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Vertebrate embryos define an anatomic plane of bilateral symmetry by establishing rudimentary anteroposterior and dorsoventral (DV) axes. A left-right (LR) axis also emerges, presaging eventual morphological asymmetries of the heart and other viscera. In the radially symmetric egg of Xenopus laevis, the earliest steps in DV axis determination are driven by microtubule-dependent localization of maternal components toward the prospective dorsal side. LR axis determination is linked in time to this DV-determining process, but the earliest steps are unclear. Significantly, no cytoskeletal polarization has been identified in early embryos capable of lateral displacement of maternal components. Cleaving Xenopus embryos and parthenogenetically activated eggs treated with 2,3-butanedione monoxime (BDM) undergo a dramatic large-scale torsion, with the cortex of the animal hemisphere shearing in an exclusively counterclockwise direction past the vegetal cortex. Long actin fibers develop in a shear zone paralleling the equator. Drug experiments indicate that the actin is not organized by microtubules, and depends on the reorganization of preexisting f-actin fibers rather than new actin polymerization. The invariant chirality of this drug response suggests a maternally inherited, microfilament-dependent organization within the egg cortex that could play an early role in LR axis determination during the first cell cycle. Consistent with this hypothesis, brief disruption of cortical actin during the first cell cycle randomizes the LR orientation of tadpoleheart and gut.
Fig. 1. BDM exposure late in the first cell cycle produces counterclockwise torsion of blastomeres during first cleavage. (A) Xenopus embryos were immersed in 20 mmol/l BDM at 75 minutes post-fertilization (0.83 NT). In every embryo, each blastomere rotates relative to the other in a counterclockwise direction, resulting in a characteristic chiral pattern. (B) The counterclockwise twisting of the pigmented animal cortex of each blastomere relative to the animal-vegetal axis of the embryo (black arrows) produces an invariably counterclockwise torsion between blastomeres as they cleave (blue arrows).
Fig. 2. Normal first cleavage in Xenopus is chiral and not mirror-image symmetric. Both fixed (A-C) and live (D-E) embryos, untreated, reveal a slight counterclockwise torsion of the two blastomeres during cleavage furrow advance. The apex of each furrow margin, i.e. the site at which furrowing began, becomes offset relative to the corresponding point in the opposite blastomere (arrow bases). By midcleavage (C), the offset between devitellinated blastomeres may be as much as 150 μm. Although the asymmetry is accentuated by dejellying (D) or removal of the vitelline envelope, even embryos with undisturbed jelly coats (E) display the same chirality. The offset normally becomes obscured as cleavage proceeds and the stress folds relax.
Fig. 3. Microfilaments bundle ectopically in cortex following BDM treatment.Xenopus zygotes were exposed to 20 mmol/l BDM beginning at 0.38 NT (A) or 0.94 NT (B) after fertilization. The earlier BDM exposure caused extensive contraction and irregular blebbing in the animal cap (A). Rhodamine-phalloidin staining of the surface, showing long, branching ectopic microfilament bundles (A′). The later BDM exposure induced torsion between the two cleaving blastomeres (offset indicated by paired arrowheads in B) and extensive bundling of ectopic actin fibers that branched and spread across cortex from the growing edge of the contractile ring (arrows in B′). Scale bars: 10 μm in A′; 75 μm in B.
Fig. 4. Cortical torsion occurs in parthenogenetically activated eggs. (A-F) Eggs were parthenogenetically activated and, from 75 minutes post-activation (∼0.79 NT) onward, bathed continuously in 20 mmol/l BDM. Activated egg, viewed from the side through two cleavage cycle equivalents (frames approximately 10 minutes apart), developed a spiraled pigment pattern in the animal hemisphere as the surface rotated left to right (counterclockwise).
Fig. 5. Cortical torsion does not require interaction with microtubules. The eggs were incubated continuously for 4 hours (about four cell-cycle equivalents) in the presence of 20 mmol/l BDM and 10 μmol/l nocodazole. The cortical pigment pattern indicates counterclockwise rotation through several revolutions.
Fig. 6. A broad shear zone develops at the equator of BDM-treated activated eggs. The frame from a side-view time-lapse sequence spanning a 20 minute period at the beginning of torsion (see Movie S3 in the supplementary material). Motion of individual pigment granules (marks 1-6) traced via time-lapse is indicated by colored tracks. The colored bar indicates a 20 minute time interval (blue: start of tracking; red: +20 minutes).
