexa. This approach will allow us to create a computerised genetic network which is involved in early generation of HSCs. In the longer term, this model will be a useful resource for testing the role of other genes in development of HSCs.

The adult haematopoietic system is maintained by definitive haematopoietic stem cells (HSCs), a rare population of cells capable of self-renewing and multi-lineage lymphoid and myeloid differentiation. The haematopoietic system emerges from the mesodermal germ layer during embryo development and subsequently, at least partly via the haematogenic endothelium. However, precise origin and also the sequence of events leading to specification and expansion of definitive HSCs remains a controversial and extensively debatable issue.
We have recently developed a powerful novel system, which replicates in vitro the specification and expansion of in vivo development of definitive HSCs in the embryo (Taoudi et al., 2008). This system for the first time allowed us to address the roles of individual cell populations in HSC development. We identified a population of pre-HSCs (immediate precursors to definitive HSCs) whose phenotype suggests that they are intermediates emerging from the haematogenic endothelium. Using this system we have recently identified a novel yet more immature class of pre-HSCs preceding the previously identified pre-HSCs, suggesting their intimate relationship with the embryonic endothelial compartment (unpublished).
It is thought that HSC development is a multi-step process involving complex cell rearrangements and maturation steps regulated by a cascade of genetic events. Although Flk1 and Scl have been identified as essential genes for HSC emergence, their exact points of action in HSC development remain unresolved. For example, Flk1 is expressed in a large fraction of early mesodermal, all endothelial and early haematopoietic cells. Literature data indicate that Scl is a downstream target of Flk1, but it can also regulate Flk1 expression.
Since various classes of pre-HSC can functionally be identified and characterised, we propose here to determine whether conditional ablation of Flk1 or Scl in these classes of pre-HSC would result in blockade and/ or deviation in HSC development. We will also establish how Flk1 ablation and Scl ablation in these populations would result in expression of Scl and Flk1 genes, respectively, using Scl-GFP and Flk1-GFP reporter mice. In addition, through global gene expression analysis we will map potential genetic network interactions involved in genetic regulation of the HSC lineage around the Flk1/Scl interactive axis.