Topology of cleavage in light of the Pierre Curie principle

The stages of radial, spiral, and bilateral cleavage, including blastula, are considered as polyhedrons and projections of polyhedrons onto a plane: Wenn, Schlegel, and Euler projections. The blastula spatial organization is characterized by face numerals and Euler characteristics, as well by symmetry groups. The classes of equivalence of polyhedrons have been considered: duality and eqicomposition. The correspondence between different types of cleavage has been established by shift transformation on Schlegel projections and turn on a spherical noneuclidean surface. Determination of the prospective significance of blastomeres during cleavage was compared with the dichotomous division of the general notion in logic. This in view, the Wenn diagram of four figures has been reflected onto the sphere surface. Blastula faceting is interpreted as a reflection of hereditary information about the prospective significance of blastomeres onto a spherical surface. Topologically, the reflection represents a transformation of the linear orderliness of information contained in the genome into a two-dimensional orderliness of prospective properties on the blastula noneuclidean surface. Therefore, the elements of blastula symmetry can be considered as self in the sense of Pierre Curie principle.     

A fine structural study of cytodifferentiation during cleavage, blastula, and gastrula stages of Fundulus heteroclitus

The fine structure of cleavage, blastula, and gastrula stages of Fundulus heteroclitus was investigated. Cleavage blastomeres are relatively unspecialized, containing few or poorly developed organelles. Beginning in blastula stages, signs of differentiation were noted, including development of the endoplasmic reticulum and Golgi apparatus and appearance of a primary nucleolus and polyribosomes. More extensive structural specializations occur in gastrula stages, including further development of the endoplasmic reticulum and appearance of a granular component in the nucleolus. These changes are associated with cell differentiation and an increased capacity for protein synthesis, and may be preparatory to subsequent histogenesis. The periblast is a continuous syncytial cytoplasmic layer located between the blastodisc and yolk and is formed during late cleavage by incomplete division of the cytoplasm of the blastodisc. Cytoplasmic projections extend from the periblast (and from the basal region of cleavage blastomeres prior to formation of the periblast) into the yolk and function in uptake of yolk material in the absence of pinocytosis. Yolk material appears to be digested by the periblast and transferred into the segmentation cavity where it is available to the blastomeres. Protein granules, lipid droplets, glycogen, crystalline arrays, and multivesicular bodies are related to food storage and utilization by blastomeres. The yolk gel layer enclosing the yolk sphere was found to be a thin layer of cytoplasm continuous with the margin of the periblast and is renamed the yolk cytoplasmic layer.     

An electron microscopic study of the development of amphioxus,Branchiostoma belcheri tsingtauense: Cleavage

Development of the Asian amphioxus, Branchiostoma belcheri tsingtauense, was investigated by scanning and transmission electron microscopy (SEM and TEM) from the fertilized egg through the blastula stage. The fertilized egg is spherical (mean diameter 115 μm after SEM preparation) and is covered with microvilli. Throughout cleavage, the second polar body remains attached to the animal pole. The cleavage type in this species is essentially radial, as revealed by SEM observations. At the third cleavage or 8-cell stage, and at later stages, a size difference between blastomeres in the animal and the vegetal halves is clearly discernible, but less marked than that reported for the European amphioxus, B. lanceolatum. During the period spanning the third to the fifth cleavage (8–32-cell) stages, blastomeres are arranged in tiers along the animal-vegetal axis. After the sixth cleavage, or 64-cell stage, the tiered arrangement of the blastomeres is no longer seen. At the 4-cell stage, the blastocoel or cleavage cavity is seen as an intercellular space, opening to the outside. The blastocoel remains open at the animal and the vegetal poles in later stages. Throughout early development, the cytoplasm of the blastomeres includes yolk granules, mitochondria, Golgi complexes, and rough and smooth endoplasmic reticulum. Chromatin in the interphase nucleus is not clearly demonstrated, and chromosomes in the mitotic phase are also extremely difficult to detect. As yet, regional differences have not been found in distribution and organization of cytoplasmic components with respect to prospective ectodermal, mesodermal, and endodermal areas in the fertilized egg and later cleaved embryos, although there are possibly fewer yolk granules in the region of the animal pole than in the vegetal polar zone.     