Fig. 7. Long microfilament bundles parallel the equatorial shear. A parthenogenetically activated egg treated with BDM/nocodazole observed to be undergoing torsion (A) was fixed and stained with rhodamine-phalloidin and examined via confocal microscopy (B). Bundles of cortical microfilaments parallel the direction of shear in the equatorial region. The region indicated by the colorized box is shown at higher magnification (C). A small post-fixation tear in the surface (dark spot) reveals relatively little organized f-actin beneath the cortex. Scale bar in C: 30μ m.
Fig. 8. Cortical microfilament bundles slide past each other, paralleling the direction of shear. (A) Equatorial view via confocal microscopy of an egg matured from an oocyte injected with GFP-actin mRNA. The egg was parthenogenetically activated and incubated in BDM/nocodazole. White bars indicate the orientation of the animal-vegetal axis. (B) The colorized region is examined at higher magnification, showing GFP-decorated microfilaments oriented in a band paralleling the equator. (C) The fluorescence intensity profile along a white transect in B paralleling the animal-vegetal meridian. The distribution of intensity maxima indicates relatively uniform spacing of GFP-actin microfilament bundles. Scale bar in B: 15 μm.
Fig. 9. BDM treatment can randomize cardiac and gutleft-right patterning. (A) An untreated stage-47 tadpole displays normal situs of heart and gut coiling. (B) A tadpole exposed to 20 mmol/l BDM for 20 minutes during the first cell cycle displays reversed cardiac and gut organizations, with otherwise normal body patterning. Insets show outlines of normal (A) and reversed (B) cardiac outflow tract and ventricle.
Fig. 10. Hyperdorsalization results from radialized vegetal microtubule array. (A) Embryos treated with 20 mmol/l BDM for 20 minutes during the first cell cycle displayed a range of conjoined-twin and radialized hyperdorsal morphologies. (B) A normal vegetal-cortical microtubule array, seen from the side in a whole-mount embryo immunostained forαβ -tubulin via confocal microscopy. The specimen is oriented with the animal pole up, sperm entry point to the right, and thus prospective dorsal to the left. (C) A vegetal-cortical microtubule array in a BDM-treated sibling displaying the extreme hyperdorsal phenotypes seen in A. The specimen orientation is the same as that for B.
Fig. 11. Cortical f-actin in the dorsal marginal zone reorganizes to form a contractile equatorial band following BDM treatment. (A) Embryos were exposed to 20 mmol/l BDM beginning at 55 minutes post-fertilization and fixed 5, 10 and 20 minutes later (60, 65, 70 minutes post-fertilization; A1-A3, respectively). Each develops a horizontal sulcus, indicating surface contraction, on the side opposite the SEP (prospective dorsal marginal zone) (arrows in A2, A3). (B) Phalloidin-stained zygote, fixed at 75 minutes (20 minutes after BDM exposure) viewed in whole-mount via confocal microscopy; same orientation as zygotes in A. Rhodamine-phalloidin (red) stains equatorial sulcus brightly. Green reflection mode provides background, primarily to indicate that cortical pigment is not obscuring the view of cortical actin. (C) Detail of the surface at the edge of the sulcus, showing equatorially aligned microfilaments. Scale bar: 60 μm. (D,E) Animal and vegetal surfaces, respectively, of untreated zygote, fixed 75 minutes post-fertilization and stained with rhodamine-phalloidin, showing enrichment of cortical f-actin on the dorsal marginal zone.
Fig. 12. Symmetry breaking accompanies DV axis specification during the first cell cycle. Top: side view. Bottom: vegetal view. (A) Before fertilization, deep (blue) and shallow (red) determinants are localized concentrically about the animal-vegetal axis. (B) After fertilization, the spermcentrosome initiates orientation of the vegetal-cortical microtubule array, producing a shift of dorsalizing components toward the incipient dorsal marginal zone, and a concomitant shift of deeper components and the yolk mass (yellow) toward the sperm entry point. The axial separation of the deep and shallow components defines the plane of bilateral symmetry. (C) An actin-dependent shift of surface counterclockwise relative to the deeper components produces significant material differences across the embryonic midline.
