ULTRASTRUCTURE AND SYNTHESIS OF THE EXTRACELLULAR-MATRIX OF PISASTER OCHRACEUS EMBRYOS PRESERVED BY FREEZE-SUBSTITUTION

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preserva tion of the cells and extracellular matrix (ECM) is excellent. The bas al lamina, although thicker and less well defined than in conventional ly fixed embryos, demonstrates a region of decreased density just belo w the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed tissues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the stru cture of the basal lamina is different in different regions of the emb ryo. The ECM of the blastocoel consists of thin ''twig-like'' elements that form a loose meshwork evenly distributed throughout the blastoco el. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the e sophagus crossing the region between it and the ectoderm. Immunocytoch emical work with three different monoclonal antibodies shows that glyc oprotein molecules, synthesized in the Golgi apparatus, are also secre ted here and form part of the matrix structure. The results suggest th at the blastocoel is filled with a gel-like material reinforced with b undles of 20-nm fibers. The manner in which the observed arrangement c ould contribute to the development and maintainence of the shape of th e embryo is discussed. (C) 1997 Wiley-Liss, Inc.