Essays on the application of nonclassical symmetries to natural biomorphs

The following symmetries are conventionally termed "nonclassical": conformal symmetry, inversion symmetry, Mikheev homology, curvilinear symmetry, colour symmetry, and antisymmetry. These symmetries are applicable to biomorphs differing in linear dimensions and proportions (such as the shells of bivalves, crustaceans, diatoms, etc.) and to such three-dimensional biomorphs as gastropod shells. These three-dimensional objects used to be substituted so far with two-dimensional images. The shift transformation causing inhomogeneous deformations is discussed in the framework of Mikheev homologies. Antisymmetry and colour symmetry are discussed by the example of flowers, duckweed plants, and the crustacean chela. Early stages of cleavage, like colloid crystals, may be represented as polyhedrons. They have Euler characteristics and face symbols, and, because of this, stages of cleavage may have symmetries of crystals. Mastering nonclassical symmetries may promote the progress of biosymmetrics. The potential of discrete (arithmetical) biomorphology in taxonomy and the potential of continuous (geomertical) biomorphology in biosymmetrics are discussed.     

Mechanism of an Alternate Type of Echinoderm Blastula Formation: The Wrinkled Blastula of the Sea Urchin Heliocidaris erythrogramma

While most indirect-developing echinoderms (possessing a feeding larval stage) form a hollow, smooth-walled blastula, most direct-developing species form a wrinkled blastula. The process of wrinkled blastula formation was examined in the direct-developing sea urchin, Heliocidaris erythrogramma . Approximately 5 hrs after fertilization the blastula epithelium contains folds along one, two or three orthogonal planes, which superficially appear like 2-, 4- or 8-cell stages, respectively. Microinjection of fluorescent dye into individual blastomeres of 2-, 4- and 8-cell embryos revealed that the wrinkles correspond with the first, second and third cleavage planes. Two factors appear to generate the wrinkled blastula epithelium. First, blastomeres undergo a partial separation along the first, second and third cleavage planes during early cleavage. Subsequent cell divisions are oriented such that the blastula epithelium is constructed with deep creases along these planes of cell separation. Second, there is no room for the expansion of the developing blastoderm within the tightly fitting fertilization envelope. Prior to hatching from the fertilization envelope, wrinkles in the blastula epithelium disappear, due to an increased packing and elongation of the cells. In addition, a substantial volume of cellular material is removed as lipids are secreted into the blastocoel in an apocrine fashion.     

Analysis of the embryonic development of Pogonophora in connection with the problems of phylogenetics

The development of the mesoderm in the Pogonophora being a point of argument, some stages of their ontogenesis are analyzed. The cleavage of Siboglinum caulleryi is considered as a modified spiral cleavage with the demonstration of the prospective significance of blastomeres. All mesoderm in Pogonophora is formed in enterocoelic mode from the anterior quadrant B. The spiral cleavage of pogonophores is compared to that of the Polychaeta and other animals. Some aspects of the formation and structure of the telosoma in the larva and adults are analyzed with a discussion of the nature of its segmentation. Some general problems of the evolution of the spiral cleavage are considered. The division of the Coelomata into 5 superphyla is confirmed, the Pogonophora being one of them.     

Indeterminate cell lineage of the zebrafish embryo

We have examined the clonal progeny descended from individual blastomeres injected with lineage-tracer dye in the zebrafish embryo. Blastomeres arising by the same cleavages in different embryos generated clones in which the types and positions of cells were highly variable. Several features of early development were correlated with this diversity in cell fate. There was no fixed relationship between the plane of the first cleavage and the eventual plane of bilateral symmetry of the embryo. By blastula stages the cleavages of identified blastomeres were variable in pattern. Moreover, cell fate was not easily related to the longitudinal and dorsoventral position of the clone in the gastrula. These results establish that single blastomeres can potentially generate a highly diverse array of cell types and that the cell lineage is indeterminate.     

