Gavity and Microtubules in Dorsoventral Polarization of The Xenopus Egg

In Xenopus laevis , the dorsoventral axis of the embryo is specified by a 30° relative rotation between the cortex and the cytoplasm of the fertilized egg, and a cortical array of parallel microtubules may be part of the rotation machinery (7). The parallel microtubules are aligned with the sperm entry point in most of the eggs as expected, since the dorsoventral axis is usually defined by the sperm entry point. We show that gravity can play two roles in the formation of the dorsoventral axis. First, a simple 90° tilt off-axis before the start of the rotation overcomes the influence of the sperm and determines the orientation of the parallel microtubules. Second, a 90° tilt off-axis can specify the dorsoventral axis even in the absence of the parallel microtubules. Therefore, gravity can affect dorsoventral polarity by orienting the parallel microtubules or by moving cytoplasm directly without microtubules.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification

The amphibian egg undergoes a rotation of its subcortical cytoplasm relative to its surface during the first cell cycle. Nile blue spots applied to the egg periphery move with the subcortical cytoplasm and make rotation directly observable (J.-P. Vincent, G.F. Oster, and J. C. Gerhart (1986). Dev. Biol. 113, 484). We have previously shown that the direction of rotation accurately predicts the orientation of the embryonic axis developed by the egg. This suggests an important role for subcortical rotation in axis specification. In this report, we provide two kinds of experimental evidence for the essential role of rotation, and against a role for other concurrent cytoplasmic movements such as the convergence of subcortical cytoplasm toward the sperm entry point in the animal hemisphere. First, dispermic eggs develop only one embryonic axis, which is oriented accurately in line with the direction of the single rotation movement and not with the two convergence foci that form in the animal hemisphere. Rotation probably modifies the vegetal, not animal, hemisphere since axial development is normal in dispermic eggs despite highly altered animal subcortical movement. Second, we show that the amount of rotation correlates with the extent of dorsal development. UV irradiation of the vegetal hemisphere, or cold shock of the egg, inhibits rotation effectively. When there is no rotation, there is no dorsal development. On average within the egg population, increasing amounts of rotation correlate with the increasingly anterior limit of the dorsal structures of the embryonic body axis. However, individual partially inhibited eggs vary greatly in the amount of axis formed following a given amount of movement. Furthermore, the egg normally rotates more than is necessary for the development of a complete axis. These findings suggest that rotation, although essential, does not directly pattern the antero-posterior dimension of the body axis, but triggers a response system which varies from egg to egg in its sensitivity to rotation. This system is artificially sensitized by exposure of the egg to D2O shortly before rotation. We show that D2O-treated eggs produce extensive axes despite very limited rotation, often developing into hyperdorsal embryos. However, like normal eggs, they depend on rotation and cannot form dorsal structures if it is eliminated.     

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. II. Experimental dissociation by lateral compression of the egg

In eggs of Xenopus laevis, the meridian of sperm entry (SEP meridian), the direction of subcortical rotation, and the first cleavage furrow have been used to predict, with varying degrees of accuracy, the position of the plane of bilateral symmetry of the embryo. We show here that altering the shape of the uncleaved egg by lateral compression disrupts some of these topographical relationships in a reproducible way. The neural groove, which identifies the embryonic dorsal midline, usually forms at either of the two narrow ends of the compressed egg, regardless of the position of the SEP meridian, whereas the first cleavage furrow divides the compressed egg across its shorter dimension, regardless of the position of the SEP meridian. Thus the positions of the SEP meridian, the cleavage plane, and the embryonic bilateral plane can be completely uncoupled from each other. In contrast, the direction of subcortical rotation is usually parallel to the plane of compression and predicts the position of the neural groove in all cases. Since the direction of subcortical rotation and the plane of bilateral symmetry still correlate under conditions of compression, we conclude that subcortical rotation is the crucial early step in the process of axis specification.     

Axis determination in polyspermic Xenopus laevis eggs

Polyspermic Xenopus laevis eggs can be identified easily because of regions of pigment accumulation and white stripes, which arise by a nocodazole-sensitive process. Eggs containing up to four sperm are capable of forming a single embryonic axis. Dispermic eggs display two regions of pigment accumulation, one around each sperm entry point (SEP), and one white stripe between the SEPs. Such eggs with a 180 degree separation between the SEPs were bisected before first cleavage along the white stripe, creating dorsal and ventral halves in many cases. Each half cleaved and formed a tadpole. When eggs were bisected early in the period of cytoplasmic reorganization (0.5-0.6 normalized time), each half could form a complete tadpole. When eggs were bisected after the period of reorganization (0.8-0.9), often one half formed a tadpole with a complete head but reduced or absent tail and the other half formed a tadpole with a complete tail but reduced or absent head. These results demonstrate that sperm cooperate to give a single embryonic axis in polyspermic eggs and the development of dorsal and ventral egg halves differs after egg reorganization before first cleavage.     

