Commitment of a pluripotent hemopoietic stem cell (HSC) to the lymphoid lineage and its sustained differentiation to mature immunocompetent lymphocytes is partly controlled by transcription actors. Temporally expressed or activated transcription factors, that turn on expression of lineage specific Genes and initiate or arrest cell cycling of pluripotent HSC or multipotent lymphoid progenitors, serve the role of master regulators for lymphocyte commitment and differentiation. From our studies on transcription factors that control lymphocyte development we have isolated and characterized the Ikaros gene. The early hemopoietic and lymphopoietic expression of the Ikaros gene together with its ability to activate transcription in a lymphocyte independent manner, make it a strong candidate to control lymphocyte specification and differentiation. To study the role of the Ikaros gene in lymphocyte development two functionally distinct domains in the Ikaros proteins were targeted for deletion in the mouse germ line. Mice homozygous for a mutation that abrogates the DNA binding properties of the Ikaros proteins and generate functionally null proteins, exhibit a very early arrest in lymphocyte development. No T, B and NK cells or their earliest identifiable progenitors are present in these mice. In contrast, a second line of mice with a deletion at the C-terminus of the Ikaros proteins have alpha/Beta T cells, but lack B cells and their earliest progenitors, gamma/delta T and NK cells. Therefore, embryonic and adult T and B lymphoid progenitors are differentially affected in Ikaros C-I- mice. The goal of this grant is to characterize the specific effects of the C-terminal mutation in the ontogeny and subsequent development of different lymphoid lineages. In the following studies we will perform an extensive phenotypic and functional analysis of the emerging embryonic and adult lymphocyte populations in Ikaros C-/-mice. Expression of Ikaros mutant isoforms will be determined at the mRNA and protein levels. The DNA binding properties and postranslational modifications of these mutant Ikaros forms will be studied. These studies will allow us to determine the effects of the C- terminal mutation on the function of Ikaros isoforms and give us insight on the role of these proteins in lymphoid lineage determination. In addition, the developmental properties of lymphoid progenitors isolated from Ikaros C-/- mice will be investigated in in vitro organ cultures. Finally, target genes for the Ikaros proteins, important in the development and maturation of lymphoid progenitors, will be investigated by studying differential mRNA expression in the Ikaros C-/mutant lymphoid and HSC populations.
