Developmental changes in localization of the main ganglioside during sea urchin embryogenesis

Maiko Nezuo1, Hidehiko Shogomori2, Motonori Hoshi3, Toshihiro Yamamoto4, Tadashi Teshima4, Tetsuo Shiba4 and Kazuyoshi Chiba

Department of Biology, Ochanomizu University, 2–1–1 Otsuka, Bunkyo-ku, Tokyo, 112–8610, Japan, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, 3Center for Life Science and Technology and Department of Chemistry, Keio University, Hiyoshi 3–14–1, Kouhoku-ku, Yokohama 223–8522, Japan, and 4Peptide Institute, Inc., Protein Research Foundation, 4–1–2 Ina, Minoh-shi, Osaka, 562–8686, Japan

Received on May 9, 2000; revised on July 10, 2000; accepted on July 13, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ganglioside M5 (NeuGc{alpha}2–6Glcß1–1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.

Key words: ganglioside/endocytosis/extracellular matrix


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Gangliosides exist predominantly on the outer leaflet of the plasma membrane, and play important roles in cell–cell or cell–ECM interaction (Hakomori, 1981Go, 1990, 1993; Fishman, 1986Go). Ganglioside M5 (M5: previously called M5 ganglioside; Kubo and Hoshi, 1990Go; Kubo et al., 1990Go; Shogomori et al., 1993Go, 1997) is the main ganglioside in sea urchin and sand dollar eggs, constituting more than 90% of the egg gangliosides and 0.8% or more of the egg dry weight (Kubo et al., 1990Go). In unfertilized eggs, ganglioside M5 has been shown to localize in the plasma membrane, endoplasmic reticulum (ER) and yolk granules (Shogomori et al., 1993Go, 1997). At least half of M5 localizes in the yolk granules and associates with yolk lipoprotein (Shogomori et al., 1997Go). However, the distribution of M5 during embryogenesis has been unclear. Revealing the distribution of M5 during embryogenesis is an important step in evaluating the significance of gangliosides in early development.

In sea urchin eggs, yolk granules have been thought to provide the developing embryos with building materials or nutrients. Recent studies show that yolk granules store the components of the embryonic ECM. A 32 kDa protein that is a component of the sea urchin embryonic ECM is stored in yolk granules of unfertilized eggs (Mayne and Robinson, 1998Go). Also, a 22S glycoprotein in sea urchin yolk granules, called toposome, is released to the cell surface of the blastula (Gratwohl et al., 1991Go). Similarly, in Xenopus embryos, a 43–45 kDa lectin, detected in association with yolk platelets (Roberson and Barondes, 1983Go), is secreted into the ECM in embryos through gastrulation (Outenreath et al., 1988Go). Galectins, a family of lectins from Rana catesbeiana oocytes, which are mainly distributed extracellularly in several organs, are also found in yolk platelets of growing oocytes in the ovary (Uchiyama et al., 1997Go). Thus, we hypothesized that M5 in yolk granules is released to the ECM with these proteins. To examine this possibility, we performed fluorescent immunocytochemistry using an anti-M5 antibody and embryo cryosections. As expected, bright fluorescence was observed at the outer surface of embryos. Immunoelectron microscopy confirmed that M5 was localized in the embryonic ECM. These results also indicate that released M5 interacts with the ECM.

Fluorescent-labeled gangliosides are used to visualize the localization of gangliosides in living cells. In this study, we used NBD-M5 to observe M5 in the ECM during embryogenesis and found that it was incorporated into endocytotic vesicles.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Localization of ganglioside M5 in embryos
To study the localization of M5 in blastula and gastrula embryos, we stained cryosections of embryos with the anti-M5 monoclonal antibody. As shown in Figure 1, the outer surface of the embryos (or the apical surface) and the cytoplasm were intensely labeled with the antibody. In unfertilized eggs, M5 is not detected at the outer surface, although it is clearly demonstrated in the cytoplasm (Kubo and Hoshi, 1990Go; Shogomori et al., 1993Go). Thus, it is likely that M5 was partly released to the outer surface during embryogenesis. To reveal the ultrastructural localization of M5 in blastula embryos, we performed immunoelectron microscopy. Colloidal gold particles were observed throughout the electron-dense structures of the ECM (Figure 2), indicating that M5 was transported to the cell surface during embryogenesis. Also, these results suggest that M5 interacted with the ECM.



