SOMITE CHONDROGENESIS A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54-60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse se~um (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utihzed to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 A) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200-800 A protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many celts die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitie mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most noninduced cultures fed medium containing FCS and FI2X There is more ceil death, less mamx accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites, I N T R O D U C T I O N The aggregates of primary mesoderm known as somites undergo chondrogenic, fibrogenic, and myogenic differentiation, and the chondrogenic pathway of somite differentiation is influenced by the inducer tissues notochord and spinal cord (cf. Grobstein and Parker, 1954, Lash et a l , 1957) Being easily isolated from other embryonic tissues, somites provide a convenient system for studying the effects of such environmental factors as inducer tissues, culture substrate, or nutrient media on the Tan JOVnNAL OF C~LL BIOLOGY • VOLUU~ 56, 1978 • pages ~7-50 27 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom chondrogenic response of embryonic mesoderm (cf. Lash, 1967; Ellison et al., 1969, Ellison and Lash, 1971). Histological and biochemical studies of cultured somites have suggested that chondrogenic differentiation in these cultures is similar to that which occurs during vertebral chondrogenesis (Grobstein and Parker, 1954, Lash et a l , 1957, Lash, 1968) The cartilage formed in cultured somites is histologically similar to vertebral cartilage, and in both cartilages the protein-bound polysaccharides are almost exclusively chondroitin-4-sulfate (CSA), a n d / o r chondroitin-6-sulfate (CSC) (FrancoBrowder et a l , 1963; Marzullo and Lash, 1967, Kvist and Finnegan, 1970, Abbott et el., 1972). In cartilage matr ix chains of the chondroitin sulfates are linked to a linear protein backbone (Mathews and Lozaltyte, 1958, Hascall and Sajdera, 1969). These molecules form large proteoglycan (chondromucoprotein) complexes (Hascall and Sajdera, •969; Rosenberg et a l , 1970), and the synthesis of the chondroitin sulfates is inseparable from the synthesis of the complete protein polysacchande molecule (de la Haba and Holtzer, 1965, Telser et el., 1965). Since detectable amounts of these chondroitin sulfates are only found in cartilage in 10-day chick embryos (Marzullo and Lash, •967), biochemical studies of the chondrogenic response ofsomites have used either labeled sulfate incorporation into matr ix proteoglyeans, or the synthesis and accumulation of chondroltin sulfates, as criteria of chondrogenic expression in cultured somites (rater aha Lash, •968, Ellison and Lash, 1971, Gordon, 1971) There was, however, a lack of information about collagen synthesis, and the patterns of organization of matrix components, in these cartilages. The structural components of cartilage matrix are collagen which is present in the form of fibrils, and the large proteoglycan complexes which are present in fixed cartilage in the form of 200700 ~ protein polysaccharide matrix granules (/vIatnkas et a l , 1967; Smith, •970; Anderson and Sajdera, 1971). This study has therefore utilized electron microscopy to compare the patterns of accumulation of these matrix components in cultured somites with that which occurs durmg vertebral chondrogenesis (of. Strudel, 1971; Olson and Law, 1971). In addition, the effects of the inducer tissues (notochord and spinal cord), as well as the effects of different nutrient media, on structural differentiation in cultured somites have been examined. This comparison of in vivo and in vitro chondrogenesis has shown that structural differentiation in cultured somites is similar to vertebral chondrogenesis. The structured matrix components in both cartilages are thin unbanded collagen fibrils, and prominent protein polysaccharide granules These components have a recognizable structure, which is easily distmguished from the structured matrix of fibrous tissues derived from