The long-term goals of the proposed research are to dissect the signal transduction pathways mediating early B lymphocyte development, and to prepare the candidate to become an independent investigator by providing rigorous training in molecular biology and immunology. The candidate s sponsor, Dr. Frederick Alt, has a longstanding interest in molecular and developmental immunology, and has created several murine models for immunodeficiency through targeted mutagenesis. In recombinase activating gene (RAG)-2-deficient mice, lymphoid development is blocked at the pro-B and CD4+ CD8+T cell stages. Elimination of the heavy chain J (JH) regions also arrests B lymphopoiesis at the pro-B cell. These mutant mice emphasize the importance of the mu heavy chain in driving B cell development. The mu heavy chain-surrogate light chain complex appears crucial to several checkpoints in B lymphopoiesis, including precursor expansion, stage-specific regulation of surface markers, allelic exclusion, and onset of light chain rearrangement. Experiments are proposed to elucidate the signaling mechanisms which facilitate these developmental transitions. Previous work from the Alt laboratory demonstrated that expression of a rearranged heavy chain gene in RAG-1- or JH-1-backgrounds causes the generation of B lineage cells resembling pre-B cells. Our initial results indicate that a constitutively active form of the Ha-ras proto- oncogene bypasses the requirement for mu in these backgrounds, yielding pre-B cells expressing surface markers usually associated with even later stages of B cell maturation. Further experiments will assess the expression of developmentally regulated genes and in vitro proliferative responses. Additional studies in Ha-ras-JH-1- B lineage cells will examine effects on light chain rearrangement, and Ha-ras expression in the wild-type background will assess whether Ras influences mu rearrangement. Signaling cascades downstream of Ras, such as the MAP kinase and stress-activated/cell morphology pathways will be similarly analyzed for their ability to mediate B cell development. Signaling proteins upstream of Ras will also be examined; specifically, the influence of an activated Syk tyrosine kinase gene will be assesed in RAG-1- and JH-1- B lymphocytes, and the role of Syk in the regulation of allelic exclusion will be tested through expression in the B lineage of wild-type mice. Use of the RAG-2-deficient blastocyst complementation system, developed in the Alt laboratory, will allow these signaling proteins to be assessed singly and in combinations. These experiments should have implications not only for B cell development, but also for the understanding of immunodeficiency syndromes and lymphoid malignancies.
