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Ultrastructural studies on the centrosome-attracting body: Electron-dense matrix and its role in unequal cleavages in ascidian embryos
Article first published online: 25 DEC 2001
Development, Growth & Differentiation
Volume 41, Issue 5, pages 601–609, October 1999
How to Cite
Iseto, T. and Nishida, H. (1999), Ultrastructural studies on the centrosome-attracting body: Electron-dense matrix and its role in unequal cleavages in ascidian embryos. Development, Growth & Differentiation, 41: 601–609. doi: 10.1046/j.1440-169x.1999.00457.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- ascidian embryo;
- centrosome-attracting body;
- electron-dense matrix;
- germ plasm;
- unequal cleavage.
In ascidian embryos, three successive unequal cleavages occur at the posterior pole, generating a specific cleavage pattern. A recently reported novel structure designated the centrosome-attracting body (CAB) has been suggested to play essential roles in the unequal cleavages attracting centrosomes and the nucleus towards the posterior pole. To examine the morphological features of the CAB, the ultrastructure of the CAB of two ascidian species, Halocynthia roretzi and Ciona intestinalis was observed by transmission electron microscopy. Detailed observations clarified that the electron-dense matrix (EDM) was a CAB-specific component that was commonly observed in the CAB of both species but was not found in other areas of the embryo. Further observations of the CAB in various staged embryos revealed that the ultrastructure was quite stable, with no difference between points of a cell cycle or between each stage from the 8- to 64-cell stage when unequal cleavage occurred. Observations of extracted embryos implied that the EDM was the extraction-resistant component of the CAB and was tightly anchored to the plasma membrane. It has been proposed that the EDM functions as a physical attachment site at the cell cortex for microtubules emanating from centrosomes and provides a scaffold for the centrosome-attracting machinery. Interestingly, the ultrastructure of the CAB resembled germ plasm reported in other animals, raising the possibility that the CAB-containing posterior-most blastomeres are germ- line precursors.
The positioning of the cell division plane is one of several important issues in cell and developmental biology. Stereotyped cleavage patterns are observed in embryos of various kinds of organisms. The orientation of cleavages determines the overall organization of the embryos. The positioning of the cleavage planes also plays important roles in cell fate determination in two ways ( Nishida et al. 1999 ). First, it ensures the proper partitioning of localized developmental determinants, resulting in asymmetric cell divisions ( Freeman 1979). Second, it generates proper spatial arrangement of interacting cells.
The cleavage pattern of ascidian embryos is unique and invariant ( Conklin 1905; Satoh 1979). In ascidian embryos, three successive unequal cleavages occur at the posterior-most blastomere pairs from the fourth to sixth cleavages, always producing a smaller blastomere posteriorly. Hibino et al. (1998) reported that the occurrence of these unequal cleavages was closely related to the existence of a novel structure termed the centrosome-attracting body (CAB), which exists in the posterior cortex of the unequally cleaving blastomeres. At interphase, microtubules emanated from one centrosome are focused on and attached to the CAB. The centrosome-nucleus complex is attracted towards the CAB by means of shortening of the microtubule bundle. Consequently, the mitotic apparatus is formed eccentrically, resulting in unequal cleavage. Anti-kinesin antibody recognizes the CAB, suggesting that some kinesin-like molecule exists in the CAB ( Nishikata et al. 1999 ). The precursors of the CAB are first recognizable as many granules at the posterior cortex as early as the 2-cell stage. These granules aggregate gradually and form the CAB in the posterior (B4.1) blastomere pair at the 8-cell stage. The CAB are then present at least until the gastrula stage ( Hibino et al. 1998 ). The posterior vegetal cytoplasm (PVC) of fertilized eggs that has completed ooplasmic segregation directs the formation of the CAB, and is consequently involved in unequal cleavages ( Nishida 1994; Nishikata et al. 1999 ). Removal of PVC causes loss of the CAB and unequal cleavage, while transplantation of this cytoplasm results in formation of the ectopic CAB and in ectopic unequal cleavage. Experiments using cytoskeletal inhibitors revealed that the process of formation of the CAB is independent of microtubules, while maintenance of the integrity of the CAB requires microfilaments ( Nishikata et al. 1999 ).
