Morphology and histochemistry of the extracellular matrix of embryos following freeze substitution of the starfish Pisaster ochraceus
Creators: name: Cambell, Stephen Sean
Publisher: name: University of British Columbia
Year: 2010

description: All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pisaster ochraceus, consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which fills the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the fluid-filled blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a fluid with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The initial objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pisaster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask filled with liquid nitrogen, a small cup was filled with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fluid with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, justifies its use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies.