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Fig. 1. Novel microtubule (MT) structures in cytoplasmic and cortical reorganization. Schematic drawing of cytoplasmic and cortical reorganization. In ascidian eggs, cytoplasmic and cortical reorganization have pivotal roles for the translocation of myoplasm (yellow), and actin filaments (red) and MTs (green) drive these movements. Photographs represent novel MT structures (red rectangles; DEM, TAF, and CAMP: Ishii et al., 2017). Embryos were stained for MT (green) and myoplasm (red), which was detected by the mitochondrial marker, anti-MnSOD antisera, because the myoplasm is mitochondria-rich. Scale bar, 50 µm.
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Fig. 2. Changes of microtubule (MT) structures during the first cell cycle. (A) Changes of meiotic and mitotic apparatuses and the nucleus during the first cell cycle in a fertilized Ciona intestinalis egg. Embryos were fixed at various stages and stained for MT (green) and chromatin (blue). Three-dimensional (3D) models rendered from confocal images are shown. Scale bar, 20 µm. (B, B') Progression of meiotic and mitotic cell cycle events were quantitatively analyzed and the duration of each event was estimated. PNfo, PNm, PNfu, and the suffix 1 denote pronuclear formation, pronuclear migration, pronuclear fusion, and mitosis of the first cell cycle, respectively. Three independent experiments were carried out and in total, more than 60 embryos were analyzed at each time point. Error bars represent standard deviation (SD; n = 3). Dotted line of Prometa 1 in B' indicates duplicated sperm centrosomes. (C) The concurrence of cell cycle events and novel MT structures was quantitatively analyzed. The population of embryos that showed each novel MT structure in each embryo stage were indicated. The experiment was the same as that described in B. At least 30 embryos were counted in each stage. Error bars represent SDs (n = 3) and the average of each experiment is represented as a dot.
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Fig. 3. Changes of deeply extended microtubule (MT) meshwork (DEM) and transiently accumulated fragments (TAF) along with meiotic progression. (A) Scheme for counting the number of MT fibers in the cortical meshwork. A regular octagon inscribed inside the egg cortex is drawn. Among this octagon, only 4 sides (chords; red lines), which do not interact with the meiotic apparatus or sperm aster are selected. Tubulin staining intensity along the selected chord is measured and the peaks in line profile of each chord are recognized as MT fibers. Such analysis was performed on 5 optical sections around mid-plane. (B) Embryos were stained for MT and the equatorial region of the mid-plane of the rendered 3D model is shown. Red line indicates an example of line profiling for tubulin staining. Scale bar, 20 µm. (C, C’) Graphs show examples of line profiling of MT staining. C and C’ are unfertilized egg and Telo I embryo, respectively. Line profiling was represented by relative intensities, which were normalized to the median of the entire egg and smoothened by moving average. The peak of the profile above the relative intensity value of 2.0 was recognized as an individual MT fiber (yellow arrow heads). (D) Dot plots show average number of MT fibers in each egg (n = 120: 4 chords on the 5 sections in 6 eggs). Significance was evaluated by a two-tailed T-test (*p < 0.01). (E) Changes of sperm aster and TAF (arrowheads) were observed in the mid-plane optical section and vegetal view of the 3D model (transparent). Note that the sperm aster seemed to start expansion in Meta II, when the TAF disappeared, and formed a significantly large well-developed sperm aster in Ana II. Scale bar, 20 µm. (F) Procedures for quantifying total MT length in the sperm aster. Six serial-optical sections containing the sperm aster were selected from confocal images and rendered 3D model. The sperm aster region was cropped to a 70-µm diameter circle. The image was edited to enhance microtubule bundles by the contrast enhancement and bandpass filter. Image J plugin “Ridge detection†(Steger, 1998) was applied to the edited image. (G) Sum of MT length in each stage is shown in dot plots with error bars (SD; n = 6). Significance was evaluated by two-tailed T-test (*p < 0.01, †p = 0.07 versus Ana II).
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Fig. 4. Changes of sperm aster and cortical MT array in the posterior vegetal cortex (CAMP) during the pronuclear stage and mitosis. Embryos were stained for microtubules (MT) and the low-magnification 3D models are shown (left). Upper and lower images are side view and posterior view, respectively. Enlarged images of the red rectangle in left image are shown (right). Arrow, small arrow heads, and arrowhead indicate the sperm aster, attached point of astral MT to the cortex, and cortical MT focus, respectively. Scale bars, 50 (left image) and 20 (right image) μm.
