Melvin 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.

Cell adhesion during gastrulation. A new approach.

Abstract In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.

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.

Protein composition of the hyaline layer of sea urchin embryos and reaggregating cells

The protein composition of the sea urchin embryo hyaline layer has been studied by 125I surface labeling. The electrophoretic patterns of iodinated proteins indicate that the composition of the hyaline layer is species and stage-specific. Dissociation of iodinated embryos removes some labeled proteins of apparent molecular weights of 290,000, 175,000, 145,000, 110,000, 70,000, and 50,000. The electrophoretic pattern of labeled proteins found in the intact embryo is reestablished on the surface of aggregates following reaggregation. The possible function of these proteins with respect to cell adhesion, cell-sorting behavior and morphogenesis is discussed.

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.

Wheat germ agglutinin binding to the micromeres and primary mesenchyme cells of sea urchin embryos

Abstract Fluorescein isothiocyanate-conjugated wheat germ agglutinin (WGA-FITC) binds exclusively to the primary mesenchyme cells when the lectin is microinjected into the blastocoels of living Lytechinus pictus and Strongylocentrotus droebachiensis embryos. WGA-FITC binding increases throughout the period of primary mesenchyme cell migration and aggregation. Similar binding is observed in embryos cultured in sulfate-free seawater (SFSW) but not in seawater (ASW) containing tunicamycin. The temporal expression of WGA-FITC binding sites in vivo is also correlated with the pattern of binding observed in vitro. Sixteen-cell stage Arbacia punctulata embryos were dissociated in Ca2+ and Mg2+-free seawater (CMFSW) and the micromeres isolated using sucrose gradients. Arbacia micromeres, cultured in ASW containing calf serum, first bind WGA-FITC during the period when primary mesenchyme cell ingression occurs in control embryos. Micromeres cultured in the presence of tunicamycin do not develop WGA binding sites. The temporal expression of WGA-FITC binding in micromere cultures is unaffected by the absence of sulfate, but the size and morphology of aggregates cultured in SFSW differ from that of the controls.

Synthesis of RNA by dissociated cells of the sea urchin embryo

Abstract The incorporation of radioactive uridine into RNA by micromeres, mesomeres and macromeres of sea urchin embryos was studied, employing methods for separating the cell types in pure suspension. At the 16-cell stage, the 3-cell types, on a per genome basis, synthesized RNA at approximately the same rate although on a per mg protein basis the micromere-RNA synthetic rate was considerably higher than either mesomeres or macromeres. At the 32-cell stage, incorporation of radioactive uridine by micromeres decreased relative to mesomeres and macromeres. It was demonstrated that radioactive uridine could not be effectively washed or diluted out of the cells of 16-cell stage embryos. Experiments on reaggregating cells did not detect any transfer or transport of radioactivity from micromeres to the other cells. Possible explanations for these findings versus the disparate results of previous investigators were presented.

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.