In the present report, we observed the structure of the CAB of Halocynthia roretzi and Ciona intestinalis. The CAB represents a unique cytoplasmic structure that exists just beneath the plasma membrane and is composed of an electron-dense cytoplasm that is not bounded by a limiting membrane. We compared the CAB of Halocynthia and Ciona, which are distantly related ascidian species in phylogeny. Some common components among the two species that are thought to be general features of the CAB have been identified. We propose that one component specific to the CAB is the electron-dense matrix (EDM), and that it is important to CAB function. We also examined the existence of temporal change in the CAB structure during a cell cycle and during cleavage stages. The ultrastructure of the CAB was quite stable at all stages observed.
Materials and Methods
Adult ascidians, H. roretzi, were collected near the Asamushi Marine Biological Station of Tohoku University, Aomori, Japan, and Otsuchi Marine Research Center of Tokyo University, Iwate, Japan. Naturally spawned eggs were artificially fertilized with sperm of other individuals. Eggs are about 280 μm in diameter. Fertilized eggs were devitellinated by treatment with seawater containing 1% sodium thioglycolate and 0.05% actinase E at a pH of about 10 for ∼ 10 min ( Mita-Miyazawa et al. 1985 ). After washing, eggs were reared in agar-coated plastic dishes filled with Millipore-filtered (pore size, 0.45 μm) seawater containing 50 μg/mL streptomycin sulfate and 50 μg/mL kanamycin sulfate at 13°C. Embryos reached the 8-cell stage at 3.5 h after fertilization.
Adult C. intestinalis were collected at Tokyo Bay, and in the vicinity of the Onagawa Fisheries Laboratory of Tohoku University, Onagawa, Japan. Eggs and sperm were obtained by dissection of the gonoducts. The eggs were about 130 μm in diameter. Fertilization, devitellination and culture of the embryos were conducted as described earlier. Embryos were reared at 18°C.
Extraction of embryos
Extraction of embryos was carried out as previously described in Hibino et al. (1998) . Devitellinated embryos were rinsed twice with Ca2+-free artificial seawater containing 1 m M ethyleneglycol-bis-(β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and then transferred to an extraction buffer composed of 50 m M MgCl2, 10 m M KCl, 10 m M EGTA, 2% Triton-X100, 20% glycerol, and 25 m M imidazole at pH 6.9 until the embryos became transparent after ∼ 2–3 h. Extracted embryos were mounted in glycerol and observed under Nomarski optics.
Devitellinated embryos were fixed for 1–2 h at 13°C in 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M PIPES buffer (pH 7.4) with 0.45 M sucrose, then postfixed in 1.0% OsO4 in the same buffer for 1 h on ice. They were dehydrated in graded ethanol and embedded in Spurr’s resin. Ultra-thin sections were obtained with an LKB V ultramicrotome, and stained with uranyl acetate and lead citrate. They were observed with a JEOL 1200EX transmission electron microscope.
CAB in Ciona embryos
The CAB was at first found in the embryos of H. roretzi as a small cytoplasmic structure that was relatively resistant to detergent extraction and recognizable under a Nomarski optical microscope ( Hibino et al. 1998 ). Ciona intestinalis is a cosmopolitan ascidian species. Halocynthia belongs to the order Pleurogona, Ciona to Enterogona. Therefore, we chose Ciona, which is distantly related to Halocynthia in phylogeny, for comparison of CAB structure in order to extract common features of the CAB. To elucidate whether the CAB also exists in Ciona in similar fashion to Halocynthia, we extracted Ciona embryos and observed them with a Nomarski microscope. The CAB was found in all embryos from the 8- to 64-cell stage ( Fig. 1A–D) and its position was almost the same as that in Halocynthia. The CAB existed on both sides of the midline of the embryo at the posterior pole. A small difference was noticed in the 8-cell stage embryo in which the CAB was present slightly vegetal to that in Halocynthia ( Fig. 1A; cf. Fig. 5a in Hibino et al. 1998 ). Essentially, similar events that have been reported in Halocynthia embryos ( Hibino et al. 1998 ) were also observed in unequal cleavages in Ciona embryos. Nuclei migrated towards the CAB at interphase, and one pole of the mitotic spindles was attached to the CAB during mitosis.