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Fig. 1. Localization of M5 labeled with the anti-M5 antibody, SUG-44. Frozen section of sea urchin blastula after hatching (A, B) and gastrula (C, D) labeled with SUG-44 were imaged by fluorescent microscopy (A, C). Differential interference contrast images (B, D) are also shown. A-2 and C-2 are higher magnification of boxed areas in A-1 and C-1, respectively. Bright fluorescence is seen in the outer surface of the embryo (A-1, C-1; arrows). As was reported previously in unfertilized eggs, anti-M5 antibody stained the whole cytoplasm (A-2, C-2; CY), but not the nucleus (A-2, C-2; N), of all cells in the embryos. Scale bar, 20 µm.

 


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Fig. 2. Electron micrograph of sea urchin blastula embryo. Using rapid freezing, freeze substitution, and postembedding labeling technique, the ultrathin section was labeled with SUG-44 and a gold-conjugated anti-mouse IgG secondary antibody. Immunogold labeling for M5 (*) appears to be associated with the ECM. PM, Plasma membrane. Scale bar, 0.5 µm.

 
Uptake and intracellular transport of fluorescent ganglioside M5 analogue
To examine the fate of extracellular M5, we used fluorescent-labeled M5 (NBD-M5: Figure 3). Fertilized eggs were bathed in artificial sea water (ASW) containing NBD-M5 for 20 min and washed with NBD-M5-free ASW. When they were observed with a confocal microscope, strong signals in the ECM (hyaline layer) were detected (Figure 4C,D). Plasma membrane was also likely to be stained by the M5 analogue. On the other hand, weak signals were observed in the plasma membrane and/or the vitelline layer of unfertilized eggs treated with NBD-M5 (Figure 4A,B). These results suggest that the M5 analogue as well as the endogenous M5 strongly interacted with ECM formed after fertilization. The fluorescence was also seen in "dotlike" structures scattered throughout the subcortical region (Figure 4C, inset; arrow), indicating that NBD-M5 was transported from the ECM and/or the plasma membrane to the subcortical region of fertilized eggs. On the other hand, in unfertilized eggs, uptake of NBD-M5 did not occur (Figure 4A). Thus, incorporation of NBD-M5 is likely to be triggered by fertilization.



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Fig. 3. Structure of fluorescent-labeled M5.

 


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Fig. 4. Unfertilized and fertilized sand dollar eggs labeled with NBD-M5. Unfertilized or fertilized eggs were incubated with NBD-M5 for 20 min, and then washed with ASW. The same field of the unfertilized egg was examined under the confocal microscope (A) and the bright-field microscope (B). A similar experiment was performed using fertilized eggs (C and D). NBD-M5 was localized in the hyaline layer (H), plasma membrane (P) and intracellular vesicles (C, inset; arrow). F, Fertilization envelope. Scale bar, 10 µm.

 
Incorporation of NBD-M5 into endocytotic vesicles
It is well known that a burst of exocytosis at fertilization is followed by a burst of endocytosis. Therefore, we hypothesized that the punctate fluorescence in Figure 4 was a consequence of the incorporation of NBD-M5 into endocytotic vesicles. Since the endocytotic vesicles after fertilization are distinguished by a dye uptake assay, using an extracellular fluid-phase dye, Rh-dex (Whalley et al., 1995Go), we treated the fertilized eggs with NBD-M5 (green) and tetramethylrhodamine dextran (Rh-dex, red) simultaneously. If the punctate fluorescence of NBD-M5 was due to endocytosis, the distribution of NBD-M5 should be similar to that of Rh-dex. As expected, when a confocal optical section of the subcortical region of the egg bathed in ASW containing NBD-M5 and Rh-dex was captured on two channels of a confocal microscope, the distribution of green structures in the cytoplasm (Figure 5A, arrow and arrowhead) and that of red ones were almost identical (Figure 5B, arrow and arrowhead). The composite image of Figure 4A and Figure 4B showed yellow spots (Figure 5C, arrow and arrowhead) confirming the colocalization of Rh-dex and NBD-M5 in the endocytotic vesicles. Some NBD-M5 fluorescent structures were "ringlike" (Figure 5A, arrowhead), while those of Rh-dex were "dotlike" (Figure 5B, arrowhead). Similar ringlike images are reported to be due to endosomal vesicles originating from the plasma membrane labeled with the lipid fluorescent dye (Terasaki, 1995Go; Whalley et al., 1995Go). Thus, the "ringlike" vesicles stained with NBD-M5 represent plasma membrane endocytosis. Spots or "dotlike" vesicles may contain ECM stained with NBD-M5.



