Enzymatic events during cartilage differentiation in the chick embryonic limb bud

Abstract

Chondroitin sulfate and an unidentified sulfated mucopolysaccharide insensitive to hyaluronidase are synthesized by the prospective limb region of stage 15–16 chick embryos and stage 17–18 limb buds more than 2 days before the first appearance of metachromatic material. Sulfate-binding mucopolysaccharide material with the same electrophoretic mobility as chondroitin sulfate becomes entirely sensitive to hyaluronidase in stage 19 and older limb buds. The activity of four enzymes presumed to be involved in the pathway of chondroitin sulfate biosynthesis has been demonstrated before histological evidence of cartilage in both cell-free extracts of stage 19 chick limbs and in cultured aggregates of dissociated cells from the same source. The developmental kinetics of three of these enzymes, UDPG dehydrogenase, UDPGNAc-4-epimerase, and the sulfate-activating enzymes, have been examined; the first two enzymes are involved in the synthesis of the polysaccharide backbone of chondroitin sulfate, and the last system is responsible for sulfation of the polysaccharide chain. Although all three enzymes are present in low levels in limb bud tissue before chondrogenic differentiation, the rise in their specific activity is related to the development of cartilage both in vivo and in vitro. The fourth and most specific enzyme catalyzing the formation of a polysaccharide from UDPGNAc and UDPGA has merely been identified in both fresh stage 20 limb buds and aggregates cultured for more than 6 days. The products of the four enzymatic reactions have been identified. The relationship of these results to a theory of biochemical differentiation based upon restriction or enhancement of preexisting metabolic potentialities has been discussed.