Intrinsic chiral properties of the Xenopus egg cortex: an early indicator of left-right asymmetry?

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 tadpole heart and gut.     

A transient array of parallel microtubules in frog eggs: potential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis

The dorsoventral axis of the frog embryo is specified by a rotation of the egg cytoplasm relative to the cortex. When eggs undergoing the cortical/cytoplasmic rotation were examined by immunocytochemistry and electron microscopy, an extensive array of parallel microtubules was found covering the vegetal hemisphere of the egg. The microtubules were 1-3 microns deep from the plasma membrane and were aligned parallel to the direction of rotation. They formed at the start of rotation and disappeared at its completion. Colchicine and uv irradiation, inhibitors of the rotation, prevented the formation of the parallel microtubules. Based on these properties, we suggest that the parallel microtubules serve as tracks for the cortical/cytoplasmic rotation which specifies the dorsoventral axis of the embryo.     

Heat-Induced Reversal of Dorsal-Ventral Polarity in Xenopus Eggs

Heat-treatment of fertilized Xenopus laevis eggs at 30°C induced; 1. conspicuous concentration of the pigment toward the sperm entry point (SEP), 2. eccentric first cleavage furrow formation, and 3. reversal of the dorsal-ventral polarity of the embryos. The optimal treatment was for 2.5 min applied at 20 min postfertilization (p.f.). The rotation movement of the Nile-blue stained spots in the vegetal hemisphere of the heated eggs accurately located the future dorsal midline as in untreated embryos (ref. 22). Exposure of eggs to D₂O also reversed the dorsal-ventral polarity of the embryo suggesting that stabilization of microtubules is involved in the dorsal-ventral axis reversal.     

Two UV-sensitive targets in dorsoanterior specification of frog embryos

Previous work has shown that ultraviolet (UV) irradiation of fertilized frog eggs yields embryos that lack dorsal and anterior structures. The eggs fail to undergo the cortical/cytoplasmic rotation that specifies dorsoventral polarity, and they lack an array of parallel microtubules associated with the rotation. These eggs can be rescued by tilting with respect to gravity, and normal dorsoanterior development occurs. We find here that UV irradiation of Xenopus prophase I oocytes or Rana metaphase I oocytes also causes the dorsoanterior deficient syndrome, but the UV target is different from that in fertilized eggs. Tilting eggs, irradiated as oocytes, with respect to gravity, does not rescue dorsoanterior development, although lithium treatment does. The UV dose required to produce dorsoanterior deficiency for Rana metaphase I oocytes is much less than that for fertilized eggs, and the oocytes can form the array of parallel microtubules and undergo the cortical/cytoplasmic rotation after fertilization. Despite these features of normal development, no dorsoanterior structures form. While the UV target in fertilized eggs is thought to be the parallel microtubules (Elinson & Rowning, 1988; Devl Biol. 128, 185-197), the UV target in the oocytes may be a dorsal determinant.     

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.     

Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation

In amphibians, the cortical rotation, a translocation of the egg cortex relative to the cytoplasm, specifies the dorsoventral axis. The cortical rotation involves an array of subcortical microtubules whose alignment is mediated by Kinesin-related proteins (KRPs), and stops as M-phase promoting factor (MPF) activation propagates across the egg. To dissect the role of different motor proteins in the cortical rotation and to analyse their regulation, we have developed an open cell assay system involving reactivation of microtubule movement on isolated cortices. Microtubule movements were dependent on ATP and consisted mainly of wriggling and flailing without net displacement, consistent with a tethering of microtubules to the cortex. Reactivated movements were inhibited by anti-KRP and anti-dynein antibodies perfused together but not separately, the KRP antibody alone becoming fixed to the cortex. Neither antibody could inhibit movement in the presence of MPF, indicating that arrest of the cortical rotation is not due to MPF-dependent inhibition of motor molecules. In contrast, D(2)O treatment of live eggs to protect microtubules from progressive depolymerisation prolonged the cortical rotation. We conclude that the cortical rotation probably involves cytoplasmic dynein as well as cortical KRPs and terminates as a result of local MPF-dependent microtubule depolymerisation.     