Comparison of ultrastructure of CAB in Halocynthia and Ciona
Details as to the ultrastructure of the CAB of two ascidian species, Halocynthia and Ciona were obtained by transmission electron microscopy.
In Halocynthia 16-cell embryos, the CAB were clearly recognized as electron-dense cytoplasmic masses in the posterior cortex of the posterior-most blastomeres (B5.2 blastomeres; see Fig. 1B for Ciona embryo) at low magnification ( Fig. 2A). The CAB was not bounded by a limiting membrane ( Fig. 2B). Some protrusions were observed on the cell surface underlined by the CAB ( Fig. 2B, arrows). Mitochondria, reported to be abundant in the posterior cytoplasm known as myoplasm ( Reverberi 1956; Mancuso 1962, 1964, 1969; Zalokar & Sardet 1984) were found around but never within the CAB. Three types of matrices that showed low, medium and high electron density were distinguishable within the CAB ( Figs 2B,3A). Granules with the appearance of ribosomes ( Fig. 3A, arrowhead) were mainly embedded in the matrix of medium density. Endoplasmic reticulum (ER)-like membranous structures (arrows) were also abundant in low- and medium-density matrices. In addition to these, several types of vesicles were sometimes observed in the CAB ( Fig. 2B, asterisk).
We could not visualize the microtubules in the present study, although they have been shown to connect the centrosome and CAB by staining with anti-α- tubulin antibody ( Hibino et al. 1998 ). We tried several fixation methods, but did not obtain good results. Microtubules in ascidian embryos were unable to be fixed by the methods we used.
In Ciona 16-cell embryos, the CAB were recognized as distinctive cytoplasmic masses from which mitochondria were excluded, like in Halocynthia ( Fig. 2C,D). The fixation seemed much better in Ciona embryos for unknown reasons. The structure of the CAB showed overall similarity to Halocynthia CAB. Three kinds of matrices were distinguished ( Figs 2D,3B). In detail, the high-density matrix did not seem homogeneous and many electron-dense spots were observed within the matrix in Ciona, although those spots were obscure. Many ribosome-like granules mainly in medium-density matrix and ER-like structures in low- and medium- density matrices were present ( Fig. 3B). However, the vesicles observed in Halocynthia CAB were not found in Ciona CAB.
Common morphological features of the CAB in these two species were the three types of matrices, ER-like membranous structures and ribosome-like granules. Among the matrices, those of low and medium density were not specific to the CAB, but rather also present in the cytoplasm surrounding the CAB ( Fig. 2), as well as being contiguous in and out of the CAB at the boundary ( Fig. 4). Similarly, ER-like membranous structures and ribosome-like granules were also present in adjacent cytoplasm ( Fig. 4). By contrast, a high- density matrix was not observed in the cytoplasm other than in the CAB region. It seems that the only CAB- specific component is the high-density matrix.
CAB in extracted embryos
The CAB was first found in extracted embryos under light microscopy ( Hibino et al. 1998 ). It was thought that some components of the CAB were resistant to extraction. Therefore, extracted embryos were observed by electron microscopy. The extracted embryos were almost transparent. As large Halocynthia embryos were much easier to handle, the ultrastructure of the CAB in extracted embryos was observed in Halocynthia. In extracted embryos at the 16-cell stage, most of the cytoplasm was removed and only remnants of organelles including the nucleus and mitochondria were observed throughout the embryo ( Fig. 5A). At the posterior pole, the CAB was clearly distinguishable. It remained at the cortex although surrounding cytoplasm was extracted. Most components of the CAB were also extracted ( Fig. 5B). However, the matrix of high density that was observed in non-extracted embryos looked resistant to extraction treatment. Many ribosome-like granules were stuck to the surface of the matrix.
Ultrastructure of the CAB at various cell cycle stages
The centrosome-nuclear complex translocates towards the CAB at interphase. During mitosis, one pole of the mitotic apparatus is still anchored to the CAB, and unequal cytokinesis occurred. Therefore, it was expected that morphological changes to the CAB might occur during each cell cycle at the ultrastructural level. We observed Ciona CAB at different points during the 16-cell stage.
