Mutations of both the murine c-kit and steel gene sequences result in profound lethal hematologic deficiencies. Understanding the interaction of these proteins in the hematopoietic microenvironment (HM) is of fundamental interest to hematologists and developmental biologists. The HM serves as a source for multiple regulators of hemapoiesis, including a growth factor, termed stem cell factor (SCF) (also called mast cell growth factor, kit-ligand, or steel factor) which is the product of the steel gene and the ligand for the c-kit gene product (a receptor tyrosine kinase). In the HM the interaction of SCF and c-kit is a potent proliferative signal to cells in the hematopoietic stem/progenitor cell compartment. The protein sequence of SCF predicts both a membrane- associated protein and secreted protein. The presence of two forms of SCF suggests the possibility that SCF presentation in the HM may play a key role in hematopoietic cell proliferative behavior. This possibility is further strengthened by observations that the viable murine mutation called steel-Dickie (S1) represents a genomic deletion of sequences encoding the membrane spanning of severe hematologic deficiencies in S1.S1 mice implicates the importance of the membrane-associated form of SCF in hematopoiesis. We have demonstrated that hematopoietic cells respond differently to membrane-activation and half-life of c-kit protein differs significantly depending upon stimulation by either membrane- associated or secreted forms of SCF. The hypothesis to be studied in work proposed in this grant is that hematopoietic progenitor cell proliferation and differentiation is affected in a differential fashion by the isoform of SCF presented in the HM. These derived from gene targeted embryonic stem cells which express either form of SCF) and in vitro (using genetically modified bone marrow-derived stromal cells) and will be correlated with changes in the signal transduction pathway from c-kit through mitogen activated protein (MAP) kinases.