description) Hematopoiesis is a regulated developmental cascade which initiates with a self-renewing, pluripotent stem cell (HSC) that differentiates into at least eight distinct lineages of the blood and lymph. The migration of HSC during ontogeny, as well as the ability of HSC to properly repopulate ablated hematopoietic microenvironments, implies a specific mechanism for homing and colonization. Adhesion molecules, particularly integrins, have been implicated in homing of hematopoietic cells to the appropriate microenvironment. Stromal cells, which make up the microenvironment, must express cognate ligands for the adhesions on hematopoietic cells. The cellular interplay between HSC and stromal cells may well be required to retain long-term pluripotency. The transcription factor PU.1 regulates expression of a large number of integrins such as CD11b (Mac-1). Fetal hematopoietic stem cells express PU.1, and are CD11b positive. To understand the function of PU.1 in the mouse, the gene was targeted via homologous recombination in embryonic stem (ES) cells, and """"""""knock-out"""""""" mice were generated. Analysis of the hematopoietic system revealed that lymphoid and myeloid development was completely abrogated in mutant embryos (Scott et al., 1994). Subsequent experiments have shown that PU.1-/-embryos contain HSC which are capable of generating erythroid and megakaryoid lineages, but not lymphoid or myeloid cells in the fetal liver. PU.1-/-embryos die at the stage when bone marrow hematopoiesis initiates during ontogeny (E17-18). Chimeric animals, generated with PU.1-/-ES cells, contain mutant hematopoietic progenitors in the fetal liver, but not in the bone marrow. PU.1-/- HSC lack a number of integrins normally expressed in the fetal liver (i.e. CD11b). One or more of these adhesion molecules may be required for HSC homing to the bone marrow. Therefore, PU.1-/- animals and ES cells provide an unique and valuable tool to address questions of hematopoietic stem cell homing. These investigators propose to use this system to explore the cellular and molecular basis for stem cell - stromal cell interactions. Specifically they plan to:
Aim I. Determine the effect of the PU.1 mutation on HSC homing and stromal adherence.
Aim II. Determine the ability of PU.1-/- stroma to support wild-type hematopoietic progenitors.
Aim III. Determine the role of adhesion molecules in PU.1-/- HSC function.
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