Structural and ultrastructural analysis of embryonic development of Prochilodus lineatus (Valenciennes, 1836) (Characiforme; Prochilodontidae)

This survey was performed to characterize the embryogenesis of Prochilodus lineatus. Seven stages of embryo development were identified--zygote, cleavage, blastula, gastrula, segmentation, larval and hatching--after a period of incubation of 22 h (24 degrees C) or 14 h (28 degrees C). The following cleavage pattern was identified: the first plane was vertical (2 blastomeres); the second was vertical and perpendicular to the first (4 blastomeres); the third was vertical and parallel to the first (4 x 2); the fourth cleavage was vertical and parallel to the second (4 x 4); the fifth was vertical and parallel to the first (4 x 8); and the sixth cleavage was horizontal (64 blastomeres). At the blastula stage (3.0-4.0 h (24 degrees C); 1.66-2.0 h (28 degrees C)) irregular spaces were detected and periblast structuring was initiated. At the gastrula stage (4.0-8.0 h (24 degrees C); 3.0-6.0 h (28 degrees C)) the epiboly, convergence and cell movements, as well as the formation of embryonic layers, had begun. The segmentation stage (10.0-15.0 h (24 degrees C); 7.0-10.0 h (28 degrees C)) was characterized by a rudimentary formation of organs and systems (somites, optic vesicle and intestinal delimitation). The embryo at the larval stage (16.0-21.0 h (24 degrees C); 11.0-13.0 h (28 degrees C)) showed a free tail, more than 25 somites, an optic vesicle and a ready-to-hatch larval shape. The blastomeres at cleavage stage had disorganized nuclei indicating high mitotic activity. At gastrula, the blastomeres and the periblast had euchromatic nuclei and a large number of mitochondria and vesicles. The yolk was organized into globose sacs, which were dispersed into small pieces prior to absorption.     

Unequal distribution of otx1 mRnas among cleavage stage blastomeres in the teleost,Leucopsarion petersii (shiro-uo)

The otx genes belong to the orthodenticle gene family and play important roles in anterior brain development in vertebrates. We isolated two cDNA sequences, one homologous to human and zebrafish otxl and another homologous to zebrafish otx3, from the teleost Leucopsarion petersii (shiro-uo), which belongs to the family of gobies in the Perciformes. During embryogenesis of shiro-uo, otx1 and otx3 were expressed in the fore- and mid-brain throughout development in a manner similar to that observed in other vertebrates so far studied. However, otx-1 mRNA was also present at earlier stages and we obtained unique results using in situ hybridization and RT-PCR analysis demonstrating that otx-1 signals showed a distinct increase in the upper blastomeres, but not in the lower blastomeres, at the 8-cell stage. These stronger signals were maintained in the animal pole blastomeres during the 16-cell to 64-cell stages, followed by a gradual decrease during blastula stages. Such unexpected unequal distribution of otx1 mRNA revealed that blastomeres at early cleavage stages already showed non-equivalence in the embryogenesis of shiro-uo.     

Surface activity and locomotion of Fundulus deep cells during blastula and gastrula stages

The surface activity and locomotion of deep cells of the Fundulus blastoderm were studied in vivo with time-lapse cinemicrography. During late cleavage, the surfaces of the blastomeres begin to undulate gently. By early blastula, these undulations increase gradually in amplitude and hemispherical surface protrusions called blebs appear. These blebs form and retract rapidly, and at mid blastula some may be seen adhering to the surfaces of other cells. At the same time, they often expand into elongate lobopodia. Cell locomotion is first evident in mid blastula and continues throughout gastrulation. During locomotion, the leading edge of a deep cell behaves in various ways. When blebs and lobopodia adhere to a substratum (other deep cells, the undersurface of the enveloping layer, or the periblast) and retract, the cell may move in the direction of the shortening cell process. Alternatively, blebs and lobopodia may adhere, but not shorten. Locomotion is accomplished rather by protoplasmic flow into the protrusion. Blebs and lobopodia also may flatten and spread on the substratum as lamellipodia. Variations in the contact and locomotory behavior of deep cells and in the rate of their movement during blastula and gastrula stages are described in detail.     

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.     

Mesoderm formation by isolated and cultivated 8-cell stage blastomeres of the teleost, Leucopsarion ptersii (shiro-uo).

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.     

