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.     

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.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

GBP binds kinesin light chain and translocates during cortical rotation in Xenopus eggs

In Xenopus, axis development is initiated by dorsally elevated levels of cytoplasmic beta-catenin, an intracellular factor regulated by GSK3 kinase activity. Upon fertilization, factors that increase beta-catenin stability are translocated to the prospective dorsal side of the embryo in a microtubule-dependent process. However, neither the identity of these factors nor the mechanism of their movement is understood. Here, we show that the GSK3 inhibitory protein GBP/Frat binds kinesin light chain (KLC), a component of the microtubule motor kinesin. Upon egg activation, GBP-GFP and KLC-GFP form particles and exhibit directed translocation. KLC, through a previously uncharacterized conserved domain, binds a region of GBP that is required for GBP translocation and for GSK3 binding, and competes with GSK3 for GBP. We propose a model in which conventional kinesin transports a GBP-containing complex to the future dorsal side, where GBP dissociates and contributes to the local stabilization of beta-catenin by binding and inhibiting GSK3.     

Structural basis of mechanochemical coupling in a hexameric molecular motor

The P4 protein of bacteriophage phi12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small approximately 1 A changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNA-binding site that moves along the mechanical reaction coordinate. The lever is flanked by gamma-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.     

Turing's next steps: the mechanochemical basis of morphogenesis

Nearly 60 years ago, Alan Turing showed theoretically how two chemical species, termed morphogens, diffusing and reacting with each other can generate spatial patterns. Diffusion plays a crucial part in transporting chemical signals through space to establish the length scale of the pattern. When coupled to chemical reactions, mechanical processes - forces and flows generated by motor proteins - can also define length scales and provide a mechanochemical basis for morphogenesis. forces and flows generated by motor proteins - can also define length scales and provide a mechanochemical basis for morphogenesis.     

Cytoskeleton in Xenopus oocytes and eggs

The Xenopus egg is a huge cell divided into compartments with distinct characteristics. The organization of the cytoskeleton reflects both the size of the egg and its regional differences. We review the information concerning the deployment and function of cytoskeletal elements during the changes in cellular organization accompanying oogenesis, oocyte maturation, and following fertilization.     

Fertilization induces endocytosis in Xenopus eggs

Fertilization-induced endocytosis in Xenopus eggs was shown by direct visualization of fluorescent dye in semithin sections. The eggs were incubated in a medium containing 0.1% Lucifer yellow CH for 20 min before, during and after fertilization and then fixed at different times after fertilization. The eggs incubated during or immediately after fertilization contained fluorescent vesicles in the cortex. These vesicles were mainly distributed in the animal hemisphere.     

DNA synthesis in a multi-enzyme system from Xenopus laevis eggs

Cytoplasm from unfertilized eggs of the frog Xenopus laevis was separated by DEAE-cellulose column chromatography into nine fractions. Supercoiled pXir 11 DNA molecules (pXir 11 is a Col El-based recombinant plasmid containing part of the Xenopus laevis 18S and 28S ribosomal genes and transcribed spacer region) were incubated with each fraction singly and in various combinations. After incubation for 4 hr at 26 degrees C, the pXir 11 DNA was reisolated and examined by electron microscopy. Using appropriate reaction conditions (pH 7.2, 10 mM Mg2+, 250 micron NTP, 50 50 micron dNTP, 50 MM KCl, fractions III and IV or VI), at least 5-10% of the input DNA was converted to theta structures (presumed intermediates in DNA replication).     

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.     

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep- etch analysis of its modification at fertilization

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.     

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions’ distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.     

Parthenogenesis in Xenopus eggs requires centrosomal integrity

Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

The coelomic envelope of Xenopus laevis eggs: a quick-freeze, deep-etch analysis

The extracellular matrix of Xenopus laevis oocytes was analyzed before and after meiotic maturation using quick-freeze, deep-etch, rotary-shadow electron microscopy. The perivitelline space (PS) of the meiotically immature oocyte contains a filamentous network which connects microvilli (MV) and follicle cell macrovilli to the folded oocyte surface below. The envelope overlying the PS is composed of bundles of large fibers which course between the tips of the MV. Spaces between these bundles contain smaller fibrils which secure the egg envelope to the microvillar tips. Meiotic maturation is accompanied by flattening of the oocyte plasma membrane, formation of an orderly array of MV, and elevation of the egg envelope. In the coelomic eggs, the reorganized envelope is composed of loosely bundled large fibers which course above the microvillar tips rather than between them. The spaces between these bundles contain small fibers similar to those seen in the meiotically immature oocyte. This reorganized envelope, however, will not bind sperm; further modifications must transpire during passage through the oviduct to render it sperm receptive.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Recombinant DNA formation in a cell-free system from Xenopus laevis eggs

A cell-free system is described which formed very high levels of recombinant DNA structures in 4 hr at 26°C. It consisted of a single fraction of a high speed supernatant prepared from an extract of unfertilized eggs of the frog Xenopus laevis. This fraction eluted at 0.16?0.18 M Tris homogenization buffer from a DEAE-cellulose column. When two partially homologous supercoiled DNA molecules of different contour lengths were incubated simultaneously in this system, high levels of heterologous figure eight DNA structures were formed and observed by electron microscopy. Subsequent cleavage of the newly formed figure eight structures with Bam HI and Eco RI restriction endonucleases gave rise to “α structures” and “χ structures.” The observed figure eight structures presumably represent the recombination intermediate predicted by the Holliday model for genetic recombination.