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Translocation of the maternal dorsalizing activity. (A-D) Translocation of the dorsalizing activity leads to β-catenin stabilization and the formation of the dorsal organizer. Between the time of fertilization and the first embryonic cell division, a maternally deposited dorsalizing activity (red) moves from (A) the vegetal pole to (B) the prospective dorsal region. (C) By the two-cell stage, maternal β-catenin (yellow) has become asymmetrically stabilized in the region that has received the dorsalizing activity. (D) Stabilized β-catenin activates genes of the dorsal organizer (green circle; also called the Nieuwkoop and Spemann organizers) in the dorsal equatorial region, as shown in an early gastrula embryo. (E,F) The dorsalizing activity translocates in the same direction as cortical rotation. (E) The dorsalizing activity (red) resides in the shear zone, an area of looser cytoplasm that forms between the outer cortex of the egg and the dense core cytoplasm following fertilization. The black bars at the vegetal pole mark the starting positions of the core and the cortex early in the first cell cycle. (F) During the first cell cycle, the cortex rotates relative to the core, moving about 30° towards the dorsal side in the same direction as the dorsalizing activity. This process is called cortical rotation, as represented by the displacement of the outer black bar. D, dorsal; V, ventral.
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Formation of the microtubule (MT) array. (A-C) Vegetal views of microtubule array formation during the first cell cycle. Times shown are normalized times (NT), with 0.0 NT representing fertilization and 1.0 NT representing the first cleavage division. (A) At 0.4 NT, short disorganized MT polymers have started to appear in the vegetal shear zone. (B) By 0.5 NT, more MTs are present, but are not yet aligned. (C) By 0.7 NT, during peak cortical rotation, the vegetal shear zone is populated by a parallel array of MT bundles that are aligned along the axis of rotation. (D-F) MTs of the vegetal array arise from several sources. (D) The sperm centriole introduces polarity by acting as a minus-end MT-organizing center (-). The resulting radial array of MTs is called the sperm aster. (E) MTs from the sperm aster grow toward the periphery of the egg, as do additional MTs from unknown sources in the core cytoplasm. In addition, short disorganized MT polymers arise in the vegetal shear zone. (F) During rotation, MTs from deep in the cytoplasm bend into the vegetal shear zone and align with peripheral MTs to form the parallel array, with the plus-ends (+) of the growing MTs pointing towards the future dorsal (D) side of the embryo. V, ventral. (A-C) Reproduced, with permission, from Cha and Gard (Cha and Gard, 1999). (D-F) Adapted, with permission, from Houliston and Elinson (Houliston and Elinson, 1991b).
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The β-catenin destruction complex and its regulation by GBP and Dsh. (A) The destruction complex contains the large proteins Axin and APC, which bring β-catenin (β) into the complex and into close proximity to GSK3 and CK1α. These kinases phosphorylate (p) N-terminal residues inβ -catenin and target it for degradation by the ubiquitin-proteasome pathway. (B) GBP and Dishevelled (Dsh) cooperatively inhibit the phosphorylation of β-catenin by the destruction complex. Dsh binds Axin and may help recruit its binding partner GBP to the destruction complex. GBP removes GSK3 from Axin, thereby disrupting the ability of GSK3 to phosphorylate β-catenin. Unphosphorylated β-catenin accumulates and enters the nucleus to activate the transcription of its target genes in a complex with TCF/LEF box transcription factors.
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Translocation of kinesin light chain (KLC)-GFP particles during cortical rotation. Xenopus KLC-GFP was expressed and imaged with a scanning confocal microscope in the vegetal shear zone of immobilized live eggs during the first cell cycle. (A) KLC-GFP particles (green) are observed in the vegetal shear zone during peak rotation. Yolk platelets of the core cytoplasm are stained in red. The starting positions of four KLC-GFP particles are circled, and three neighboring yolk platelets are marked with asterisks. (B) Time-lapse image showing the same field of view ∼38 seconds later than in A. Owing to the immobilization of the egg, the core rotates opposite to the normal direction of cortical rotation, as seen by the displacement of the three core yolk platelets (Y) from right to left. The KLC-GFP particles have translocated a longer distance in the opposite direction (i.e. in the same direction cortical rotation would normally move), from left to right. Scale bar: 5 μm. Reproduced, with permission, from Weaver et al. (Weaver et al., 2003).
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Model for alignment of the MT array and the transport of the dorsalizing activity. (A) The sperm centriole (-) organizes microtubules (MTs) near the sperm entry point in the animal hemisphere. As cortical rotation initiates, disorganized MTs populate the vegetal shear zone, arising from both deep and peripheral sources. MTs are shown as red lines, and plus-end directed kinesin-related motor proteins (KRPs) attached to the cortex are shown as dark blue circles. (D-F) Enlargements of boxed regions shown in A-C. (D) Early in the first cell cycle, dorsalizing particles nucleate in the vegetal shear zone of the embryo. GBP, which is perhaps associated with other dorsalizing proteins such as Dishevelled (Dsh), interacts with the plus-end-directed motor protein kinesin by binding its cargo-carrying subunit, kinesin light chain (KLC). (B) As cortical rotation progresses, plus-end-directed KRP motor proteins tethered to the moving cortex associate with the MTs and move along their length, which serves to align them so that their plus-ends grow in the same direction that the cortex is moving. (E) As the MT array aligns, kinesin carries particles quickly towards the MT plus-ends, which are directed towards the future dorsal region near the equator. Some particles are transported more slowly in the same direction by associating with the rotating cortex. (C) Towards the end of the first cell cycle, the MT array depolymerizes, and cortical rotation and kinesin-based transport cease. (F) In the dorsal region, GBP dissociates from kinesin in favor of binding to GSK3. The interaction of GBP and GSK3 may be facilitated by Dsh binding to Axin, which would bring GBP to the destruction complex. GBP removes GSK3 from Axin, allowingβ -catenin (β) to be stabilized in the dorsal region (also indicated in C). Stabilized β-catenin enters dorsal nuclei and activates transcription of dorsal organizer genes, ultimately resulting in the formation of the DV and AP axes. D, dorsal side; KHC, kinesin heavy chain; NT, normalized time; V, ventral.
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