Mechanism of formation, ultrastructure, and function of the cytoplasmic bridge system during morphogenesis in Volvox

The cytoplasmic bridge system that links all cells of a Volvox embryo and plays a crucial role in morphogenesis is shown to form as a result of localized incomplete cytokinesis; sometimes bridge formation occurs before other regions of the cell have begun to divide. Vesicles, believed to be derived from the cell interior, align along the presumptive cleavage furrow in the bridge-forming region. Apparently it is where these vesicles fail to fuse that bridges are formed. Conventional and high voltage transmission electron microscopy analyses confirm that bridges are regularly spaced; they possess a constant, highly ordered structure throughout cleavage and inversion. Concentric cortical striations (similar to those observed previously in related species) ring each bridge throughout its length and continue out under the plasmalemma of the cell body to abut the striations of neighboring bridges. These striations are closely associated with an electron-dense material that coats the inner face of the membrane throughout the bridge region and appears to be thickest near the equator of each bridge. In addition to the parallel longitudinal arrays of cortical microtubules that traverse the cells, we observed microtubules that angle into and through the bridges during cleavage; however, the latter are not seen once inversion movements have begun. During inversion, bridge bands undergo relocation relative to the cell bodies without any loss of integrity or change in bridge spacing. Observation of isolated cell clusters reveals that it is the sequential movement of individual cells with respect to a stationary bridge system, and not actual movement of the bridges, that gives rise to the observed relocation.     

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.     

Intercellular communication in the eight-cell stage of Xenopus laevis development: a study using dye coupling

We report an investigation of dye coupling in the eight-cell stage of Xenopus laevis development. Our results indicate that fluorophors injected into micrometers at this stage pass only to sister cells (the corresponding macromeres) and that detectable dye transfer occurs only via cytoplasmic bridges, which persist for about the first two-thirds of the fourth cell cycle. We had previously shown that the dorsoventral polarity of the Xenopus embryo is regulated by a cell interaction that occurs at the end of the fourth cell cycle and we conclude that this cell interaction probably does not require cytoplasmic bridges or gap junctions.     

COLONY DEVELOPMENT IN EUDORINA ELEGANS (CHLOROPHYTA,VOLVOCALES)1

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.     

A kinesin,invA, plays an essential role in volvox morphogenesis

In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.     

VOLVOX POCOCKIAE,A NEW SPECIES WITH DWARF MALES1

Volvox pocockiae is described as the second species in the section Janetosphaera. The somatic protoplasts are connected by cytoplasmic strands approximately the same diameter as flagella, and the construction of the spheroid is identical to that of V. aureus. Asexual reproduction by the division of gonidia differs from that in V. aureus in the enlargement of the gonidium prior to its division to form the embryo. Sexual reproduction is very similar to that in V. spermatosphaera, a species in the section Merrillosphaera without cytoplasmic connections. Dwarf males are formed in the posterior end of the parental spheroid, and, as in V. spermatosphaera, the dwarf males are composed exclusively of androgonidia with no sterile somatic cells. Females are facultatively asexual spheroids, the gonidia of which function as eggs. The single biflagellate zoospore produced by the germinating zygote undergoes cleavage to form a germling spheroid. The differentiation of gonidia in the asexual embryo and in the germling spheroid is evident only after inversion and enlargement of the spheroid have begun.     

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.     

红松胚胎发育及其淀粉的动态

本文观察了红松胚胎发育及其淀粉的分布全过程。红松合子位于颈卵器中上部,分裂时形成的纺锤体与颈卵器长轴呈一定角度,第一次有丝分裂产生2个游离核,它们边下沉边进行第二次有丝分裂,形成4个游离核排列成一层.接着进行第三次有丝分裂产生8个游离核,排成两层并形成细胞壁。第四次分裂产生16个细胞排成四层,组成了原胚,经一个半月的生长发育及胚胎选择阶段,7月中旬进入幼胚的发育时期。8月上旬幼胚已经进行组织和器官分化,此时淀粉逐渐由珠孔端向合点端方向推移,9月末种子成熟。     

Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.     

Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma

Embryos of the indirect developing sea urchin, Heliocidaris tuberculata, and of Heliocidaris erythrogramma which develops directly without the formation of a pluteus larva, were bisected at the two- and four-cell stages. Paired half-embryos resulting from the bisection of H. tuberculata embryos along either the first or the second cleavage plane develop identically into miniature prism stage larvae. As in other indirect developing sea urchins, no differential segregation of developmental potential takes place as a result of the first and second cleavage divisions. Although half-embryos resulting from bisection along the second cleavage plane differentiate all cell types and develop equivalently in H. erythrogramma, the isolated first cleavage blastomeres do not. One of these two cells always forms significantly more mesodermal and endodermal cells. These patterns of differentiation are consistent with fate-mapping studies indicating that most mesodermal and endodermal cells are derived from the prospective ventral blastomere. Therefore, a differential segregation of developmental potential takes place at the first cleavage division in H. erythrogramma. When embryos of H. erythrogramma were bisected during the eight-cell stage, isolated tiers of animal blastomeres typically formed only ectodermal structures including the vestibule, whereas vegetal embryo halves formed all differentiated cell types. We propose that animal-vegetal cell determination and differentiation takes place along an axis which has been shifted relative to the pattern of cell cleavages in the embryos of H. erythrogramma. Vegetal morphogenetic potential for the formation of mesodermal and endodermal structures has become more closely associated with the prospective ventral side of the embryo during the evolution of direct development in Heliocidaris.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

Reproductive cell specification during Volvox obversus development

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.     

Morphogenesis in Volvox: analysis of critical variables.

