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Figure 1:. Schematic representation of sea urchin embryonic development. The times in blue indicate hours postfertilization. The sea urchin oocyte is organized along an animal–vegetal (A/V) primary axis, which is observable in P. lividus due to the presence of a subequatorial cortical pigmented band visible under blue light (arrow in A). Fertilization is followed by first and second cleavages, which are meridional (parallel to the A/V axis) and perpendicular to each other (B, C). The third cleavage is equatorial, perpendicular to the first two cleavage planes, and separates the animal and vegetal hemispheres from one another (D). During the fourth cleavage, animal blastomeres divide equally to produce eight mesomeres, and the vegetal blastomeres divide unequally to produce large macromeres and small micromeres located at the vegetal pole of the embryo (E, F). Six hours after fertilization the sea urchin embryo enters the early blastula stage with an empty central cavity called a blastocoel (G). The cells start developing cilia on their outer surface to form a swimming blastula (H). Approximately 10–12 h after fertilization, the midblastula, composed of ∼600 cells, hatches out of the fertilization envelope (not shown). At the animal pole, the cilia are longer but do not beat. This “apical tuft” (I) provides directionality to swimming, as embryos almost always move with the apical tuft region forward. In the late blastula stage the embryo becomes thickened at the vegetal pole, forming the vegetal plate (I). This represents the gastrulation site where the primary mesenchyme cells (PMCs), which are derived from the micromeres and located in the center of the vegetal plate region, migrate into the blastocoel (I, J). The vegetal cells will continue to ingress in order to form the archenteron, led by filopodia extending from the secondary mesenchyme cells (SMCs), which eventually contact the animal pole at the future site of mouth formation (K). A prism (not shown) and finally a feeding pluteic larva will be formed 24 h later around an endoskeleton, which contains two spicules made of calcium carbonate secreted by the PMCs (L). A few days later, this pluteus will metamorphose into a tiny male or female adult urchin.
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Figure 2:. I. Domain structure of sea urchin aPKCs. (A) The short isoform found so far in P. lividus and S. purpuratus encodes an aPKC that lacks the PB1 protein interaction domain. Kinase: serine threonine kinase domain; C1: cysteine-rich domain, which binds InsPtd(3,4,5)P3. The three urchin species examined so far (P. lividus, H. pulcherrimus, and S. purpuratus) also contain a long isoform that is similar to most other known aPKCs in its domain composition (see Supplemental Figure S1 for alignment). II. Two maternal aPKC proteins that localize in the cortex in early embryos of P. lividus. (A) Western blot with the SC216 anti-aPKC antibody on P. lividus extracts from (a) unfertilized eggs, (b) 2-cell stage, (c) 8-cell stage, (d) 16- to 32-cell stage, (e) swimming blastula, (f) early gastrula, (g) prism, (h) plutei. Equal amounts of protein were loaded in each lane. (i) Standard molecular weight markers from top to bottom: 200, 120, 100, 70, 50, 37, and 20 kDa. (B–F) Immunolocalization of aPKC in early sea urchin embryos observed by confocal microscopy. (B) Unfertilized egg, (C) 2-cell stage, (D) 8-cell stage, (E, F) two confocal sections of the same 16-cell embryo showing (E) the interior and (F) the surface. Scale bar, 20 μm.
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Figure 3:. aPKC is associated with microtubule structures during mitotic divisions in the early sea urchin embryo. Sixty-four–cell stage embryos were fixed and labeled with DM1A antitubulin antibody (red) and anti-aPKC antibody (green). aPKC associates with the MTOC (arrows in B and C) during prophase (A–C); decorates spindle microtubules during metaphase (D–F); starts to concentrate in the spindle midzone (arrow) during anaphase (G–I), and ends up in the midbody (arrow) during telophase (J–L). The nucleus (in C) and chromosomes (in F, I, and L) are stained in blue with Hoescht stain. Bar, 5 μm.
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Figure 4:. aPKC localization in ciliated swimming sea urchin embryos. (A, B) Dark-field videomicroscopy showing (A) short, active cilia and (B) the “apical tuft” made of a few long and immobile cilia at the animal pole. (C–L) Confocal sections of swimming embryos fixed and labeled for microtubules (red) and aPKC (green). (C) Early gastrula showing the longer cilia (in red) and the concentration of aPKC at the animal pole (arrow). (D–F) Swimming blastula. (G–I) Late gastrula. (J–L) Pluteus larva; arrows: ciliary band in K and stomach in L.
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Figure 5:. Sea urchin aPKC localizes to a disk situated between the basal body and the elongating axoneme of motile cilia. (A–E, G–I) Confocal sections of cells on the surface of swimming blastulas stained for microtubules (red) and aPKC (green). (A, B, D, E) Surface views; (C, G–I) sagittal views. The arrowhead and arrow in C show the absence of aPKC in the basal body and in the elongating axoneme, respectively. (D′, E) Higher-magnification views of aPKC ring-shaped labeling at the bases of two different cilia. (F) Relative intensity of the aPKC signal observed in (E) using the surface plot quantitation function of the ImageJ program. (J) Relative quantitation of intensity of the fluorescence signals (y-axis) over distance (x-axis) obtained in the green (top) and red (bottom) channels along the line drawn in I using the plot profile function of the ImageJ image processing program. All bars, 2 μm.
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Figure 6:. Sea urchin embryos treated with aPKC inhibitors do not swim and exhibit short cilia. (A–C) Dark-field videomicroscopy images taken from time-lapse sequences of late gastrulas control (A) or treated with the PKC inhibitors GF109203X, 10 μM (B) and Gö 6976, 20 μM (C). White arrows indicate the presence (A, C) or the absence (B) of the apical tuft at the animal pole. Yellow arrowheads and yellow arrows indicate the archenteron and the forming spicules, respectively. (D–I) Confocal surface views of gastrula-stage embryos either (D–F) untreated or (G–I) after treatment with the myristoylated aPKC pseudosubstrate inhibitor. Fixed embryos were labeled for tubulin (D, G) or aPKC (E, F, H, I). F and I are higher-magnification views of the regions boxed in E and H.
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Figure 7:. Cilia length is proportional to level of aPKC inhibition. Confocal surface views of embryos fixed and labeled for tubulin. Embryos were deciliated by osmotic stress and then allowed to reciliate in the absence (A) or in the presence (B–D) of myristoylated aPKC pseudosubstrate inhibitor. Inhibitor final concentrations used are indicated.
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Figure 8:. The inhibition of aPKC and its effect on ciliogenesis can be reversed. (A–C) Control embryos were deciliated and left to reciliate for 2 h before fixation and staining for microtubules (red) and aPKC (green). The arrows show the apical tuft and the accumulation of aPKC at the animal pole. (D–F) Embryo deciliated and left to reciliate in the presence of 2.0 μM pseudosubstrate, fixed, and stained like the control. (G–I) Embryo treated as in D, then washed to remove the inhibitor and allowed to reciliate for 2 h. The arrows show the reformation of the apical tuft and apical accumulation of aPKC when the kinase is no longer inhibited.
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