Measurements of Collagen and GAG Production

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534//~m 2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300//zm 2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation with z5SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibrohlasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma. The first event in avian corneal morphogenesis is the laying down of an acellular stroma of orthogohal layers of collagen fibrils embedded in glycosaminoglycan (GAG); z both the collagen and GAG i Abbreviations used in this paper are: GAG, glycosaminoglycan; ECM, extracellular matrix; IMP, intramembranous particle; CAM, chorioailantoic membrane; ES, extracellular cell surface; SR, sarcoplasmic reticulum. are products of the epithelium (14, 31). Subsequently, the endothelium migrates into place under the stroma and secretes hyaluronate into its interstices, bringing about, directly or indirectly, a remarkable swelling of the matrix (21, 52, 55). The neural crest cells which will become corneal fibroblasts (24) migrate into the acellular, primary corneal stroma on day 6 of embryogenesis in the chick, moving between the orthogonal layers of THE JOURNAL OF CELL BIOLOGY VOLUME 72, 1977 " pages 667-686 667 on Jauary 8, 2018 jcb.rress. org D ow nladed fom collagen fibrils that seem to serve as their substratum (4, 21). From the time of their appearance at day 6, the presumptive corneal fibroblasts actively migrate and proliferate in the stroma, so that by day 14 only the subepithelial stroma remains uninvaded. During the period between days 6 and 14, these relatively undifferentiated mesenchymal cells acquire abundant secretory organelles (endoplasmic reticulum, Golgi complexes) and begin to add collagen and GAG to that present in the original stroma (21). After day 14, fibroblast migration and cell division cease and the stroma condenses under the influence of the thyroid hormone that now appears in the embryo. By day 18, all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form (12, 13, 29). Thus, there are at least three relatively discrete but overlapping phases in corneal fibroblast differentiation: one in which the major activity of the cells is migration and proliferation, a second when the cells settle down to the business of secreting extraceUular matrix (ECM), and a third when fibroblast proliferation ceases and the cornea condenses and becomes transparent. We have used the freeze-fracture technique to study developmental changes in fibroblast plasmalemma ultrastructure during the three phases of cytodifferentiation described above. It is now generally accepted that this technique offers a unique view of membrane ultrastructure by splitting the lipid bilayer along its hydrophobic interior (40, 51). Most information to date indicates that the globular, particulate ulaits which freeze-fracture reveals embedded in the cleaved membrane are proteins or lipoproteins and that the flattened areas surrounding these intramembranous particles (IMP) represent the "haif-bilayer" lipid domain (8, 22, 28, 47). Morphologic alterations of particulate and lipid domains occur in different functional states (37, 49, 53). Moreover, intraand inter-membrane interactions of protein and lipid components can be expressed in morphologically recognizable forms, such as tight and gap junctions (9, 17). Thus, it seemed reasonable to expect that distinct morphologic changes in the fibroblast plasmalemma might be detected during embryogenesis. In this paper, we describe the general appearance of the corneal fibroblast and surrounding stroma in freeze-fracture replicas and the changes that occur in fibroblast plasmalemma ultrastructure during corneal morphogenesis. In addition, biochemical studies of matrix synthesis and studies employing thiouracil and thyroxine are reported which were performed to find out whether the increase that was observed in IMP number in the third phase of corneal development was related to the level of synthesis of GAG and collagen or to the initiation of thyroid function and accompanying stromal condensation. MATERIALS AND METHODS