The studies proposed in this application will characterize the morphogenetic properties of the Pbx proteins, a novel class of proto- oncogenic proteins implicated to function as cofactor for Hox proteins. The oncogenic properties of this divergent group of homeodomain proteins are vividly illustrated by the involvement of Pbx1 in protein fusions induced by t(1;19) chromosomal translocations in a subset of childhood pre-Bell cell acute lymphoblastic leukemias. A potential developmental role for Pbx proteins is suggested by genetic studies of a Drosophila homolog, extradenticle, which has the unusual property of inducing homeotic transformations without altering the expression of homeotic selector proteins. Biochemically, both exd and Pbx proteins form cooperative DNA-binding complexes with select Hox/HOM proteins. The genetic and biochemical studies provide strong support for the thesis that Pbx/exd proteins function as molecular cofactors for Hox/HOM proteins in the regulation of cellular identity. In contrast to the fly, no developmental or morphologic abnormalities have yet been attributed to germline mutations of the PBX genes in mice or humans thereby limiting an examination of the role of Pbx proteins in different developmental stages or tissues. This proposal outlines a systematic approach for elucidating the developmental roles of mammalian Pbx proteins by creating an allelic series of defined germline mutations of the mouse PBX genes. Using gene targeting techniques in embryonic stem cells, mice that are null for expression of individual Pbx proteins will be created and PBX (-/-) mice will be characterized for developmental defects. Initial studies will focus on knockout of the PBX1 gene to be followed by creation PBX2 (-/-) and PBX3 (-/-) mice. In the event that there is considerable functional redundancy in the PBX gene family, different PBX null mice will be crossed to obtain double knockout strains. The contributions of Pbx proteins to murine development will also be addressed by examining their expression profiles using in situ hybridization and immunohistochemical techniques. Forced heterodimers between various Pbx and Hox proteins will be investigated as an approach to specifically complement or induce developmental defects in genetically manipulated mice. The combination of genetic and biochemical approaches in flies and mice has clearly demonstrated that the Pbx/exd pathway is critically important for pattern formation, regulation of cell death/survival, and induction of oncogenesis. Although the intersection of Pbx with Hox proteins is likely to be an important nodal point for various signalling pathways, little is known about their upstream regulators or downstream target genes. The creation of an allelic series of mouse Pbx mutants will provide an indispensable resource for elucidating the details of how this distinctive family of homeodomain proteins contributes to normal and neoplastic cellular processes.
