Environmental factors (extracellular macromolecules) possibly operating in somite differentiation were examined by using an in vitro system with myogenesis as the end point. It was found that differentiation depended on the time of removal of the somitic tissue from the host, i.e. between stages 17 and 26 (2½ to 5 days of age), and the question was raised as to the relation of these observations and the appearance of glycosamino-glycans (mucopolysaccharides). A review of the literature revealed that no information was available on this subject so that an examination of the time of appearance, distribution, and nature of the glycosaminoglycans and neutral polysaccharides in the axial region (dermatome, myotome, scleratome, neural tube and notochord) of the developing chick embryo during early somite differentiation was necessary. It became apparent that both histochemical and biochemical analysis were required to identify, quantify, and localize the glycosaminoglycans since histochemical techniques alone limit the interpretations possible because interference from proteins and glycoproteins could not be ruled out. Histochemical analysis indicated that there was very little sulphated anionic glycosaminoglycan present in the early embryonic stages examined. The cytoplasm of cells in all axial areas contained strongly acidic material, but extracellularly, sulphated anionic glycosaminoglycans were almost all confined to the notochordal sheath. The extracellular matrix in all areas contained weakly acidic anionic glycosaminoglycans. With development, the weakly acidic anionic glycosaminoglycans increased in concentration in most areas, but most noticeably in the neural tube and scleratome. The concentration of sulphated anionic glycosaminoglycans also increased and they began to appear in the extracellular matrix in all areas although never attaining the deep staining intensity demonstrated by the weakly acidic anionic glycosamino-glycans. By stage 25, however, the extracellular matrix of the scleratome around the notochord was mostly sulphated anionic glycosaminoglycans. Testicular hyaluronidase digestion suggested that most of the stainable material was either hyaluronic acid or chondroitin 4- and/or 6-sulphate (chon-croitin sulphates A and/or C). A small amount of strongly acidic anionic glycosaminoglycan present in the scleratome, neural tube and notochord was polysulphated. Biochemical analysis confirmed that the weakly acidic anionic glycosaminoglycan was hyaluronic acid and that the sulphated anionic glycosaminoglycan was mainly chondroitin 4- and/or 6-sulphate (chondroitin sulphate A and/or C). Only trace amounts of dermatan sulphate (chondroitin sulphate B) were present. A small amount of heparin could be present since some glucosamine was present in the sulphated fractions. This heparin could account for the polysulphated material observed with histochemical staining. On a quantitative basis, the hyaluronic acid concentration (uronic acid/gm dry wt. of tissue) was at a peak between stages 21 to 25 and was greater than the chondroitin sulphate concentration up until stage 25. After that stage, the chondroitin sulphate concentration began to increase very rapidly, concomitant with the formation of cartilage around the notochord, and the hyaluronic acid concentration began to decline slowly. Thus, whereas the hyaluronic acid content was 2½ times greater than the chondroitin.sulphate content in stage 17 embryos, this ratio was almost completely reversed by stage 28 due to the rapid increase in chondroitin sulphate. Histochemical staining supported these findings. It would seem that the increase in sulphated anionic glycosaminoglycans is directly related to cartilage formation while the high hyaluronic acid content present during stages 21 to 25, a time in development when myotube formation and scleratome cell aggregation and orientation are occurring, may play some more general developmental role in somite differentiation. / Science, Faculty of / Zoology, Department of / Graduate