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.     

Regional Difference in Mechanical Properties of the Cell Surface in Dividing Echinoderm Eggs*

Stiffness of the cell was surface was determined in fertilized sea urchin and starfish eggs by measuring the mechanical resistivity of the cell surface against negative pressure applied to a restricted part with a micropipette in contact with the cell surface at its tip (elastimetry). In both sea urchin and starfish eggs, the stiffness of the cell surface changed almost in parallel between the presumptive furrow and polar surfaces before the onset of the first cleavage, and the stiffness of the furrow surface became larger than that of the polar surface when cleavage started, although temporal changes in the stiffness were different between sea urchin and starfish eggs. The stiffness of the cell surface changed almost in parallel between the surfaces at the equator and at the animal pole in starfish eggs before the onset of polar body formation. The stiffness of the cell surface around the forming polar body increased during the formation of the polar body and remained at a high level after the polar body formation. It seems that the stiffness difference responsible for the formation of the contractile ring develops simultaneously with rather than prior to the formation of the cleavage furrow.     

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.     

Effect of protein phosphatase inhibitors on cleavage furrow formation in newt eggs: Inhibition of normal furrow formation and concomitant induction of furrow-like dents

The effects of three protein phosphatase inhibitors, okadaic acid, calyculin A and tautomycin, on the formation of cleavage furrows and the induction of furrow-like dents in the egg of the newt, Cynops pyrrhogaster , were examined. Solutions of the individual compound were injected into the animal hemisphere of one of the two presumptive blastomere regions of the embryo during the first cleavage. Injection of a solution containing any of the chemicals often disturbed the formation of a normal furrow in the injected blastomere at second cleavage. Injection with okadaic acid or calyculin A often induced furrow-like dents on the surface of the injected blastomere at the same time as second cleavage in control embryos, while that with tautomycin usually did not induce them. In an injected blastomere, formation of dents started in the animal half and moved towards the vegetal half as the furrow in its counterpart blastomere extended from the animal half towards the vegetal. Dents gradually became slightly deeper and formed cytoplasmic projections that later degenerated, leaving a surface scar. Cytological observations on blastomeres injected with calyculin A revealed that nuclear division occurred normally.     

CORTICAL AND SUBCORTICAL CHANGES PRECEDING FURROW FORMATION IN THE CLEAVAGE OF NEWT EGGS

In the first cleavage of the egg of the newt, Cynops(Triturus) pyrrhogaster, some sort of preparation for the furrow formation in the cortical and subcortical cytoplasm precedes the advancing tip of the cleavage furrow. This is shown by the following facts: (1) Incisions made close to the tip of the cleavage furrow do not stop the progress of furrowing, allowing the furrow to cross the incisions and appear on the farther sides, while incisions made far enough from the furrow tip always prevent the further travelling of the furrow, (2) Displacement of the subcortical cytoplasm ahead of the furrow by rubbing with a hair loop makes the furrow bend corresponding to the width of the rubbed area, and (3) Transplantation of the subcortical material of the furrow tip to lateral parts in the same egg causes a depression in the overlying cortex at the transplanted position. The linear extension of the prepared area for the furrow formation is the longest in the animal hemisphere and it decreases in gradient towards the vegetal pole.     

A subcortical, pigment-containing structure in Xenopus eggs with contractile properties

An accumulation of insoluble, finely granular material has been observed under the pigmented surface of Xenopus eggs by a specialized "dry fracture" technique and scanning electron microscopy. Cortical granules and pigment granules can be recognized with the techniques and can be seen to be embedded in the material. Thin sections show that the region also contains mitochondria and membranous vesicles or reticula. Yolk platelets are largely excluded from the heaviest accumulations of the material. The substance is most dense just under the cortex and grades off gradually into the more diffuse, yolk-containing network of the endoplasm. The accumulation of material is much thicker in the animal hemisphere of the egg than in the vegetal hemisphere, and the pigment embedded in it defines the pigmented area of the animal hemisphere. In the pigmented area the material excludes yolk for a thickness of 3-7+ microns from the surface. In the vegetal hemisphere there is no such accumulation and yolk platelets can be found almost touching the plasmalemma. Cortical contractions have been experimentally induced in eggs. Their relative strength correlates with the relative thickness of the finely granular, subcortical material. During contraction the material accumulates to much greater thicknesses, excluding yolk from thicknesses of 15-30+ microns from the surface. The contracting entity is, or is in, the finely granular material. Injection of cytochalasins into the eggs inhibits cleavage furrow operation but does not inhibit the induced cortical contractions. The thus do not seem to be dependent on actin microfilamentogenesis as is the operation of the contractile ring of the cleavage furrow. The differential sensitivity to cytochalasins of the contractile ring and the system responding in the induced cortical contractions, suggests a two-component system for cortical contractions in the egg. A model is presented which accommodates the available data.     

