Cell proliferation in the ectoderm of the Xenopus embryo: development of substratum requirements for cytokinesis

The requirements for cell division in ectodermal blastomeres of the early Xenopus embryo were studied. Isolated blastomeres divide autonomously on nonadhesive agar in a simple salt solution up to the midblastula stage. After the midblastula transition, cell-cell contact is required for blastomere division. In isolated blastomeres of that stage, cytokinesis fails, but nuclear division continues normally for some time. Cell-cell contact as a prerequisite for blastomere division can be replaced by culturing blastomeres on an appropriate substratum. Clonal growth of isolated blastomeres is supported by a variety of protein substrata, indicating rather unspecific substratum requirements. Different substrata which do not support blastomere division can affect different steps in cytokinesis.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Asymmetrical rotations of blastomeres in early cleavage of gastropoda

Summary The movements of blastomere surfaces marked with carbon particles during cytokinesis of the Ist–IVth cleavage divisions in the eggs of the gastropodsLymnaea stagnalis, L. palustris, Physa acuta and Ph. fontinalis have been studied by time-lapse cinematographic methods. The vitelline membrane was removed with trypsin. At 2- and 4-cell stages shifts of nuclei have also been studied.Symmetrical as well as asymmetrical surface movements (in respect to the furrow plane) have been revealed. Symmetrical surface movements at the beginning of cytokinesis consist mainly in contraction of the furrow zone and in expansion of the more peripheral regions; between these there is a stationary zone. After the end of cytokinesis the furrow region expands.Considerableasymmetrical surface movements have also been observed in all four divisions. From anaphase until the end of cytokinesis each of the two sister blastomeres rotates with respect to the other in such a way, that if viewed along the spindle axis, the blastomere nearest to the observer rotates dexiotropically in a dextral species and laeotropically in a sinistral species (primary rotations). After the completion of cytokinesis the blastomeres may rotate in a reverse direction. The latter rotations are less pronounced in the IInd and IIIrd divisions and most pronounced in the IVth division. Blastomeres with the vitelline membrane intact retain a slight capacity for primary rotations. In normal conditions nuclei of the first two blastomeres shift mainly laeotropically in dextral species, but dexiotropically in sinistral species, being carried along by the reverse surface rotations.The invariable primary asymmetrical rotations of blastomeres seem to be the basis of enantiomorphism in molluscan cleavage. They are assumed to be determined by an asymmetrical structure of the contractile ring carrying out the cytokinesis.     

Dislocation of gold particles in the cytoplasm of mouse zygotes during cleavage

Summary Gold particles were introduced into mouse zygotes before the first cleavage. At cleavage, dislocation of particles within the cytoplasm was monitored. Cytoplasmic movement occurs in the central region of dividing zygotes as judged from the centripetal dislocation of particles there. Cytoplasm in polar regions appears less mobile. No particle crosses a vertical axis of the zygote during cytokinesis, which suggests that the cytoplasm of the arising blastomeres is not mixed. Our general conclusion is that the cytoplasmic territories of the mouse egg appear to be passively segregated between two blastomeres.     

The blebbing response of 2-4 cell stage mouse embryos to cytochalasin B.

In light and electron microscopic examination of 2–4-cell stage mouse embryos, the embryos showed transitory blebs on the blastomere surface when cytochalasin B (CB) was applied shortly before or after mitosis. A similar effect was obtained in blastomeres removed from the drug at these stages but not in blastomeres maintained in the continued presence of the drug. CB also promoted the precocious deposition of crystalloid bodies and inhibited the formation of intercellular contacts. Microvilli and coated pits were not affected.     