Complementary roles for dynein and kinesins in the Xenopus egg cortical rotation

Aligned vegetal subcortical microtubules in fertilized Xenopus eggs mediate the "cortical rotation", a translocation of the vegetal cortex and of dorsalizing factors toward the egg equator. Kinesin-related protein (KRP) function is essential for the cortical rotation, and dynein has been implicated indirectly; however, the role of neither microtubule motor protein family is understood. We examined the consequence of inhibiting dynein--dynactin-based transport by microinjection of excess dynamitin beneath the vegetal egg surface. Dynamitin introduced before the cortical rotation prevented formation of the subcortical array, blocking microtubule incorporation from deeper regions. In contrast, dynamitin injected after the microtubule array was fully established did not block cortical translocation, unlike inhibitory-KRP antibodies. During an early phase of cortical rotation, when microtubules showed a distinctive wavy organization, dynamitin disrupted microtubule alignment and perturbed cortical movement. These findings indicate that dynein is required for formation and early maintenance of the vegetal microtubule array, while KRPs are largely responsible for displacing the cortex once the microtubule tracks are established. Consistent with this model for the cortical rotation, photobleach analysis revealed both microtubules that translocated with the vegetal cytoplasm relative to the cortex, and ones that moved with the cortex relative to the cytoplasm.     

Move it or lose it: axis specification in Xenopus

A long-standing question in developmental biology is how amphibians establish a dorsoventral axis. The prevailing view has been that cortical rotation is used to move a dorsalizing activity from the bottom of the egg towards the future dorsal side. We review recent evidence that kinesin-dependent movement of particles containing components of the Wnt intracellular pathway contributes to the formation of the dorsal organizer, and suggest that cortical rotation functions to align and orient microtubules, thereby establishing the direction of particle transport. We propose a new model in which active particle transport and cortical rotation cooperate to generate a robust movement of dorsal determinants towards the future dorsal side of the embryo.     

Local inhibition of cortical rotation in Xenopus eggs by an anti-KRP antibody

The dorsal-ventral axis of amphibian embryos is specified by the "cortical rotation," a translocation of the egg cortex relative to the vegetal yolk mass. The mechanism of cortical rotation is not understood but is thought to involve an array of aligned, commonly oriented microtubules. We have demonstrated an essential requirement for kinesin-related proteins (KRPs) in the cortical rotation by microinjection beneath the vegetal cortex of an antipeptide antibody recognising multiple Xenopus egg KRPs. Time-lapse videomicroscopy revealed a striking local inhibition of the cortical rotation around the injection site, indicating that KRP-mediated translocation of the cortex is generated by forces acting across the vegetal subcortical region. Anti-tubulin immunofluorescence showed that the antibody disrupted both formation and maintenance of the aligned microtubule array. Direct examination of rhodamine-labelled microtubules by confocal microscopy showed that the anti-KRP antibody provoked striking three-dimensional flailing movement of the subcortical microtubules. In contrast, microtubules in antibody-free regions undulated only within the plane of the cortex, a significant population exhibiting little or no net movement. These findings suggest that KRPs have a critical role during cortical rotation in tethering microtubules to the cortex and that they may not contribute significantly to the translocation force as previously thought.     

Evidences for direct involvement of microtubules in cleavage furrow formation in newt eggs

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.     

Dorsal downregulation of GSK3beta by a non-Wnt-like mechanism is an early molecular consequence of cortical rotation in early Xenopus embryos

Cortical rotation and concomitant dorsal translocation of cytoplasmic determinants are the earliest events known to be necessary for dorsoventral patterning in Xenopus embryos. The earliest known molecular target is beta-catenin, which is essential for dorsal development and becomes dorsally enriched shortly after cortical rotation. In mammalian cells cytoplasmic accumulation of beta-catenin follows reduction of the specific activity of glycogen synthase kinase 3-beta (GSK3beta). In Xenopus embryos, exogenous GSK3beta) suppresses dorsal development as predicted and GSK3beta dominant negative (kinase dead) mutants cause ectopic axis formation. However, endogenous GSK3beta regulation is poorly characterized. Here we demonstrate two modes of GSK3beta regulation in Xenopus. Endogenous mechanisms cause depletion of GSK3beta protein on the dorsal side of the embryo. The timing, location and magnitude of the depletion correspond to those of endogenous beta-catenin accumulation. UV and D(2)O treatments that abolish and enhance dorsal character of the embryo, respectively, correspondingly abolish and enhance GSK3beta depletion. A candidate regulator of GSK3beta, GSK3-binding protein (GBP), known to be essential for axis formation, also induces depletion of GSK3beta. Depletion of GSK3beta is a previously undescribed mode of regulation of this signal transducer. The other mode of regulation is observed in response to Wnt and dishevelled expression. Neither Wnt nor dishevelled causes depletion but instead they reduce GSK3beta-specific activity. Thus, Wnt/Dsh and GBP appear to effect two biochemically distinct modes of GSK3beta regulation.     

Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.