): The goal of this proposal is to further develop the research skills of the principal investigator by studying the role of the proto-oncogene Pbx1 in normal hematopoietic development. The candidate's predoctoral research experience in transcriptional regulation, and clinical interest in high-risk leukemia led him to the laboratory of Dr. Cleary at the Stanford University School of Medicine. This environment is highly conducive to the objectives of this proposal due to the existence of core facilities for making transgenic and knockout mice, as well as molecular genetic tools to study their phenotypes. Collaborators with expertise in these areas are readily available at Stanford and the nearby University of California, San Francisco. Through continued work with Dr. Cleary and consultants, the candidate will gain experience in the molecular genetic manipulation and differentiation of embryonic stem (ES) cells and in the use of mouse model systems for the study of hematopoietic disease. He will apply this knowledge in his future independent research career, which will focus on the use of ES cells and mouse model systems to study the biology of infant and leukemia. The focus of this application on Pbx1 arises from its involvement in chromosomal translocations in a subset of high-risk pediatric leukemia. Biochemical studies implicate Pbx1 as a cofactor of Hox proteins required for binding to specific DNA sequences. Our preliminary studies show that mice lacking Pbx1 fail to sustain definitive hematopoiesis during embryogenesis. To identify developmental pathways requiring Pbx1, hematopoietic progenitors from Pbx1 null mouse embryos will be transplanted into irradiated recipients and their contribution to hematopoiesis will be determined. In addition to transplantation experiments, the contribution of Pbx1 null ES cells to hematopoiesis will be studied in chimeric mice to identify defects in myeloid and lymphoid differentiation and self-renewal. The candidate proposes to develop a model system for the study of Pbx1/Hox cooperation in hematopoietic differentiation. The hematopoietic differentiation of Pbx1 null ES cells in vitro will be compared to that of normal ES cells. The effect of over- expressing Hox genes on the hematopoietic development of normal and Pbx1 deficient progenitors and ES cells will be studied. Differences between normal and Pbx1 deficient ES cells in terms of in vitro differentiation or susceptibility to Hox over-expression will form the basis of an assay for Pbx1/Hox function. By defining the specific requirements for Pbx1 function in normal hematopoiesis, these experiments will further our understanding of the molecular pathogenesis of leukemia, aplastic anemia and myelodysplasia.
