In situ distribution of concanavalin A-binding sites in mesenchyme blastulae and early gastrulae of the sea urchin Lytechinus pictus☆
Abstract
The spatial distribution of concanavalin A (conA)-binding sites was studied in situ in order to investigate the role of conA-binding sites in primary mesenchyme cell migration in mesenchyme blastulae and early gastrulae (gastrulation one-quarter complete) of the sea urchin, Lytechinus pictus. In normally developed embryos conA is bound around the entire basal surface of the ectoderm and to the entire surface of ingressed primary mesenchyme cells in mesenchyme blastulae. However, in early gastrulae conA-binding sites are lost on the vegetal half ectoderm and on the primary mesenchyme cells, which had returned to the vegetal half in order to aggregate for spicule formation. ConA-binding is completely inhibited in embryos incubated with α-d-mannoside, but not in those incubated with β-d-mannoside or galactose. In sulfate-deprived embryos conA-binding sites are distributed normally in spite of the deficiency in primary mesenchyme cell migration. In embryos treated with LiCl, on the other hand, the ingressed primary mesenchyme cells migrate extensively, but fail to return to the vegetal half. In these embryos the conA-binding sites on the basal surface of the vegetal half ectoderm remain even at the early gastrula stage. The results correlate the spatial distribution of conA-binding sites with the pattern of primary mesenchyme cell migration in the blastocoel.
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Accessing the embryo interior without microinjection
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Pamlin, a Primary Mesenchyme Cell Adhesion Protein, in the Basal Lamina of the Sea Urchin Embryo
1995, Experimental Cell ResearchPamlin, a primary mesenchyme cell (PMC) adhesion protein, was isolated from the blastocoel of embryos of the sea urchin Hemicentrotus pulcherrimus. PMCs isolated from mesenchyme blastulae bound exclusively to pamlin. Pamlin is a distinctive extracellular matrix (ECM) component from reported ECM molecules in sea urchin embryos in its motility on SDS-PAGE gels both with and without 2-mercaptoethanol and histological localization. A monoclonal antibody was raised against pamlin, and this protein was immunoaffinity purified. The Mr of pamlin shown by SDS-PAGE gel analysis under nonreduced conditions was 255 kDa. Under reduced conditions, pamlin was cleaved to 180-, 52-, and 23-kDa subunits, indicating the 255-kDa protein is an SS-bonded heterotrimer. PMCs bound exclusively to the 52-kDa subunit. Mannose residues occur in the larger two subunits, but not in the smallest subunit. Pamlin does not run into 4% nondenatured PAGE gels, suggesting that the native glycoprotein forms a large polymeric supramolecular configuration in vivo. Immunohistochemistry showed that pamlin is seen on the entire basal lamina in the blastocoel and hyaline layer of mesenchyme blastulae. In vitro PMC migration assays on pamlin show that an optimum amount of pamlin for PMC migration was 2.5 μg/ml and that a synthetic RGDS peptide inhibited PMC migration dose dependently.
Primary mesenchyme cell migration in the sea urchin embryo: Distribution of directional cues
1994, Developmental BiologyThe directional migration of the primary mesenchyme cells (PMCs) of the sea urchin embryo is a critical step in the process of gastrulation. Although interactions between the migrating cells and the blastocoel environment are necessary for guiding the PMCs to their subequatorial target site, the nature of these interactions and the localization of guidance cues involved in directing the cells are not yet known. Previous studies have suggested that PMC migration is the result of random exploration and selective trapping at the target site by a pattern of adhesiveness in the ectoderm or basal lamina. To better characterize the distribution of guidance cues in the blastocoel we used a combination of timelapse microscopy microsurgery and fluorescence photoablation to study the behavior of the migrating cells. By using fluorescence time-lapse microscopy and a two-dimensional random-walk analysis of cell trajectories we demonstrated that fluorescently labeled PMCs injected near the animal pole move in a directed fashion over a relatively long distance to reach the target site. This suggests that guidance cues are distributed globally throughout the embryo and are not restricted to the immediate ring area. To further test this hypothesis we investigated the migratory behavior of PMCs that were prevented from interacting directly with the target site. First we examined the behavior of PMCs injected into animal embryo fragments lacking the target site. We found that PMCs move to the vegetal-most area of such embryo fragments regardless of their size. Second we studied the effects of photoablating a stripe of ectoderm between PMCs injected at the animal pole region (APR) and the target site. PMCs were found to accumulate along the ablated stripe and were unable to cross it for up to 6 hr after ablation. We also examined the migratory behavior of endogenous PMCs in embryos treated with lithium a vegetalizing agent which shifts the position of the PMC ring toward the animal pole. We found that PMCs accumulated along an ablated stripe of ectoderm positioned below the shifted target site suggesting that endogenous PMCs follow a set of directional cues to the target site which may be similar to those used by PMCs injected into the APR. As a whole these results suggest that migrating PMCs follow a set of directional cues that are widely distributed throughout the blastocoel and that maybe arranged in a gradient.