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Fig. 5. Colocalization of NBD-M5 and Rh-dex. Fertilized sand dollar eggs were incubated in ASW containing both NBD-M5 (0.1 mg/ml) and Rh-dex (0.3 mg/ml). Using a confocal microscope, separate photographs of a given field were obtained to visualize NBD-M5 (A) and Rh-dex (B). The composite image of (A) and (B) is shown in (C). NBD-M5 was concentrated in the ECM (A*), indicating a high affinity of M5 to the ECM, while the concentration of Rh-dex in the ECM was the same as that of the extracellular area without ECM (B). The inner area of the ECM corresponds to the cytoplasm, where "dotlike" (arrow) and "ringlike" (arrowhead) structures are seen (A). Scale bar, 10 µm.

 
Localization of NBD-M5 during development
To observe the localization of NBD-M5 in living embryos, fertilized eggs prelabeled with NBD-M5 were grown in ASW without the dye (Figure 6). At the end of mitosis (first cell division), the thick ECM (hyaline layer) near the cleavage furrow was stained (Figure 6A arrow). Similar thick layers of staining were observed in 2-, 4-, and 8-cell stage and blastula embryos (Figure 6C,E,G,I, arrows). In the 2-cell stage embryo, the thin layer probably corresponding to the plasma membrane was clearly visible (Figure 6C, arrowhead). Similar membranes were also observed in the 8-cell stage embryos (Figure 6G, arrowhead) and the blastula (Figure 6I,K, arrowheads). Also, many vesicles in the cytoplasm were stained by the dye (Figure 6K). These vesicles probably originated from endocytotic vesicles as shown in Figure 5. The strong NBD-M5 labeling can be observed in the apical side throughout the early development but not in the basal side of embryos, even after a long incubation. This indicates that the NBD-M5 does not diffuse from the apical side to the basal side, suggesting that M5 specifically and strongly associates with the apical ECM.



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Fig. 6. Detection of NBD-M5 in early development with a confocal microscope. Sand dollar embryos at the end of mitosis (first cell division) (A and B), 2-cell stage (C and D), 4-cell stage (E and F), 8-cell stage (G and H) and before hatching (IK). (K) is a higher magnification of the cortical region of (I). The localization of NBD-M5 did not change. (B, D, F, H, J) are corresponding bright-field images. Scale bar, 10 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
After fertilization, cleavage occurs and the contents of yolk granules are partly released to the surface of cells (Outenreath et al., 1988Go; Gratwohl et al., 1991Go; Uchiyama et al., 1997Go; Mayne and Robinson, 1998Go). Then the yolk granules disappear in the feeding pluteus stage embryos (Scott et al., 1990Go). M5, however, is quantitatively constant during embryogenesis (Hoshi and Nagai, 1970Go). Thus, M5 in yolk granules is likely to be transported to other cellular components during development.

In this study, using a specific antibody against M5, we showed that M5 was distributed in the embryonic ECM. Since M5 exists in unfertilized eggs in yolk granules (Shogomori et al., 1997Go), we concluded that it was transported from the yolk granules to the ECM and/or the plasma membrane after fertilization.

A number of studies have shown the role of gangliosides as mediators in the interaction of various cells with the ECM. Cheresh et al. (1986)Go demonstrated that anti-GD2 and GD3 monoclonal antibodies inhibit cell-substratum interaction in melanoma and neuroblastoma cells. Furthermore, Cheresh and Klier (1986)Go showed that GD2 is localized directly on the microprocesses that make contact with the fibronectin substrate on human melanoma M21 cells. Spiegel et al. (1985)Go demonstrated that mouse fibroblasts (NCTC 2071A cells), which are deficient in gangliosides and cannot retain endogenously synthesized fibronectin at the cell surface, can recover the ability of fibronectin organization when exogenous gangliosides are added to the culture medium. Also, they reported that exogenous fluorescent gangliosides are distributed on the fibrillar network of fibronectin, indicating a high affinity of gangliosides to fibronectin. Since the sea urchin embryo is surrounded by several ECM components, such as molecules similar to vertebrate fibronectin, collagen, and laminin (Wessel et al., 1984Go), M5 may be involved in the organization of these molecules, although it remains to be determined whether M5 actually interacts with these proteins. Hyalin is the major protein in the embryonic ECM and is sensitive to Ca2+ for its polymerization in sea urchin (Kane, 1973Go; McClay and Fink, 1982Go). To determine whether M5 interacts with hyalin, we treated embryos with Ca2+-free ASW. M5, however, was not detected significantly in the Ca2+-free ASW extract (data not shown), suggesting that M5 interacts with the ECM component(s) that are insensitive to Ca2+. It has been reported that the apical and basal ECM consist of different components (Matese et al., 1997Go). The strong labeling by the antibody and fluorescent analogue is observed only in the apical area, suggesting the polarized localization of M5 in sea urchin embryos and the specific interaction with components of the apical ECM. The polarized localization of sphingolipids has also been reported in epithelial cells (reviewed in Simons and van Meer, 1988Go).

