Hematopoietic stem cells attract wide interest in biomedical research and basic science alike. They are used in transplantation experiments involving cancer patients and are popular targets for introducing genes in protocols aimed at gene therapy. Blood cell differentiation is one of the major paradigms for understanding the molecular processes involved in tissue diversification. In spite of a wealth of information about multipotent progenitors and the different cell types they generate, little is known about what happens in between. Here we propose to study the intermediates and pathways taken during differentiation using a novel in vivo labeling approach that allows us to analyze cells from various lineages without the need for staining procedures. This will be achieved by creating mice in which myeloid, megakaryocytic, erythroid and eosinophilic cell populations are labeled with various forms of fluorescent proteins, using homologous recombination and transgenic approaches. These mouse lines will be crossed to generate bi- or tri-colored lines and their bone marrow analyzed, using a combination of fluorescence microscopy, FACS and in vitro colony assays. To directly visualize the formation of differentiated cells, multipotent progenitors from the various mouse lines will be seeded in culture and recorded by time-lapse fluorescence microscopy using different filters. Using similar approaches we will also determine whether the transcription factor GATA-1 is capable of reprogramming hematopoietic cells in vivo.
