Activity of aminopeptidases (amino naphthylamidases) during early limb bud differentiation in the chick embryo

Abstract

The morphologic changes in the differentiation of chick embryo limb buds from Hamburger-Hamilton stages 19 through 34 were correlated with biochemical measurements of hydroxyproline biosynthesis and aminopeptidase enzyme activity. The distribution and quantitative approximations of collagen, mucopolysaccharides and aminopeptidase activity were observed by histochemical methods. Aminopeptidase enzyme activity increased after four days of incubation (stages 24-25) concomitantly with the formation of precartilage masses (development of lakes of intercellular mucopolysaccharides). Histochemical preparations revealed the mesodermal cells in the precartilage masses to have an increased aminopeptidase enzyme activity. It was also noted that the enzyme activity in the ectodermal ridge was in excess of that found in the subjacent mesodermal cells or adjacent epithelium. Wet weights of limb buds revealed a constant rapid rate of growth throughout the period of study. However, aminopeptidase enzyme activity was not biochemically detectable until the onset of increased mucopolysaccharide synthesis, when cartilaginous dserentiation begins. At five days (stage 26), hydroxyproline was &st detectable biochemically and collagen histochemically. This was concomitant with the onset of hydroxyproline production in the embryo in toto, reported previously. The curve of acceleration of hydroxyproline production per milligram of bud mesoderm was less than the acceleration of aminopeptidase activity. The aminopeptidase enzyme activity in the premuscle masses and ectodermal-mesodermal interface, where histochemically detectable collagen was located, was at the same level as the intervening tissues. After stage 19, limb bud mesoderm was cultured in vitro for seven days. Hydroxyproline production was not biochemically detectable and collagen not stainable, though mucopolysaccharide production was extensive. No muscle differentiation occurred and cell proliferation was minimal. Aminopeptidase enzyme activity had increased 20-fold. It is suggested that at least part of the aminopeptidase enzyme activity of limb buds is associated with mucopolysaccharide synthesis. There was no evidence that it was associated with mesodermal cell proliferation, muscle differentiation, or hydroxyproline production.