Early development of the chick embryo

The ultrastructure of the early chick embryo was investigated, using scanning (SEM) and transmission electron microscopy (TEM). Eggs were obtained from the shell gland by injecting hens intravenously with a synthetic prostaglandin or arginine vasopressin. Embryos were examined during late cleavage (stages IV–VI, Eyal-Giladi and Kochav, '76), formation of the area pellucida (stages VII–XI), and formation of the hypoblast (stages X–XIV). SEM highlighted the reduction in cell number at the underside of the embryo during formation of the area pellucida although it became apparent that the thickness of the embryo is not reduced to a single layer of cells at stage X. In addition, blastomeres at the perimeter of embryos (stages V–VI) project filopodial extensions onto a smooth membrane that separates the sub-embryonic cavity from the yolk. During hypoblast formation, epiblast cells generate stellate projections at their basal aspect, thus providing a meshwork for the advancing secondary hypoblast cells. By stage XII the epiblast was one cell thick and reminiscent of a columnar epithelium when viewed transversely. Cells of the deep portion of the posterior marginal zone were distinguished morphologically in the stage XII embryo by their many cell surface projections and ruffled appearance. Blastomeres at the perimeter of stage V–VI embryos projected filopodial extensions onto a smooth membrane which separates the sub-embryonic cavity from the yolk. This membrane is presumed to be confluent with the cytolemma. Evidence is presented demonstrating the presence of intracellular membrane-bound droplets which are hypothesised to contain sub-embryonic fluid. © 1993 Wiley-Liss, Inc.     

Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis

The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of lambda-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.     

Role of specialized microvilli and the fertilization envelope in the spatial positioning of blastomeres in early development of embryos of the starfish Astropecten scoparius

In the eggs of a wide range of animal species, various factors that determine the blastomeres' presumptive fate are known to locate unevenly within the egg. In the embryos of these animals, cleavage occurs not just to increase cell numbers, but also to distribute the factors to the respective blastomeres, resulting in cell specialization at the later stages. In the early cleavage stages, before the establishment of a device such as desmosomes to directly join the blastomeres, some other means is needed to keep the blastomeres together and maintain the relative positions among them. In this study, we found that the embryos of the starfish Astropecten scoparius lack the hyaline layer seen in sea urchin embryos and that blastomeres adhere to the fertilization envelope (FE) via filamentous cellular projections (fixing processes). Electron microscopy revealed the fixing processes to be specialized microvilli formed, after the elevation of the FE, by the elongation of short microvilli that pre-exist in unfertilized eggs. After the first cleavage, the two blastomeres separate from each other and finally attach to the FE. In the subsequent cleavages, the blastomeres undergo repeated cell division without separating from the FE. Between the blastomeres and the FE, only shortened fixing processes were observed. Destruction of the fixing processes caused release of the blastomeres from the FE and disturbance of the relative positions of the blastomeres, resulting in abnormal development of the embryos. These observations suggest that the fixing process is a device to keep the egg placed centrally in the FE up to the first cleavage, and after the first cleavage and beyond to anchor the blastomeres to the FE so that the FE can be used as a scaffold for morphogenesis. Electron microscopy also suggests that the inner layer of the FE, which is derived from the contents of cortical granules, reinforces the adhesion of the fixing processes to the FE. Immuno-electron microscopy, using an antibody against sea urchin hyaline layer, showed that the inner layer of the FE of starfish eggs and the hyaline layer of sea urchin eggs, which are both derived from cortical granules, contain some common elements.     

A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

Electrical Characteristics of Triturus Egg Cells during Cleavage

The membrane potential in the blastomeres of dividing Triturus egg cells increases progressively from the first cleavage to the late morula stages. Both the animal and vegetal poles show the same increasing trend in potential; there is no significant potential difference between them. Upon first cell cleavage, the total resistance of the egg cell surface in contact with the exterior decreases to about one-tenth of its value before cleavage, and then remains rather constant up to the late morula stage. The specific resistance of this membrane surface drops rather abruptly upon first cleavage, and rises progressively during the morula stage. The resistance of the junctional membrane surface of the blastomeres, that is, the membrane formed at the former planes of cleavage, is small in relation to that of the cell surface in contact with the exterior. As a result, the blastomeres are electrically coupled throughout all stages of embryonic development examined.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

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.