Effect of 5-bromodeoxyuridine on ultrastructure of developing limb-bud cells in vitro

Abstract

Limb-bud cells differentiate into chondrocytes, myocytes, fibrocytes and epithelial cells in vivo. While detailed morphological descriptions of developmental events have been presented, mechanisms governing differentiation of cells in vivo are not yet apparent. Limb-bud cells can be grown in tissue culture and will differentiate into the same cell types as occur in vivo. Chondrogenesis in vitro appears to mimic differentiation in vivo as evaluated both biochemically and ultrastructurally. Incorporation of H 2 35SO 4 into glycosaminoglycans increases markedly on the fourth day in culture. At the same time, cells assume the morphology of mature chondrocytes and synthesize extracellular matrix composed of 250–400Å proteoglycan granules and poorly cross-banded collagen fibers. Myocytes can be detected as early as 21 hr after initiation of cell cultures and contain giant polysomes and thick and thin filaments. When early embryonic limb-bud cells are treated with 5-bromo-2′-deoxyuridine (BrdUrd), myogenesis is diminished and differentiation into chondrocytes is prevented irreversibly. Incorporation of H 2 35SO 4 remains depressed throughout the culture period despite removal of the drug after the second day of growth. Development of organelles associated with synthesis of extracellular material (rough endoplasmic reticulum and Golgi apparatus) is not prevented by treating limb-bud cells with BrdUrd. Extracellular matrix consists primarily of bundles of collagen fibers.