Since the contents of yolk granules are released to the ECM during embryogenesis, lipoprotein is also likely to be released to the ECM. If this is the case, lipoprotein may be the component of the embryonic ECM that plays a role in a ganglioside reservoir.

In this study, localization of endogenous M5 in the ECM was mimicked by NBD-M5, which encouraged us to examine the fate of extracellular M5 using NBD-M5. After fertilization, NBD-M5 in the ECM was internalized in the cells by endocytosis, suggesting that extracellular M5 was transported from the ECM to endocytotic vesicles. Although the fate of endocytotic vesicles of sea urchin embryo is still unknown, ganglioside M5 in the vesicles is likely to be used to make the body of the embryos or adult organs. Indeed, M5 is found in the somatic cells of adult esophagus and testis (Kubo et al., 1990Go). It is also possible that M5 in the endocytotic vesicles may be metabolized and change their structure in the recycling pathway. Further studies are necessary to reveal the metabolic and recycling pathway of the ganglioside.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Animals and gametes
Sea urchin Hemicentrotus pulcherrimus and sand dollar Clypeaster japonicus from the Pacific coast of Chiba prefecture, Japan were used. Eggs and sperm were obtained by intracoelomic injection of 10 mM acetylcholine. Eggs were kept in artificial sea water (ASW; JAMARIN-U, Jamarin Laboratory, Osaka, Japan) and sperm were collected dry. For fertilization, eggs were perfused with ASW containing a 1:1000 dilution of sperm. The embryos were cultured in ASW at 20°C.

Monoclonal antibody
The culture supernatant of the hybridoma was used as the source of the anti-M5 antibody (SUG-44, IgM). The characterization of this antibody was performed previously (Shogomori et al., 1997Go).

Immunofluorescence microscopy
Embryos were fixed with 4% paraformaldehyde, 0.5% glutaraldehyde and 7% sucrose in ASW for 2 h on ice. After washing with ASW, fixed embryos were sequentially transferred to 5%, 10%, and 15% sucrose in phosphate-buffered saline (PBS) for 30 min each and 20% sucrose/PBS:O.C.T. Compound (Miles Laboratories) (2:1) overnight on ice. Embryos were quickly frozen in liquid nitrogen and stored at –20°C. The embedded embryos were cut into 6 µm sections with a Minotome (Damon/IEC Division) at –20°C. The sections were incubated with SUG-44 and then with rhodamine-conjugated anti-mouse IgM Ab (Cappel, Malvern, PA).

Immunoelectron microscopy
Fixed embryos were subjected to rapid-freezing and freeze-substitution, and then embedded in LR-White as described previously (Shogomori et al., 1997Go). Ultrathin sections were cut with an ultramicrotome (LKB), placed on Formvar-coated nickel grids (150- or 200-mesh), and labeled with SUG-44 and 15-nm diameter colloidal gold-conjugated anti-mouse IgG + IgM Ab (Cappel). Sections were stained with uranyl acetate and lead citrate. Labeled and stained sections were examined and photographed with a JEOL 1200EX electron microscope at 80 kV.

Confocal microscopy
For confocal microscopy, zygotes and embryos labeled with NBD-M5 were held between two coverslips separated by double-stick tape and observed with a confocal microscope (TCS-NT, Leica, Germany).

Fluorescent ganglioside M5
NBD-M5 was synthesized from Neu5Gc{alpha}2–6Glc derivative and phytosphingosine (Yamamoto et al., unpublished observations) and its structure is shown in Figure 3.

Labeling eggs with NBD-M5
Unfertilized or fertilized eggs were incubated with ASW containing 0.1 mg/ml of NBD-M5 for 20 min. Then they were washed three times with ASW. After incubation for the indicated period, they were observed with a confocal microscope.

Endocytosis assay
NBD-M5 (0.1 mg/ml) and tetramethylrhodamine dextran (Rh-dex; MW 3000; Molecular Probes, Eugene, OR) (0.3 mg/ml) were dissolved in ASW. Immediately after fertilization in ASW, the eggs were placed in ASW containing NBD-M5 and Rh-dex. After 20 min, the eggs were observed with a confocal microscope.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are grateful to Dr. S.Nemoto, Tateyama Marine Biological Station, Ochanomizu University, for his help in collecting the animals. This work was supported in part by grants from the Ministry of Education, Culture, Sports and Sciences, Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ER, endoplasmic reticulum; ECM, the extracellular matrix; M5, ganglioside M5; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NBD-M5, NBD-labeled-M5; ASW, artificial sea water; Rh-dex, tetramethylrhodamine dextran; PBS, phosphate-buffer saline.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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