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Fig. 5. Cortical polarity of 8-16 cell stage embryos (first unequal cleavage). (A1-A3) Living embryo double labelled for ER (red) and mitochondria (green); time-lapse confocal microscopy of eight-cell (A1), 16-cell (A2) and 32-cell (A3) stages (see Movie 6 in supplementary material). Arrowheads indicate the location of the cER/mRNA-rich CAB. Inserts show full views of embryos at the eight-cell stage (lateral view) and at the 16- and 32-cell stages (vegetal views). (B,C) Fixed embryos double labelled for Ci-PEM1 RNA by fluorescent in situ hybridization (white) and DNA (blue). Arrowheads show the mRNA-rich CAB at the beginning (interphase) of the eight-cell stage (B) and at the end (mitosis) of the eight-cell stage (C). (D) High-magnification confocal cortical section (6 µm from surface) showing Ci-PEM1 RNA signal at the end of the eight-cell stage. (E1,E2) Electron-microscopy section across the CAB of an eight-cell-stage embryo. The dotted area encircles the CAB. Enlarged view of the CAB is shown in (E1). ER tubules (cER, arrowhead) are red, mitochondria green and yolk platelets (YP) blue. Notice the presence of electron-dense matrix. Scale bars, 5 µm (E1) and 1 µm (E2). (F) Confocal observation of cortical fragments isolated from the posterior vegetal region of an eight-cell-stage embryo and labelled for cER (red). (Insert, top right) Lower-magnification view of four cortical imprints from two vegetal posterior B4.1 and two animal posterior b4.2 blastomeres. Arrowheads indicate the cER accumulations that characterize the isolated moustache-shaped CAB. (Insert, bottom right) Higher-magnification view of cER accumulation observed in F. (G) Epifluorescence observation of the CAB isolated in a more condensed state and labelled for cER (arrowheads). Dotted lines show the edges of cortical fragments corresponding to blastomeres. (H1,H2) Confocal section of isolated cortex with condensed CAB double labelled for cER (H1) and microtubules (H2). Dotted lines delimit cortical fragments. (I) Confocal section of isolated cortex labelled for microfilaments in the area of a condensed CAB (arrowheads). (J) Isolated CAB double labelled for cER (red) and ribosomes (green). Merged images show colocalization of cER and ribosomes in yellow and arrowheads indicate some rough cER tubes in continuity with the CAB. (Insert) The cER network (red) and attached ribosomes (green) normally compacted in the CAB have been stretched away from the attached cortex under the force of shear. All acquisitions were made in Phallusia except B-D, which were made in Ciona.
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