Cytoadhesive receptor-ligand mediated interactions between hemopoietic cells and their environment are thought to be responsible for their specific localization and maintenance in discrete anatomic sites as well as for their subsequent growth, differentiation, and survival. Furthermore, these specialized interactions allow emigration of specific cells types (i.e., egress of mature cells) and facilitate the settlement and colonization of IV infused donor hemopoietic cells in transplantation. Dissection of the molecular complexities that underlie these interactions in vivo has been difficult, as several adhesion molecules function in concert with a large number of other adhesion partners, and are influenced in a reciprocal fashion by several growth factor receptors and their ligands present in hemopoietic cells and/or their environment. Nevertheless, important insights have been obtained in vitro and especially in vivo from mice with genetic ablations of several genes. Through a combination of approaches the a4/fl1 integrin has emerged as a major player in the regulation of hematopoiesis. However, its specific effects on proliferation, maintenance, differentiation and migration of hematopoietic cells have not been delineated.
In Specific Aim #1, we will investigate the role of a4/fl1 integrins in fetal and adult hematopoiesis. Specifically, the impact of a4 integrin ablation on proliferation/differentiation/maintenance/trafficking on erythroid/megakaryocytic or all hemopoietic lineages will be studied using a) lineage specific or b) hemopoietic specific cre-mediated excision of a4 integrin. The impact of conditional a4 ablation, either during fetal or adult life will be studied using two approaches: a) breeding of our a4 """"""""floxed"""""""" animals will animals carrying cre-inducible promoters and b) transplantation of in vitro cre-excised a4 """"""""floxed"""""""" cells from adult bone marrow or from fetal liver to ascertain homing potential and durability of engraftment.
In Specific Aim #2 we will examine in a novel animal model the impact of cell proliferation per se on mobilization, and the kinetics and turnover of cells mobilized through the use of a mitogenic drug as compared to natural growth factors.
In Specific Aim #3, we will attempt through a systematic approach and the use of genetically deficient mice to unravel how carbohydrate-protein interactions lead to mobilization; to examine whether the post-treatment release of chemokines or the disruption of cell-stroma interactions are responsible using mice lacking chemokines or selectins; and to investigate the structural specificities that impart in carbohydrate compounds the ability to elicit mobilization.
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