Roles of beta 1 and alpha 4 integrins in development of the definitive haemopoietic system

The definitive haemopoietic system consists of a complex hierarchy of cells, all of which arise from the definitive haemopoietic stem cell. The initial generation of definitive haemopoietic stem cells in the embryo occurs in the aorta, gonads, mesonephros region. However, the establishment of definitive haemopoiesis is a multi-step process involving the migration of definitive haemopoietic stem cells to the foetal liver and finally to the bone marrow, where definitive haemopoiesis occurs during adult life. a401 integrin, or VLA-4, is an important adhesion molecule implicated in many processes during the onset and maintenance of the definitive haemopoietic system. In the absence of 131 integrin definitive haemopoiesis is completely inhibited, although it is not clear at which time this block is occurring. In the absence of ct4 integrin, definitive haemopoiesis is not completely inhibited but shows severe defects which become more apparent as development progresses. In addition, studies with blocking antibodies have shown that the 131 and ct4 integrin subunits are involved in the migration and homing of haemopoietic progenitor cells and in their differentiation and proliferation. The aim of this thesis is to further define the roles of a4 integrin and 31 integrin in the definitive haemopoietic system, more specifically in the initial stages of its establishment. We show that 131 integrin and a4 integrin are expressed on definitive haemopoietic stem cells from the embryonic day 11 aorta, gonads, mesenephorous region, day 13 foetal liver, day 13 peripheral blood and adult bone marrow, which represent the major developmental stages of the definitive haemopoietic system. In the day 13 foetal liver and adult bone marrow some u4 integrin negative definitive haemopoietic stem cells are also detected. Therefore, the expression of a4 integrin may be modulated as definitive haemopoietic stem cells mature in the later haemopoietic organs. By studying the generation of definitive haemopoietic stem cells in a4 integrin knockout embryos, a role for cr4 integrin in the correct localisation and/or development of definitive haemopoietic stem cells in the foetal liver was revealed. In addition, these studies showed that the increasing defects observed in ct4 integrin knockout mice are likely due to progressive defects in definitive haemopoietic stem cells as they develop in different haemopoietic microenvironments. Attempts to generate a reversible 131 integrin knockout which would enable identification of the time point at which (31 integrin is essential for definitive haemopoiesis is also discussed. In conclusion the data presented here supports previous studies on the role of (31 and ct4 integrin in adult and embryonic definitive haemopoiesis and gives further information as to the time points at which these molecules are essential for the establishment of the definitive haemopoietic system and during its maintenance. Chapter 1: INTRODUCTION 1.1 Mouse Haemopoiesis 1.1.1. Adult/Definitive Haemopoietic System The adult or definitive haemopoietic system is a complex hierarchy of cells, with varying degrees of maturity and lineage commitment, all of which derive from the long term repopulating haemopoietic stem cell (LTR-HSC) (Abramson et al., 1977; Morrison et al., 1995b; Orkin, 1995), see figure I.I. LTR-HSC have a number of properties: ability to self renew; high proliferative potential; pluripotency for all haemopoietic lineages; ability to stably and fully reconstitute the entire haemopoietic system of a lethally irradiated recipient (Jordan et al., 1990; Keller et al., 1985; Lemischka et al., 1986; Micklem et al., 1966; Smith et al., 1991). Although several markers have been identified that are expressed on LTR-HSCs, there is no marker, or combination of markers, that is specific to haemopoietic stem cells (Morrison and Weissman, 1994; Sprangude et al., 1988). Therefore, the ability of haemopoietic stem cells to reconstitute the haemopoietic system of irradiated recipients is used as the defining assay for their presence. Downstream of LTR-HSCs in the haemopoietic hierarchy are short term repopulating stem cells (STR-HSCs) (Zhao et al., 2000; Zhong et al., 1996) and colony forming unit spleen (CFU-S) cells (Till and McCulloch, 1961). STR-HSC are capable of reconstituting all haemopoietic lineages, and CFU-S the myeloid and erythroid lineages, of irradiated adult recipients, but progeny from these cells appear earlier than