Predetermination of cytokinesis and transition from holoblastic to partial superficial cleavage in early embryonic development of Tetrodontophora bielanensis (Collembola)

In the T. bielanensis embryo, only karyokinesis occurs during the first cleavage division, and a two-nuclear syncytial embryo forms. Then, two cytoplasmic concentrations in the form of elongated rolls perpendicular to each other develop below the periplasm at the animal pole of the egg. The second cleavage division is also associated with karyokineses only. After the embryo reaches the four-nuclear stage, cytokinesis occur at its animal pole, and two cleavage furrows perpendicular to each other develop in the periplasm above the cytoplasmic concentrations. The cell membranes forming within the furrows do not invade the cytoplasmic concentrations, but their growing tips push them into the egg interior, where they merge and form the central cytoplasmic concentration. The developing cell membranes do not invade the central cytoplasm; they band and grow above its surface. Four pyramidal blastomeres form as a result of this. The eight-blastomere embryo forms through both karyokinesis and cytokinesis, but the growing cell membranes now band below the previous ones and cut off anucleate parts of the mother blastomeres, which fuse with the central cytoplasm. Thus, during this phase of development the transition from holoblastic to partial superficial cleavage is initiated. Morphological analysis suggests that the formation of the first two cytokinesis is predetermined by and depends on factors connected with the animal pole periplasm. It also suggests that the central cytoplasm constitutes the morphological field, inducing the transition from holoblastic to partial superficial cleavage.     

Effect of Cytochalasin B on the Cytokinesis of Thuricola folliculata (Ciliophora,Peritrichida)1

ABSTRACT. Binary fission of the sessiline loricate peritrich Thuricola folliculata was examined by light microscopy. Cytokinesis, which occurs in the oral-aboral median plane, is 35-40 min in duration. Cytochalasin B (CB) was used at final concentrations of 50–100 μg/ml in dimethyl sulfoxide (DMSO). A transition point occurs at about 20 min after the beginning of binary fission and about 10 min before cytokinesis. In most cases, 50 μg/ml and 100 μg/ml CB added to cells before the transition point resulted in delays of cytokinesis which were significant compared to DMSO controls; sometimes, cytokinesis was blocked completely. If added after the transition point, CB had no effect on cells.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.     

Modification of cell division by phorbol ester in preimplantation mouse embryos

2-cell mouse embryos were treated in vitro with a 2 h pulse of phorbol myristate acetate (PMA) at 32nd, 38th and 50th h after hCG, then chased in culture for up to 46 h. Embryos were fixed at various time intervals of chasing, then stained and inspected. Some embryos were carefully inspected with a video recording system, every 1.44s and the cell divisions (cytokinesis) as well as formation of large, single blastomeres, each from two smaller ones, were recorded. PMA pulse let to the suppression of cell divisions. The rate of the suppression was time dependent: with a delay of 0-1, 12 and 18 h between the PMA pulse and time of scheduled cell division about 99, 87 and 44% of 2-cell embryos remained at this stage of development, for at least 10 h, respectively, and 90, 58 and 12% of their blastomeres revealed binuclearity. Since we found that PMA-mediated formation of binuclearity was not the effect of cell fusions, it was assumed that the inhibition of cytokinesis preceded by karyokinesis was responsible for binuclearity. PMA effect on cell divisions was reversible. PMA-treated embryos revealed formation of large, single blastomeres, each from two smaller ones. If cell division appeared after PMA pulse, in about 52% of 3- to 6-cell embryos, the large blastomere formation was recorded in the course of the subsequent 38 h. Large blastomere formation was concluded to be the result of either cell fusion or reversion of incompleted cytokinesis brought about by PMA.     

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus)

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.     

Holoblastic early cleavage of Tetrodontophora bielanensis (Collembola) eggs, with special reference to its irregularity.