At early interphase, the CAB was observed just beneath the plasma membrane with an elongated shape ( Fig. 6A). At late interphase the CAB exhibited a more compact shape at the midline of the embryo. The nuclei were very close to the CAB at this phase ( Fig. 2C). Both at metaphase and telophase the CAB became more compact at the midline of the embryo ( Fig. 6B,C). Change in the CAB shape and position corresponded to the optical observation in Ciona as well as in Halocynthia embryos ( Hibino et al. 1998 ). At higher magnification we could not detect any change in the CAB components. The three types of matrices, ER-like membranous structures, and ribosome-like granules exhibited the same pattern as in Fig. 3(B). Although the CAB changed its shape and position, the ultrastructures of its component did not differ throughout the cell cycle.
Ultrastructures of CAB at the 8- to 64-cell stage
Three successive unequal cleavages occurred between the 8- and 64-cell stage in the posterior-most blastomere pair. To elucidate whether the CAB changes its morphology during these stages, we observed Ciona CAB at various stages with transmission electron microscopy (TEM).
At the 8-cell stage, the CAB was observed at the posterior cortex of the posterior-vegetal blastomeres (B4.1 blastomeres; Fig. 7A). Some protrusions were seen on the cell surface underlined by the CAB as observed in the 16-cell stage embryo of Halocynthia ( Fig. 2B). The CAB at the 16-cell stage is shown in Figs 2(C) and 6. At the 32-cell stage, the CAB was observed in the posterior-most blastomere pair (B6.3 blastomeres) to be of compact shape near the midline of the embryo ( Fig. 7B). It seemed that the CAB maintained its position and shape observed at metaphase and telophase of the 16-cell stage ( Fig. 6B,C). At the 64-cell stage, the CAB were detected in the posterior-most blastomere pair (B7.6 blastomeres). At this stage, the CAB seemed detached from the cell cortex ( Fig. 7C). Detail observation at higher magnification did not reveal any ultrastructural differences in the components of the CAB at different stages. Those were indistinguishable from Fig. 3(B).
Electron-dense matrix is a CAB-specific component
Unequal cleavages involve the eccentric positioning of the mitotic apparatus. The physical attachment site for attraction of the spindles or centrosome-nuclear complex is thought to exist at the cell cortex. Although the presence of special cortical sites has been suggested repeatedly for various organisms ( Dan 1979; Dan et al. 1983 ; Schroeder 1987; Hyman 1989; reviewed by Strome 1993; Gönczy & Hyman 1996), no structure has been visualized so far. The CAB is the first visible structure found in Halocynthia embryos to be detected at the cortical attachment site. The CAB was also present in Ciona embryos with almost the same pattern as in Halocynthia embryos. Class Ascidiacea comprises two orders. As Halocynthia and Ciona belong to different orders (Pleurogona and Enterogona, respectively), it is likely that the CAB is a common structure at least among class Ascidiacea.
Information about the morphology of this novel structure will provide an insight into how the CAB functions. The CAB was a unique cytoplasmic structure existing just beneath the plasma membrane, and was not bounded by a limiting membrane. Observation of the CAB in Halocynthia and Ciona embryos enabled us to list three features common to the two species: (i) matrices with three types of electron density; (ii) ER-like membranous structures; and (iii) ribosome-like granules. Among them, the matrix with high electron density (electron-dense matrix, EDM) was shown to be a CAB-specific component by comparing the CAB to other regions in the embryos. Other types of matrices, ER-like structures and ribosome-like granules also existed in the cytoplasm surrounding the CAB. This result suggests that the EDM is important for CAB function.
