Undifferentiated cells are present in the adult organism. In most cases, it is not clear whether these """"""""stem cells"""""""" are capable of differentiating along multiple pathways or programmed to develop along a single pathway. Furthermore, the origin of adult stem cells is unknown. We propose to use the chick embryo as a model system with which to study these basic questions in stem cell biology. Previous experiments revealed that the skeletal muscle specific transcription factor MyoD is expressed in a small subpopulation of cells in several organs of the fetus and throughout the epiblast layer of the embryo that gives rise to all tissues of the body. When epiblast cells are placed in culture, some differentiate into muscle, chondroblasts, and neurons. These studies led to the hypotheses that stem cells for different lineages are present in the epiblast and their progeny are incorporated into a variety of mature tissues of the fetus. We will examine the epiblast and fetal organs for expression of mRNAs for Pax-l and NeuroM, transcription factors that regulate chondrogenesis and neurogenesis, respectively. In situ hybridizations will be performed with the recently developed and highly sensitive probes, fluorescently labeled DNA dendrimers. We will determine whether those cells that express MyoD in the epiblast and fetal organs are myogenic by marking them with the G8 antibody, placing them in culture for several days, and staining with an antibody to sarcomeric myosin. The potential of MyoD positive epiblast cells to differentiate into non-muscle cell types will be tested after isolating MyoD/G8 expressing cells from the quail embryo by fluorescence activated cell sorting, and implanting them into the chick embryo in the developing heart field and chondrogenic region of the limb bud. The fate of MyoD positive epiblast cells will be determined by isolating them from the quail epiblast with the G8 antibody, implanting them into the chick epiblast, culturing the embryo, and examining organs for the presence of quail cells. These studies will provide us with fundamental information regarding the extent of heterogeneity within the early epiblast and fetal tissues, the timing of development of stem cells for different lineages, and the extent to which they are committed to differentiate along a particular pathway.