The fertilized eggs of Tetrodontophora bielanensis start to cleave 6 to 8 days after oviposition and initially only karyokineses occur. The cytokinesis begins after two karyokineses, when four nuclei are observed in the ooplasm. Two cleavage furrows, perpendicular to each other, appear simultaneously at the egg poles where polar bodies are located and gradually the furrows encompass the whole egg diameter. The furrow formation is initiated by the bundle of microfilaments that contract and pull superficial fragments of the oolemma into the yolk and subsequently new membranes, separating the daughter cells, start to form. However, they do not grow towards the egg centre but bifurcate, leaving the central part of the ooplasm outside of the newly formed blastomeres. Starting from the fourth or fifth cleavage division, the bifurcations permanently occur and multiple cleavage furrows are formed on the embryo surface. Moreover, fragments of the ooplasm, enclosed within the cell membrane but devoid of cell nucleus are observed. During further development such cell fragments become reincorporated into the embryo. This mode of cleavage leads eventually to the formation of cellular blastoderm on the embryo surface. The results presented in the paper suggest that the control of cleavage in T. bielanensis acts not at the level of cytoplasmic determinants but rather at the level of positional information of blastomeres.     

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

BACKGROUND: The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. RESULTS: We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. CONCLUSIONS: We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell.     

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.     

Effects of cytochalasin B on the cleavage furrow in mouse blastomeres

Summary Blastomeres isolated from two-cell mouse embryos were cultured until they started to cleave. When the cleavage furrow developed they were subjected to cytochalasin B (CB) and were studied with the electron microscope. The initial response to CB is that the furrow is more folded and microvillous than in the control. Later the blastomeres round up. The protrusions covered with abundant long microvilli are found scattered within their equatorial surface. Extraction with glycerol solution before fixation permits visualization of condensations of felt-like filamentous material in contact with the cleavage furrow during the initial response to CB and in the protrusions of rounded cells. We consider clumping of filaments in surface protrusions to be a specific response to CB treatment of the contractile ring.Some of the previous papers by this author have been published under the name Opas     

Nuclear migration and spindle formation in the fourth cleavage of sea urchin eggs under the influence of inhibitors.

Mitosis of sea urchin eggs was inhibited when exposed to 3 micrograms/ml aphidicolin from the 2-cell stage onwards. Nevertheless the nuclei migrated to the vegetal pole at the time of the fourth unequal division in control eggs. Two or four equal or unequal asters developed. Asters in proximity to the vegetal pole were always considerably smaller than those close to the center of the two blastomeres. In contrast to colchicine, cytokinesis but not migration of the nuclei in the vegetal half was prevented by treatments with 5 microM cytochalasin B or D. Various mitotic figures were formed in the vegetal blastomeres of eggs treated with 0.4 mM colchicine or 3 microM griseofulvin after the third cleavage. In some eggs a centrally localized monaster with chromosomes in sphere-like arrangement was formed in others a monopolar mitotic figure pushed the chromosomes in bowl-like arrangements to the most vegetal cortex. In anaphase one set of chromatids migrated to the monopole leaving the scattered sister-chromatids behind. The mechanism of migration of the nuclei and of chromosome arrangement in the metaphase plate is discussed.     

Spindle pole regulation by a discrete Eg5-interacting domain in TPX2

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.     

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata

Summary Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK₂ cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were either one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK₂ cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.     

The survival of cytochalasin-induced polykaryons following exposure to cytotoxic agents.

Using CHO-K1, HeLa S3 and two Walker lines (WR and WS) differentially sensitive to cis-diamminedichloroplatinum(II) (cisplatin), the survival after exposure to cisplatin, mitomycin C, vinblastine, vincristine or cytosine arabinoside has been determined either of clonogens or of cells rendered polyploid by post-exposure incubation in the presence of cytochalasin B (CB). It is suggested that the inhibition of cytokinesis by CB permits an assessment to be made of the fraction of damage whose expression is cell division-related, possibly including that resulting from a loss or malsegregation of genetic material. It was found that the response of polykaryons in comparison to clonogens was both agent- and cell line-dependent. After cisplatin exposure, polykaryon survival (defined as the ability to accumulate at least 16C DNA) declined exponentially with dose and was qualitatively, and to some extent quantitatively, similar to that observed previously after irradiation. In HeLa S3, giant cells induced by 10-20Gy irradiation in the absence of CB exhibited a radiation dose-dependent reduction in the relative frequency of highly polyploid cells which was similar to that observed in CB-induced polykaryons.