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.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

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.     

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.     

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.     

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.     

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.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

The ultrastructural organization of the isolated cortex in eggs ofNassarius reticulatus (Mollusca)

Summary We have studied the organization of the cortex in fertilized eggs ofNassarius reticulatus by examining rotary-shadowed whole mounts of isolated cortices in the transmission electron microscope. The following components were distinguished: (a) the plasma membrane, with clathrin-coated areas and coated pits, (b) microfilaments and microtubules, and (c) a tubulovesicular network of endoplasmic reticulum. Microfilaments were identified by labeling with heavy meromyosin, and microtubules with a monoclonal anti-tubulin antibody, using both immunofluorescence microscopy and immunogold labeling for transmission electron microscopy. The microfilaments are organized in a network parallel to and closely associated with the plasma membrane, with typical Y- and X-shaped intersections. The endoplasmic reticulum is associated with this microfilamentous lattice. The microtubules also run parallel to the plasma membrane, but they are located at a greater distance, as can be inferred from stereo images. In the uncleaved egg, numerous microtubules are present in the egg cortex. Shortly before polar lobe formation, at the onset of mitosis, the microtubules disappear almost entirely. They reappear again at the end of first cleavage, as the polar lobe is being resorbed. The synthesis of cortical microtubules at this stage appears to depend on the presence of microtubule-organizing centers in the animal hemisphere of the egg, since microtubules do not reappear in isolated polar lobes. Clathrin-coated areas are present in both the animal and vegetal hemisphere before polar lobe formation. During mitosis, the clathrin-coated plaques and pits are found almost exclusively in the animal hemisphere. After resorption of the polar lobe, at the two-cell stage, no clathrin-coated areas were found at all.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

Cytokeratin intermediate filament organisation and dynamics in the vegetal cortex of living Xenopus laevis oocytes and eggs

Cytokeratin intermediate filaments are prominent constituents of developing Xenopus oocytes and eggs, forming radial and cortical networks. In order to investigate the dynamics of the cortical cytokeratin network, we expressed EGFP-tagged Xenopus cytokeratin 1(8) in oocytes and eggs. The EGFP-cytokeratin co-assembled with endogenous partner cytokeratin proteins to form fluorescent filaments. Using time-lapse confocal microscopy, cytokeratin filament assembly was monitored in live Xenopus oocytes at different stages of oogenesis, and in the artificially-activated mature egg during the first cell cycle. In stage III to V oocytes, cytokeratin proteins formed a loose cortical geodesic network, which became more tightly bundled in stage VI oocytes. Maturation of oocytes into metaphase II-arrested eggs induced disassembly of the EGFP-cytokeratin network. Imaging live eggs after artificial activation allowed us to observe the reassembly of cytokeratin filaments in the vegetal cortex. The earliest observable structures were loose foci, which then extended into curly filament bundles. The position and orientation of these bundles altered with time, suggesting that forces were acting upon them. During cortical rotation, the cytokeratin network realigned into a parallel array that translocated in a directed manner at 5 microm/minute, relative to stationary cortex. The cytokeratin filaments are, therefore, moving in association with the bulk cytoplasm of the egg, suggesting that they may provide a structural role at the moving interface between cortex and cytoplasm.     

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

Summary The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.     

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.     

Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal–vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.     

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.     

Hecate/Grip2a Acts to Reorganize the Cytoskeleton in the Symmetry-Breaking Event of Embryonic Axis Induction

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.     

Snoods: a periodic network containing cytokeratin in the cortex of starfish oocytes.

An extensive fibrous cytoskeletal component in the cortical cytoplasm of oocytes of the starfish Pisaster ochraceus reproducibly stains with anticytokeratin antibody and hence contains cytokeratin. The large-meshed network resembles a snood (hair net). Snood fibers form loops and branches throughout the cortex of a premeiotic oocyte, except at the animal pole where they emanate from a nonstaining zone surrounding the centrosomes. By immunofluorescence microscopy of isolated cortices and electron microscopy of isolated cortices and intact oocytes, snood fibers exhibit complex striations with a periodicity of approximately 0.75 micron. Snoods are not colocalized with the cortical arrays of microtubules and are unaffected by drugs that disrupt microtubules or microfilaments. Stimulation of oocyte maturation by 1-methyladenine causes snoods to disappear, presumably by disassembly, about halfway to the time of germinal vesicle breakdown. They do not reappear during meiosis, fertilization, or development to the two-cell stage, and their functional importance, if any, during oogenesis or development remains to be elucidated.     

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.