DESCRIPTION: (Investigator's abstract) Notch functions as a mediator of cell fate decisions during a variety of developmental processes, and substantial evidence indicates that this role is conserved in hematopoiesis. The capacity of Notch to act as both a cell-surface receptor and transcriptional regulator permits cell-cell signaling to directly influence gene expression, such that adjacent cells in the same environment adopt distinct fates. In general, increased Notch activity delays differentiation but leaves progenitors competent to respond to subsequent signals. The spectrum of Notch function thus includes self-renewal of multipotent progenitors as well as specification of diverse cell fates. Many of the features of Notch signaling that facilitate its unique role also complicate analysis of Notch function, particularly in a complex system such as hematopoiesis. We plan to take advantage of the unique properties of mouse embryonic stem cells (ES cells) to minimize complicating variables, while providing a physiologically relevant system to study the role of Notch in hematopoiesis. ES cells can be induced to undergo multilineage hematopoietic differentiation in vitro, and have the capacity to contribute to all cell types in chimeric mice. By transducing ES cells with Notch genes that are only expressed in the context of Cre recombination. Notch expression can be induced at defined stages of in vitro ES cell differentiation and in vivo hematopoiesis. The use of activated forms of Notch for initial in vitro studies will allow efficient screening of Notch 1-4 for effects at specific stages of differentiation, irrespective of specific ligand binding and in the context of defined cytokine stimulation. The generation of chimeric mice from ES cells expressing full length Notch receptors will permit the analysis of cell fate decisions by progenitors expressing relatively high (ES-derived) and low (blastocyst-derived) levels of Notch juxtaposed in the same developmental environment. Mice will be analyzed to determine the contribution of Notchhi-ES and Notchlo-blastocyst derived cells to various hematopoietic progenitor and lineage-committed populations. The effects of Notch on hematopoietic potential of progenitors will be further evaluated by in vitro CFU and in vivo repopulation assays. Finally, induction of Notch expression at defined developmental stages will permit assessment of Notch function during embryonic, fetal liver, and postnatal BM hematopoiesis without potential bias due to effects at earlier stages. This strategy will also facilitate the analysis of specific effects of Notch during in vitro differentiation of normal hematopoietic progenitors, without the need for genetic manipulation of primary hematopoietic cells. We hope these studies will provide insights into key aspects of Notch function that will help define the role of Notch in hematopoiesis, and ultimately apply to clinical questions in hematology, oncology, hematopoietic stem cell transplantation, and gene therapy.