ANALYSES OF FOUNDER CELLS IN THE EPIBLAST AND SOMITES

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DESCRIPTION: The George-Weinstein laboratory has demonstrated that cells from the presomitic segmental plate mesoderm and epiblast are able to differentiate into skeletal muscle and chondroblasts when isolated from surrounding tissues of the embryo and cultured in hormone and protein free medium. mRNA for MyoD and Pax-1, transcription factors that regulate myogenesis and sclerotome development, are detectable in the segmental plate by RT-PCR, and MyoD is detectable in the cells of the epiblast. Dr. George-Weinstein will test the hypothesis that (1) stably programmed founder cells for skeletal muscle and chondrogenic lineages are present in the chick embryo prior to the initiation of gastrulation; and, (2) that myogenic and chondrogenic founder cells recruit their uncommitted neighbors in the somite to form the myotome and the sclerotome. This latter event happens perhaps after receiving signals from surrounding structures such as the neural tube and the notochord. The signals are probably secreted sonic hedgehog and Wnts 1, 3, and 4. Existing data indicates that differentiation may be inhibited by tissue interactions, direct cell-cell interactions, and by nuclear proteins that affect transcription. For instance, when segmental plates are cultured as single cell suspensions, differentiation occurs readily, compared to when they are cultured as intact tissues. In order to resolve much conflicting data and to answer questions concerning the nature of uncommitted cells and the timing, location, and emergence of stably committed precursor cells, Dr. George-Weinstein proposes four specific aims. In aim 1, it will be determined if Pax-1, a factor specific for chondroblast differentiation, is expressed in the epiblasts isolated from prestreak stage embryos and from primitive streak stage embryos. This will substantiate that founder cells for chondroblasts are localized in the epiblast. In specific aim 2, the numbers and locations of cells expressing MyoD and Pax-1 mRNAs will be determined by in situ hybridization on tissue sectioned from various stage embryos. These experiments are expected to be informative because of the increased sensitivity (up to 100x) of probes using fluorescently labeled dendrimers, a high complexity nucleic acid signal network molecule developed by Polyprobe, Inc. An alternative approach of increased sensitivity will be in situ PCR followed by probing with the appropriate dentrimer. For specific aim 3, the fate of subpopulations of epiblast and segmental plate cells will be determined. The lab has previously developed 5 monoclonal antibodies that give distinct labeling patterns in unfixed prestreak and primitive streak stage embryos, each binding to a limited number of cells within the epiblast. The antibodies will be used to determine if they are, in fact, markers for myogenic and chondrogenic cells within the epiblast, and whether the epiblast cells that bind these antibodies are responsible for establishing tissues within the somite. The gold-labeled antibodies will be endocytosed by the (stage 2) embryos and then the embryo will be cultured and sampled over the next 30 hours. A second experiment involves the co-localization of antibodies with colloidal gold and fluorescent dendrimers to MyoD and Pax-1. Specific aim 4 proceeds similarly to aim 3, except those cells which bind antibody will be ablated by complement mediated lysis. The treated whole embryo will then be cultured up to 30 hours and the fate of tissue formation will be analyzed morphologically for dermomyotome and sclerotome formation, with antibodies to sarcomeric myosin, and by in situ hybridization with probes for MyoD and Pax-1.