Observation of the CAB in extracted embryos under an electron microscope revealed that some extraction-resistant components remained in the CAB region, while most components in other cytoplasmic regions were removed by extraction. This suggests that some components of the CAB are firmly anchored to the cell cortex. At higher magnification ( Fig. 5B), matrices with many ribosome-like granules stuck to their surface were observed. Interestingly, the extraction-resistant matrix quite resembles the EDM (cf. Fig. 3A). One may assume that the CAB is a sponge-like structure. The EDM corresponds to sponge material. This architecture would provide a scaffold for various CAB components. Interspace between the sponge-like EDM might be filled with ordinary cytoplasm that also surrounds the CAB and is not specific to the CAB. The ordinary cytoplasm in the CAB is likely to be extracted as well as surrounding cytoplasm. Together with the finding that the EDM is specific to the CAB, we propose that the EDM functions as a physical attachment site for attraction of the centrosome-nuclear complex to the cell cortex. Microtubule bundles emanating from centrosomes might be captured by the EDM during unequal divisions.
An antibovine kinesin antibody is known to recognize the CAB ( Nishikata et al. 1999 ). Although we have no direct evidence for the existence of kinesin-like molecules in the CAB, the microtubule motor kinesin is an attractive candidate for the molecule that associates with the EDM, contributing to the attraction of the centrosome-nuclear complex by slipping on the microtubules with itself anchored to the EDM. To understand the mechanism of unequal cleavages in ascidian embryos, the molecules anchored to the EDM and molecules composing the EDM itself must be identified.
CAB is morphologically stable throughout the succession of unequal cleavages
It was expected that the CAB would show morphological change during a cell cycle. Although the CAB changed its shape during the 16-cell stage, we could not detect any difference in the CAB at the fine structural level. This suggests that no marked change occurs in the CAB structure during the cell cycle, and that the function of the CAB is controlled at the molecular level. It was also shown that the ultrastructures of the CAB are not distinctive between the 8-, 16-, 32- and 64-cell stages. This is reasonable because the CAB seems to work in the same manner during three successive unequal cleavages. Again it was shown that the CAB is quite a stable structure during cleavage stages. At the 64-cell stage, the CAB looked detached from the plasma membrane. This observation coincides with the fact that the CAB has finished its role in unequal cleavages, because after the 64-cell stage, the CAB-containing blastomere, B7.6, does not further divide during embryogenesis ( Nishida 1987).
Ultrastructure of CAB resembles that of germ plasm in other animals
It is worth noting that the ultrastructure of the CAB looks like that of germ plasm or nuage material known to be germline-specific cytoplasm in many organisms. Germ plasm in animals such as Drosophila ( Mahowald 1962), amphibia ( Mahowald & Hennen 1971), and Caenorhabditis elegans ( Wolf et al. 1983 ), and nuage materials in animals of various phyla consist of an electron-dense cytoplasm that is not bounded by limiting membranes (reviewed by Eddy 1975). This suggests that the cytoplasm composing the CAB is germ plasm in ascidians. Moreover, a correlation between localization of germline-specific cytoplasms and cleavage plane specifications has been postulated in various organisms. In C. elegans, P granules always segregated into smaller blastomeres under experimental conditions when cleavages were perturbed ( Hill & Strome 1990). In Xenopus embryos, the germ plasm was observed to associate with one pole of the spindle in most dividing presumptive germ cells, resulting in the germ plasm segregating into one of the two daughter cells during successive divisions ( Whitington & Dixon 1975). The correlation has also been pointed out in fly and Cyclops (reviewed by Beams & Kessel 1974; Kobayashi et al. 1994 ). These facts make it plausible that the cytoplasm composing the CAB is germ plasm. The germline origin in ascidians is not yet known. The resemblance of the CAB to germ plasm raises the possibility that the posterior-most two blastomeres (B7.6 pair) are germline progenitor cells.
Localization of various maternal mRNA to CAB
It was recently reported that various kinds of maternal mRNA are localized to the posterior pole of the ascidian embryo during the cleavage stage ( Yoshida et al. 1996 ; Satou & Satoh 1997; Sasakura et al. 1998a , b). Their localization seems to coincide with the position of the CAB. It is important to know whether these mRNA are present in the CAB. The existence of many ribosome-like granules in the CAB would suggest that those mRNA are to be translated within the CAB.
We thank Dr Numakunai and all other members of the Asamushi Marine Biological Station for facilitating our work there. Thanks are also expressed to the members of the Otsuchi Marine Research Center for the supply of live materials. This work was supported by the ‘Research for the Future’ Program of the Japanese Society for the Promotion of Science (96L00404).
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