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The site of HS-uridine incorporation and the fate of labeled RNA during early embryogenesis of the newt Triturus pyrrhogaster were studied with electron microscopic autoradiography. Isolated ectodermal and mesodermal tissues from the embryos were treated in H3-uridine for 3 hours and cultured in cold solution for various periods before fixation with OsO4 and embedding in Epon. At the blastula stage, the only structural component of the nucleus seen in electron micrographs is a mass of chromatin fibrils. At the early gastrula stage, the primary nucleoli originate as small dense fibrous bodies within the chromatin material. These dense fibrous nucleoli enlarge during successive developmental stages by the acquisition of granular components 150 A in diameter, which form a layer around them. Simultaneously larger granules (300 to 500 A) appear in the chromatin, and they fill the interchromatin spaces by the tail bud stage. Autoradiographic examination has demonstrated that nuclear RNA synthesis takes place in both the nucleolus and the chromatin, with the former consistently showing more label per unit area than the latter. When changes in the distribution pattern of radioactivity were studied 3 to 24 hours after immersion in isotope at each developmental stage, the following results were obtained. Labeled RNA is first localized in the fibrous region of the nucleolus and in the peripheral region of chromatin material. After longer culture in non-radioactive medium, labeled materials also appear in the granular region of the nucleolus and in the interchromatin areas. Further incubation gives labeling in cytoplasm. INTRODUCTIONDuring the development of the amphibian embryo, the nucleoli first become visible at the time ot gastrulation (11,14,24,51) and gradually assume a typical reticular structure ("nucleolonema") (12, 24). However, no precise information has been obtained in regard to the mode of appearance and structural elaboration of the nucleolus in this cell type on a fine structure level. The formation of the nucleolus at a certain chromosomal region has been generally accepted by researchers using plant and mammalian cells (for review see Sirlin, references 42, 43). A number of electron microscope studies using various types of cells describe the behavior of the nucleolus during mitosis (30, 47), growth (20, 35), and differentiation (12,24,26). These studies also suggest a close topographical relation between nucleolar and chromosomal components. However, information on the manner in which the nucleolus originates is less detailed.During gastrulation of the amphibian embryo,

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The proliferative activities of three lines of "normal" epithelioid rat liver cells and six tumorigenic liver cell lines in the presence of a wide range of calcium concentrations were measured by a simple colony forming assay. The proliferative activities of the normal clis and, to a lesser extent, of the cells of a marginally tumorigenic line were directly proportional to the extracellular calcium concentration. The proliferative activities of the cells of the strongly tumorigenic lines, on the other hand, were either uninfluenced by or inversely proportional to the extracellular calcium concentration. Thus, the proliferative response to the extracellular calcium concentration is a sensitive indicator of the carcinogenic potential of liver cells. Extracellular calcium ions are needed for the initiation of DNA synthesis by a wide variety of cells ranging from rat hepatocytes in vivo (1-5), epithelial cells in primary cultures of mouse mammary glands (6), and several types of cultivated cells of mesenchymal origin (2)(3)(4)(5)(7)(8)(9)(10)(11)(12)(13)(14)(15) to the prokaryotic bacterium Yersinia pestis (16). On the other hand, neoplastic cells of mesenchymal origin (e.g, fibroblasts, muscle cells, and reticuloendothelial cells) need much less extracellular calcium and are able to proliferate indefinitely in low-calcium media that do not support the proliferation of their non-neoplastic counterparts (7-12, 13, 15). Extension of these observations to a clinically more important family of epithelial cells began only recently with the demonstration by Swierenga et al. (17) that neoplastic hepatocytes in primary cultures of a chemically induced transplantable rat hepatoma (Morris 5123tc) and hepatocytes of an established line (ATCC-CCL144) from another chemically induced transplantable rat hepatoma (Morris 7795) maintain their proliferative activity in a low-calcium medium much longer than do the hepatocytes in primary. cultures of fetal rat liver.In this communication we report the results of an assessment of the extracellular calcium requirements for colony formation by several "normal" and neoplastic epithelioid (i.e., epithelium-like) liver cell lines that indicates that the loss of proliferative calcium dependence is a simple, sensitive, in vitro indicator of their tumorigenic potential.