Anat Embryol (1992) 185:155 162 Anatomy and Embryology 9 Springer-Verlag 1992 Distribution of collagens and fibronectin in the subepicardium during avian cardiac development James G. Tidball Department of Kinesiologyand Jerry Lewis Neuromuscular Research Center, Universityof California, Los Angeles, CA 90024, USA Accepted July 31, 1991 Summary. The development of the layer of connective tissue between ventricular epicardium and myocardium was studied during chick morphogenesis using electron microscopy, light microscopy and immunohistochemical techniques. This layer, called the subepicardium, in- creases rapidly in volume from embryonic day 6 to 11 (E6 Ell) during mesenchymal cell invasion. Fibrous, matrix components are initially apparent at Ell to El6, and as fibrous connective tissue structures accumulate, subepicardial volume decreases. Antibody labeling shows that fibronectin is an early, prominent constituent of the subepicardium, and by ES, the subepicardium is the cardiac site most enriched in fibronectin. Collagen type III is present in circumferentially-oriented fibers at E8. During subsequent cardiac growth, collagen type III fibers become broadly distributed in the subepicar- dium, with some fibers appearing to attach myocardium to epicardium. Collagen type I fibers are not apparent until El0. At El2 collagen type I fibers are distributed circumferentially around the heart in bundles crimped into waves of low amplitude. Other collagen type I fibers are oriented radially in the subepicardium. During late cardiac morphogenesis and in fully-differentiated hearts, fibronectin and collagen types I and III are more concen- trated in the subepicardium than within the myocar- dium. These observations suggest that the composition and organization of the subepicardial connective tissue may make important contributions to cardiac mechanics from the latter half of embryonic development through adulthood. Key words: Heart - Development - Collagen Fibronec- tin Epicardium Introduction The organization of the extracellular matrix plays key roles in development and function of cardiac tissue. Fi- bronectin-mediated adhesion of embryonic cardiac myo- cytes to collagen I appears to be important in early mor- phogenesis of the heart (Icardo and Manasek 1983, 1984), and the distribution and orientation of collagen fibers in the myocardium may determine, in part, the mechanical behavior of the heart (discussed by Covell 1990). Collagen types I, III, IV and V are all present in the heart (Thompson et al. 1979; Weber et al. 1987; Egh- bali et al. 1989), first appearing in early stages of cardiac myogenesis. The distribution and function in myocardia of collagen type I has been most thoroughly examined. Recent findings show that type I collagen can interact with the lateral surface of cardiac myocytes via a recep- tor in the integrin family (Terracio et al. 1989; Gullberg et al. 1989). The collagen fibers thereby attached to myo- cytes can form additional associations with adjacent myocytes or blood vessels within the myocardium. These lateral, collagen-mediated associations are believed to maintain vessel patency throughout the cardiac cycle and to provide mechanical integration throughout the layers of myocardium (Caulfield and Borg 1979; Robin- son et al. 1983, 1987, 1988a, b). Another morphological component of the heart that may make important contributions to cardiac mechani- cal behavior is a layer of subepicardial connective tissue. This layer, called here the subepicardium, separates the simple epithelium covering the heart from the underlying myocardium. The subepicardium comprises a continu- ous connective tissue sheath surrounding the myocar- dium which may determine, in part, the elasticity and stiffness of the heart. Although previous investigations have shown that 16-week-old human fetal hearts contain both collagen types I and III in the subepicardium (Hen- drix 1981), and morphological studies have indicated that the first collagen fibrils appear in the chick heart subepicardium at about 8 days following fertilization (Manasek 1969), little is known of the development, or- ganization or composition of the subepicardium in late cardiac morphogenesis. The present investigation was directed toward analyz- ing the formation and composition of the subepicardium