An N-linked carbohydrate-containing extracellular matrix determinant plays a key role in sea urchin gastrulation
1994, Developmental BiologyDuring the process of gastrulation in the sea urchin embryo, the vegetal plate invaginates to form the archenteron, a long narrow tube that extends across the blastocoel. Rearrangement of cells within the archenteron is thought to be a key component of the process of archenteron elongation. While these cell rearrangements have been well described, the mechanism of the rearrangements and the coordination of the movements of individual cells that results in the elongation of the archenteron are not well understood. We have identified a monoclonal antibody, called ECM 1, that recognizes an N-linked carbohydrate-containing epitope on several high-molecular-weight basal lamina glycoproteins. In an attempt to block the function of this determinant in development, the ECM 1 antibody was injected into the blastocoel of living embryos and the effects on morphogenesis were determined. Injection of intact ECM 1 IgG or monovalent Fab fragments blocks cell rearrangements during secondary invagination and cell movements during segmentation of the gut. Glycopeptides derived from the glycoproteins recognized by ECM 1 also inhibit cell rearrangements during secondary invagination when injected into the blastocoel. The inhibitory activity of these peptides is eliminated by digestion with N-glycosidase F or pronase, enzymes that also disrupt the ECM 1 determinant. ECM 1 recognizes a nonuniformly distributed determinant in the basal lamina and blastocoel matrix. This determinant is stored in cytoplasmic granules in the unfertilized egg and is deposited into the basal lamina by the blastula stage. The determinant becomes concentrated in the basal lamina in the vegetal region of the embryo early in gastrulation. At the prism stage, the determinant accumulates in the basal lamina and blastocoel matrix in the ventral region of the embryo. These data indicate that the vegetally localized, N-linked carbohydrate-containing determinant recognized by ECM 1 plays an important role in cell movements during archenteron morphogenesis in the sea urchin embryo.
Developmental regulation of lectin-binding patterns in Paracentrotus lividus gonads, gametes, and early embryos
1992, Acta HistochemicaBy use of several lectins (ConA, WGA, SBA, GS I, PNA), a study was carried on gametes and developing embryos of the sea urchin Paracentrotus lividus, to investigate developmental changes in cell surface, leading to changements in cell-environment interactions.
ConA, WGA, and SBA, with high affinity, bind to the vitelline membrane of unfertilized eggs, while PNA labelling at the same site is weak; GS I-binding is only present in the cytoplasm and cortical region of the unfertilized eggs. Immediately after fertilization, no ConA-binding is present in the membrane, while WGA- and SBA-binding molecules are located in the fertilization layer. In zygotes, 40 min after fertilization, ConA affinity sites were again present in both cytoplasm and cortical region. During cleavages and gastrulation, ConA binds to the blastomere cytoplasm and cortical region, to the intercellular matrix, and to the cytoplasm of mesenchyme cells. WGA binds to the cortical region of cleaving blastomeres, including the hyaline layer, up to the unhatched blastula. Then it labels the gastrula inner and outer surfaces. SBA binds to the blastomere membranes; no GS I- and PNA-binding was detected during embryonic development. Sperms are bound by all the lectins, except GS I. Mannose and glucose conjugates are the most represented throughout the whole development of P. lividus, and their origin and locations are developmentally regulated. Galacto-residues are scarcely represented or are masked by other terminal sugars (e.g. sialic acid), and become functional during particular developmental events (cell movements).
Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules
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These results were presented in part at the 1980 meeting of the International Society for Cell Biology, in West Berlin. This work was supported in part by funds from NSF grant PCM 77-01154 to M. S.