Unequal first cleavage in the Tubifex egg: involvement of a monastral mitotic apparatus

The first cleavage in the freshwater oligochaete Tubifex hattai is unequal and meridional, and produces a smaller cell AB and a larger cell CD. This study traces the process of furrow formation, reorganization of cortical F-actin and the assembly of a mitotic apparatus during this unequal division. Cleavage furrow formation consists of two stages: (i) when eggs are viewed from the animal pole, meridionally running furrows emerge at two points of the egg's equator that are 90° apart from each other and approach the egg axis as they deepen; and (ii) at the midpoint between the equator and the egg center, the bottoms of these furrows link to each other on the animal and vegetal surfaces of the egg and form a continuous ring of constriction in a plane parallel to the egg axis. Egg cortices, isolated during the first step and stained with rhodamine-phalloidin, show that the bottoms of recently formed furrows are underlaid by a belt of tightly packed actin bundles (i.e. a contractile arc). The transition to the second stage of furrow formation coincides with the conversion of these actin belts into a continuous ring of F-actin. Whole-mount immunocytochemistry of microtubules reveals that the first cleavage in Tubifex involves an asymmetric mitotic spindle, which initially possesses an aster at one pole but not the other. This ‘monastral’ spindle is located at the egg's center and orients itself perpendicular to the egg axis. During anaphase, astral rays elongate to reach the cell surface, so that the array of astral microtubules in the plane of the egg's equator covers a sector of 270–300°. In contrast, it is not until the transition to telophase that microtubules emanating from the anastral spindle pole approach the cell margin. If eggs are compressed along the egg axis or forced to elongate, they form monastral spindles and divide unequally. In living compressed eggs, mitotic spindles, which are recognizable as bright streaks at the egg's center, appear not to shift their position along the spindle axis during division, suggesting that without eccentric migration of spindles Tubifex eggs are able to divide unequally. These results suggest that mechanisms that translocate the mitotic spindle eccentrically do not operate in Tubifex eggs during the first cell cycle. The mechanisms that generate asymmetry in spindle organization are discussed in the light of the present results.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.     

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

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.     

Cleavage furrow: Calcium as determinant of site

Direct application of EDTA at precise locations on the cortex at different times during the division of sea urchin eggs was accomplished with a capillary filled with agar-EDTA using a micromanipulator. Treatment with EDTA prior to mid-metaphase arrested cleavage irreversibly. The effects of EDTA applications during metaphase varied according to the precise site of treatment. EDTA treatment of the cleavage furrow, after it appeared at the end of anaphase, had no effect on the completion of cleavage. These results are discussed in relation to the possible role of calcium in determining the site of the cleavage furrow.     

Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow-like Dent in Amphibian Eggs

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow-like dents in eggs of the newt, Cynops pyrrhogaster , were examined.
Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.
The ability of the cortex of a cleavage-arrested blastomere to form a furrow-like dent was tested by inhibiting furrow formation of one blastomere of two-cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow-inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.
These results show that microtubule structures are directly involved in formation of a cleavage furrow.     

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

Localized calcium signals along the cleavage furrow of the Xenopus egg are not involved in cytokinesis

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.     

Early development of zooxanthella-containing eggs of the corals Pocillopora verrucosa and P. eydouxi with special reference to the distribution of zooxanthellae

Some hermatypic corals spawn eggs that contain zooxanthellae. We followed development of zooxanthella-containing eggs of two such species, Pocillopora verrucosa and P. eydouxi. We also documented changes in the distribution pattern of zooxanthellae during development. Oocytes of both species took up zooxanthellae 3 to 4 days before spawning. At first, zooxanthellae were evenly distributed in oocytes, but they later moved to the hemisphere that contained the germinal vesicle. After fertilization, early cleavage events were holoblastic, progressing by furrow formation. The first cleavage furrow started at the hemisphere that contained zooxanthellae, dividing the zooxanthellate complement of the zygote about equally into the two blastomeres. The second division divided each blastomere into one zooxanthellae-rich cell and one with few zooxanthellae. With continued cell division, blastomeres containing zooxanthellae moved into the blastocoel. The blastocoel disappeared at about 5 h after the first cleavage, and the central region of the embryo was filled with cells containing either zooxanthellae or lipid droplets, forming a stereogastrula. Our results suggest that only blastomeres that had been determined to develop into gastrodermal cells receive zooxanthellae during cleavage. This determination appears to take place, at the latest, by the second cell division at the four-cell stage.     

Localization of gamma-tubulin and cyclin B during early cleavage in physiologically polyspermic newt eggs

To understand the mechanism of the very slow block to polyspermy in physiologically polyspermic eggs of the newt Cynops pyrrhogaster, we used confocal laser microscopy to determine the distribution of gamma-tubulin and cyclin B1 in fertilized eggs. More gamma-tubulin was localized in the animal hemisphere than in the vegetal. The centrosomes of the principal sperm nucleus and the zygote nucleus had much accumulated gamma-tubulin, but little gamma-tubulin was associated with the centrosomes of the accessory sperm nuclei. These results are consistent with observations that the largest sperm aster is associated with the principal sperm nucleus. More cyclin B1 appeared in the animal hemisphere than in the vegetal at the end of interphase. The zygote nucleus had much accumulated cyclin B1, but little cyclin B1 was associated with the accessory sperm nuclei. Cyclin B1 disappeared earlier around the zygote nucleus at metaphase than around the accessory sperm nuclei. These findings correspond well with the earlier entry and exit into metaphase in the zygote nucleus than in the accessory sperm nuclei in newt eggs, supporting our maturation-promoting factor (MPF) model that accounts for the mechanism of nuclear degeneration in physiologically polyspermic eggs. Cyclin B1 began to accumulate in the nucleus during interphase in synchronous cleavage, and its greatest expression was in the centrosomes and the nucleus at prometaphase.     

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.