The experiments to be described were designed to approach the following type of question: Will the differentiated state of a tissue cell survive multiple divisions in vitro? Do the cultured progeny of differentiated cells inherit the cellular mechanisms determining the unique somatic traits of their parental cells in a manner analogous to the way in which (1) Paramecia transmit Kappa particles to their daughter cells (Sonneborn,33 34 Beale,2 Preer3), (2) bacteria inherit genes for constitutive or inducible enzymes in the absence of substrate (Novick and Weiner,28 Spiegel-man35) or (3) temperate phage particles give rise to lysogenic progeny (Jacob and Wollman20)? Information of this kind is essential if the roles of the nucleus (i.e., genes) and the cytoplasm in cell differentiation are to be defined. An unambiguous approach to these problems requires that the following five conditions be fulfilled: (1) The initial population of differentiated cells must be homogeneous. (2) The cells must divide many times during the experiment. (3) The cells must not back-differentiate to the parental type immediately after each division. (4) The initial population must consist of differentiated cells and not dividing precursor cells whose progeny differentiate and thereafter cease dividing (e.g., presumptive muscle cells, presumptive blood cells, presumptive pigment cells, etc.). (5) The somatic traits used as markers must be specific to the parent cell and readily characterized. Given this list of conditions, it is clear that many kinds of somatic cells are excluded as test material. For example, both nerve cells and keratinizing skin cells do not divide. Liver and kidney cells offer difficulties of another kind; since it is difficult to secure a homogeneous population of either type of epithelial cell, it is possible that in long-term cultures the epithelial cells are selected against and the surviving population is derived from one of the "contaminating" cell types. Cartilage cells from vertebrae of 10-day chick embryos, on the other hand, satisfy the five conditions outlined. Pieces of cartilage may be obtained which consist * This investigation was supported, in part, by Research Grants B-493 and B-1629 from the

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The epiblast layer of the chick embryo gives rise to all embryonic tissues. In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak. Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density. Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle. Some cells differentiated without replicating. MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture. Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro. Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis. These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

Autoradiographs of embryonic cartilage indicated that labeled protein accumnulated intracellularly when the tissue was incubated with tritiated proline, and when the hydroxylation of proline was inhibited by anaerobic conditions or by a chelator for ferrous iron. The labeled protein apparently corresponds to protocollagen. the polypeptide precursor of collagen which serves as a substrate for the enzymatic synthesis of hydroxyproline.

The myocardial and endocardial cell sorting out processes take place primarily between 19 and 29 hr of development in the avian embryo. This occurs in an apparent rostra1 to caudal wave through the heart forming region. During heart development considerable uncertainty exists regarding the processes that regulate cell commitments, progressive aggregation, and sorting out of the different precardiac cell populations. The question addressed in this report is whether endocardial and myocardial cells have a common origin or do the endocardial cells arise from a distinct population of cells from within the precardiac mesoderm. These cells then migrate to become localized between the developing myocardium above and the endoderm below. The distribution of preendocardial cells and premyocardial cells has been followed immunohistochemically in quail heart-forming region mesoderm explants from embryos approximately 18 hr in development and incubated for a 24-hr period. Differentiating myocardiocytes were immunostained with anti-N-cadherin and endocardiocytes with QH-1, a monoclonal antibody that recognizes an antigenic determinant on quail endothelial cells. Sparsely localized QH-1 labeled endothelial cells are localized in the stage 5 heart-forming region. These cells are often arranged in a columnar fashion in the mesoderm explants 6 hr after explantation. By 15-22 hr large patches of QH-1 expressing cells are interspersed with the N-cadherin expressing myocardiocytes. A subpopulation of cells express both N-cadherin and QH-1 antigen suggesting that endocardial and myocardial cells may arise from a common precursor population and that N-cadherin regulation may be a mechanism underlying specific cell sorting of these two cell populations during heart development. o 1993 WiIey-Liss, Inc.