Evelyn Spiegel

Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos

Fibronectin and laminin have been found in the extracellular matrix and in the basement membrane of sea urchin embryos during early development. These glycoproteins are also found on the cell surfaces of the outer epithelial layer and on the secondary mesenchyme cells within the blastocoel. The similarity of functions of the extracellular matrix and basement membrane is discussed, as is the similarity of their molecular components. These observations suggest the possibility that fibronectin and laminin form a continuous matrix surrounding the cells which links the outer ECM (hyaline layer) to the inner ECM (basement membrane). Such a network could coordinate the various activities of the embryo during early morphogenesis.

The hyaline layer is a collagen-containing extracellular matrix in sea urchin embryos and reaggregating cells.

Abstract Evidence is given to support the classification of the hyaline layer of sea urchin embryos and reaggregating cells as a collagen-containing extracellular matrix. Ruthenium red staining shows the presence of striated fibril-like structures, dense spheroids, crystalline lattice structures and filamentous material. Collagenase digestion causes disappearance of the fibril-like structures; hyaluronidase treatment causes a diminution of other matrix components, but does not affect the fibrillar structures. The hyaline layer maintains the integrity of the embryo and is also involved in morphogenesis, which are among the functions of extracellular matrices.

The biochemical identification of fibronectin in the sea urchin embryo.

We report the biochemical identification of fibronectin in the basal lamina of the sea urchin embryo. A. punctulata gastrula stage embryos were solubilized in Triton X-100 and the insoluble basal laminae extracted by incubation in buffer containing 8M urea, 2% 2-mercaptoethanol and 2% SDS. Extracted proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose filters and probed with monospecific antibodies directed against human plasma fibronectin (pFN). Incubation in 125I-labelled secondary antibody revealed a single band which co-migrates with human pFN at an apparent molecular weight of 220,000. This is the first direct biochemical demonstration of a fibronectin-like molecule in the sea urchin embryo which cross reacts with antibodies to vertebrate fibronectin.

Microvilli in sea urchin eggs: Differences in their formation and type☆

Abstract The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types.

Some observations on the ultrastructure of the hepatocyte in the metamorphosing tadpole

Abstract The principal ultrastructural changes occurring during metamorphosis in the hepatocyte of Rana pipiens tadpoles are (1) the great degree of pleomorphism in the mitochondria, (2) the appearance of glycogen in some nuclei of late stages and (3) the disappearance of cytoplasmic glycogen, presumably due to its breakdown to glucose, in late stages. The latter may be related to the inhibition of tryptophan oxygenase activity during this developmental period. Differences between the hepatocyte of the tadpole and the adult include the above, plus the appearance of pinocytotic vesicles between adjacent cells during metamorphosis which are not seen in the adult.

THE REGULATION OF LIVER TRYPTOPHAN PYRROLASE ACTIVITY DURING THE DEVELOPMENT OF RANA CATESBIANA

1. Liver tryptophan pyrrolase activity was studied in embryonic, larval, and adult Rana catesbiana. Constitutive enzyme activity appeared after Shumway stage 22, disappeared during metamorphosis, and was again detected in appreciable amounts in the adult.2. Substrate inducibility was positively correlated with constitutive enzyme activity. In no case was enzyme activity induced by substrate in the absence of detectable constitutive enzyme activity.3. Culture of tadpoles in thyroxine solutions led to the suppression of enzyme activity.4. L-histidine and hydrocortisone had no effect on liver tryptophan pyrrolase activity.5. The appearance, during metamorphosis, of tryptophan pyrrolase activity was described.6. The implications of these findings were discussed in terms of further experiments on the ontogeny of macromolecules.

Qualitative changes in the basic protein fraction of developing embryos

Abstract Acrylamide gel electrophoretic studies were carried out on basic protein extracts of developmental stages of the frog Rana pipiens, the salamander Ambystoma punctatum, and the sea urchin Arbacia punctulata. Qualitative changes were detected in early cleavage and during gastrulation in all three species. Exogastrulation experiments indicate that those proteins appearing during normal gastrulation are absent in exogastrulae. The results are discussed in relation to the findings of previous investigators.

Sorting out of sea urchin embryonic cells according to cell type

Abstract Cell types were isolated from the 16-cell stage of Arbacia punctulata embryos. Micromeres were stained with nile blue sulfate, recombined with unstained mesomeres and macromeres and cultured in normal sea water. The results support the idea that blastomeres of the early sea urchin embryo are capable of recognizing one another and of sorting out according to blastomere type during reaggregation.

A scanning electron microscope study of early sea urchin reaggregation

Summary Sea urchin embryos were observed with SEM during the first 2 h of reaggregation, following dissociation of the 16-cell stage. A dense meshwork, composed of elongated microvilli embedded in the hyaline layer, surrounds the egg during early development. The dissociation procedure strips off some of the meshwork layer leaving fewer and smaller microvilli on the cell surface. Shortly after reaggregation has begun, several types of cell extensions are formed, including filopodia, which anchor the cells to the substrate, and ruffles and pseudopods, which enable the cells to move. Possible factors involved in the behavior of dissociated cells are discussed with regard to (1) the source of additional membrane in the formation of new cell extensions; (2) the ability of the cells to move.

Cell-Cell Interactions during Sea Urchin Morphogenesis

The dissociation of sea urchin embryos into a cell suspension and their subse-quent reaggregation provide a model system for studying cell-cell interactions during morphogenesis. Herbst (1900) first showed that sea urchin blastomeres could be dissociated with calcium-free seawater and—when returned to normal seawater—could reaggregate and develop into abnormal larvae. These ex-periments demonstrated that the hyaline layer surrounding the embryo holds the cells together and that calcium plays a role in cell adhesion. Giudice and Mutolo (1970) used this system to study the role of cell-cell interactions in the appearance of new metabolic patterns in development. This chapter reports on more recent work from our own and other laboratories using reaggregating cells to study cell adhesion and other cell-cell interactions during development. It also reports on our use of intact embryos to study the extracellular matrices and cell junctions on the external and internal surfaces during sea urchin morphogenesis.