Inversion, the process by which Volvox embryos turn inside out, was analyzed by a combination of geometrical and experimental techniques. It was shown that simple geometric figures are adequate to represent cell shapes during inversion and that cell volumes remain constant as cell shapes change and the embryo inverts. The first stage of inversion, phialopore opening, results from the release of compressive forces as the embryo withdraws from its surrounding vesicle during a two-stage contraction of each cell around its radial axis. Premature phialopore opening occurs when withdrawal of the embryo from the vesicle is elicited artificially by exposure to either calcium ionophore or hypertonic solutions. The major event of inversion, generation of negative curvature, requires both microtubule-driven elongation of cells (to produce a classical "flask" shape) and cytochalasin-sensitive active migration of cytoplasmic bridges to the outermost ends of flask cells. Colchicine, cyclic GMP and isobutyl methyl xanthine (individually) block both normal elongation and bridge migration; cytochalasin D blocks bridge migration selectively. Flask cell formation and bridge migration are adequate to account for the negative curvature observed. An asymmetric bending of flask cell stalks along the ring of maximum curvature accounts for the fact that the embryo is not constricted in a "purse-string" fashion as negative curvature is generated. Inversion of the posterior hemisphere involves an elastic snap-through resulting from a combination of compressive stresses generated by inversion of the anterior hemisphere and the circumferential restraint imposed by cells at the equator. We conclude that the observed changes in cell shape and the migration of cytoplasmic bridges are the result of an ordered process of membrane-cytoskeletal interactions, and both necessary and sufficient to account for the morphogenetic process of inversion in Volvox.     

A study of intercellular bridges during spermatogenesis in the rat

A morphological evaluation of intercellular bridges was undertaken during rat spermatogenesis. The dimensions and relationships of the bridges were shown to vary during different phases of spermatogenesis. Cellular divisions of spermatogonia and spermatocytes resulted in the partitioning of pre-existing bridges by complex structures termed bridge partitioning complexes, which are described in detail, as is the process whereby new bridges are formed. The structure of premeiotic bridges was generally consistent; however, during spermiogenesis, the structure of bridges and bridge contents were modified at specific phases of their development. The plasma membrane density associated with the cytoplasmic aspect of early step 1 spermatids separated into multiple dense bands that encircled the peripheral aspect of late step 1 spermatid bridges. By step 2 of spermiogenesis, these dense bands became associated with several cisternae of endoplasmic reticulum, which later coalesced into a single saccule that completely encircled the bridge structure by step 4. At steps 10-13 of spermiogenesis, the single saccule of endoplasmic reticulum vesiculated into many smaller cisternae. Also, filament-bounded densities (measuring 10-12 nm in diameter) appeared within the bridge channel. At step 17 of spermiogenesis, the filament-bounded densities were no longer apparent, but an anastomosing network of endoplasmic reticulum, often in the configuration of a sphere, occupied the entire central region of the bridge. In step 19 spermatids, the smooth endoplasmic reticulum within the bridge channel and the multiple cisternae lining the bridge density were gradually displaced. The subsurface density of bridges gradually lost its prominence. Some cytoplasmic lobes were connected by extremely narrow (approximately 22 nm) cytoplasmic channels. Similar-appearing channels were seen on the surface zone of cytoplasmic lobes or residual bodies, this observation suggesting that channels were sites of severence of bridges. Just prior to the separation or disengagement of the spermatid from the cytoplasmic lobe, selected bridges appeared to open to form large masses. After spermiation, residual bodies were not found joined by bridges; but from the size of some of the residual bodies, it was suspected that they were formed by coalescence of more than one cytoplasmic lobe. Freeze-fracture demonstrated few intramembranous particles on either the P or E face of the plasma membrane forming the bridge; this finding suggested bridge structures restricted free lateral movement of membrane constituents across the bridge.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationship between cell size and cell fate in Volvox carteri

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.     

Abscission checkpoint control: stuck in the middle with Aurora B

At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges 'stuck' in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.     

Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres

Sponges (Porifera) are unusual animals whose body plans makeinterpreting phylogenetic relationships within the group andwith other basal metazoan taxa a difficult task. Although molecularapproaches have offered new insights, some questions requirea morphological approach using detailed ultrastructural or lightmicroscopical studies of developing embryos and larvae. Glasssponges (Hexactinellida) have perhaps the most unusual bodyplan within the Metazoa because the majority of the tissue ofthe adult consists of a single giant multinucleated syncytiumthat forms the inner and outer layers of the sponge and is joinedby cytoplasmic bridges to uninucleate cellular regions. Herewe have used serial section transmission and high-resolutionscanning electron microscopy to examine when syncytia firstform in the cave-dwelling glass sponge Oopsacas minuta. We confirmthat in O. minuta blastomeres are separate until the 32-cellstage; cleavage is equal but asynchronous until a hollow blastulais formed. The sixth division yields a collection of variouslysized micromeres at the surface of the embryo and large yolk-and lipid-filled macromeres lining the blastocoel. Syncytiathen form by the fusion of micromeres to form cytoplasmic bridgeswith each other and the fusion of macromeres to form the futuremultinucleated trabecular tissue of the larva and adult sponge.The multinucleated trabecular tissue envelops and forms cytoplasmicbridges with all uninucleate cells, covering the developinglarva with a continuous syncytial epithelium. Differentiationof tissues occurs very early during embryogenesis with the separationof uninucleate and multinucleate lineages, but all cells andsyncytia are joined by cytoplasmic bridges such that there iscytoplasmic continuity throughout the entire larva. Althoughglass sponges begin life as a cellular embryo, the unusual mechanismof syncytia formation at such an early stage in developmentdistinguishes this group of animals from their closest multicellularrelatives, the Demospongiae. Most important, however, thesedata lend support to the hypothesis that the original metazoanswere cellular, not syncytial.