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Fig. 5. The regulatory mechanisms of microtubule (MT) structures during meiosis. (A) Eggs activated with calcium ionophore A23187 (CaI, 8 µM) were reared with or without cell cycle inhibitors, roscovitine (20 µM) or U0126 (1 µM). Eggs were stained for MT and the mid-planes of the rendered 3D models are shown. The filamentous meshwork disappeared at 10 and 15 mpf upon CaI treatment, and no effect was observed in both inhibitor-treated embryos. Scale bar, 20 µm. (B) Unfertilized eggs were fertilized with or without cytochalasin B (CytB, 2 µg/ml) and reared until 20 mpf. Embryos were stained for MTs (green) and nuclei (blue), and the side view of 3D models are shown. Animal pole is up. In CytB-treated embryos, the prometaphase II female meiotic spindle (dotted arrow) became irregularly shaped owing to the inhibition of polar body extrusion, and the sperm aster (arrow) could not move to the vegetal hemisphere due to the inhibition of the first phase of reorganization. In addition, transiently accumulated fragments (TAFs) (arrowheads) could not be observed. Scale bar, 50 µm. (C) CytB- or DMSO-treated embryo, which showed TAF formation at Prophase II or Prometaphase II was counted in three independent experiments (more than 90 embryos in total). Error bars represent SDs (n = 3), and the average of each experiment is represented as a dot. Significance was evaluated by two-tailed T-test (*p < 0.01).
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Fig. 6. Effects of U0126 on cortical microtubule (MT) array in the posterior vegetal cortex (CAMP) formation. (A) Embryos treated with U0126 (1 µM) during −10–45 or 30–45 mpf were fixed at 45 mpf and stained for MTs (green) and nuclei (blue). Posterior views of the 3D model rendered from confocal images are shown. Arrow, dotted arrow and arrowheads indicate sperm aster, meiotic apparatus, and CAMP, respectively. The centriole of the sperm aster had already divided at 45 mpf. Scale bar, 50 µm. (B) U0126- or DMSO-treated embryos, which contained CAMP or sperm asters were counted in three independent experiments (more than 100 embryos in total). Percentage of embryos, which had CAMP (left) or sperm aster (right) are shown in graph. Error bars represent SDs (n = 3), and the average of each experiment is represented as a dot. (C) Embryos treated with or without U0126 from -10 mpf were fixed in each time point and stained for MT. Rendered 3D models are shown. The white rectangle shows an enlarged image of the precociously formed female pronucleus. Scale bar, 20 µm. (D) Embryos were treated with cycloheximide (0.4 or 1.0 mM) from -10 to 45 or 60 mpf. Embryos were fixed at the end of these treatments and stained for MTs and nuclei. The posterior views of rendered 3D models are shown. Arrowheads indicate CAMP. The number of embryos, which showed a similar staining pattern to these photographs, in the total number of the specimens are indicated in the left corner. Scale bar, 50 µm.
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Fig. 7. Cortical MT array formation in the artificially activated egg. (A) Schematic drawing of two series of Ca2+ oscillations and extrusion of two polar bodies in Ciona intestinalis revised from Russo et al. (1996). (B) Scheme of sample preparation (P01 to 05). The yellow blocks indicate incubation periods with CaI. (C) Unfertilized eggs were treated from 0 to 60 min with CaI (P02) and stained for MTs (green) and nuclei (blue). The 3D models rendered from confocal images were shown. Dotted arrow, open arrowhead, and arrowheads indicate meiotic apparatus, female pronucleus, and CAMP-like cortical MT array, respectively. Scale bar, 50 µm. (D) Percentage of activated eggs, which had CAMP-like structure (left) or female meiotic apparatus (right), were counted in three independent experiments (more than 100 embryos in total). Error bars represent SD (n = 3), and the average of each experiment is represented as a dot.
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Fig. 8. Effect of roscovitine treatment on cortical microtubule (MT) array in the posterior vegetal cortex (CAMP) formation. (A) Scheme of sample preparation (P06 to 10). The blue and yellow blocks indicate incubation periods with DMSO and roscovitine (20 µM), respectively. (B) Embryos treated as indicated in A were fixed at 45 mpf and stained for MTs (green) and nuclei (blue). Posterior views of rendered 3D models are shown. Arrow, dotted arrow, open arrowhead, and arrowheads indicate sperm asters, meiotic apparatus, female pronucleus, and CAMP, respectively. The white rectangle shows an enlargement of fused (P06) and female (P07-P10) nuclei. Scale bars, 10 (within white rectangle) and 50 µm. (C) Roscovitine- or DMSO-treated embryos, containing CAMP or sperm aster were counted in three independent experiments (more than 90 embryos in total). The percentage of embryos containing CAMP (left) or sperm asters (right) are shown. Error bars represent SDs (n = 3), and the average of each experiment is represented as a dot.
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Fig. 9. Schematic model of the controlling mechanisms for microtubule (MT) structures during the first cell cycle in Ciona intestinalis. The egg activation pathway, including Ca2+ signaling, MEK/MAPK signaling, and CDK1/Cyclin B (MPF) signaling, mutually regulate each other and direct cell cycle progression. MT structures including deeply extended MT meshwork (DEM), transiently accumulated MT fragments (TAF), and cortical MT array in the posterior vegetal cortex (CAMP), are regulated by the egg activation pathway concomitant with cell cycle progression. Red arrows indicate the novel regulatory mechanisms revealed in this study. Note that calcium signaling, actin contraction, and CDK activity regulate DEM breakdown, TAF formation, and CAMP formation, respectively. Dotted red lines represent processes that are still under debate.
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