Studies on the yolk granules of the silkworm,Bombyx mori L.: The morphology of diapause and non-diapause eggs during early developmental stages

Summary The morphological features during development of diapause and non-diapause eggs of the silkworm,Bombyx mori, were investigated by means of light and electron microscopy, with special reference to eggs up to 24 h after oviposition.The blastoderm and yolk cells began to be formed about 6 and 24 h after oviposition, respectively, in both the diapause and non-diapause eggs, indicating that the diapause and non-diapause eggs develop at similar rates at least until 24 h after oviposition.Specific changes in the distribution of yolk granules were observed during early development of the diapause egg. Its yolk granules gradually aggregated into clusters from the periphery toward the inside of the egg during the period of blastoderm formation. Aggregation of yolk granules was most noticeable about 12 h after oviposition and then they dispersed again before yolk cell formation. On the other hand, yolk granules of the non-diapause eggs remained dispersed during development.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.     

Mesoderm differentiation in explants of carp embryos

An analysis of carp blastoderm development was carried out in culture after isolation from the yolk cell and its yolk syncytial layer (YSL). The blastoderms were separated from the YSL at four different stages of embryogenesis: the blastula, early epiboly, early gastrula and late gastrula stages. Absence of the YSL in explants was checked by scanning electron microscopy. From observations of living embryos and histological examination of tissues which were formed in explants from all stages studied it was observed that they contained notochordal, muscle and neural tissue as signs of dorsal types of differentiation. Only in explants from the early and late gastrula stages were histotypical tissues organized in an embryonic-like body pattern. The data indicate that mesoderm differentiation in fish embryos is independent from the YSL, contrary to normal pattern formation which needs the presence of the YSL before the onset of gastrulation.     

Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer

Dye coupling and cell lineages of blastomeres that participate in the formation of the yolk syncytial layer (YSL) in the zebrafish Brachydanio rerio have been examined. The YSL is a multinucleate layer of nonyolky cytoplasm underlying the cellular blastoderm at one pole of the giant yolk cell. It forms at the time of the 10th (sometimes 9th) cleavage by a collapse of a set of blastomeres, termed marginal blastomeres, into the yolk cell. Marginal blastomeres possess cytoplasmic bridges to the yolk cell before the YSL forms, and injections of fluorescein-dextran into the cells revealed that bridges between the yolk cell and blastoderm do not persist after this time. Injections of Lucifer yellow revealed that shortly after the YSL forms the yolk cell and blastoderm are dye coupled, presumably by gap junctions, and that this coupling disappears gradually during early gastrulation. Lineage analyses revealed that not all of the progeny of early marginal blastomeres participate in YSL formation. Although some descendants of marginal blastomeres remained on the margin during successive cleavages, neither "compartment" nor "strict lineage" models are sufficient to explain the origin of the YSL. It is proposed that the position of a cell on the blastoderm margin, and not the cell's lineage, determines YSL cell fate.     

Behavior of silkworm yolk protein on phospholipid membranes

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of -70 mV for yolk and -198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.     

Magnetic resonance imaging study of the structure of the yolk in the developing avian egg

Magnetic resonance imaging (MRI) techniques were used to study the morphology of the latebra and concentric rings seen in the yolk of White Leghorn eggs during development of the avian embryo. Previous studies of the macroscopic structure of avian yolk have revealed the latebra, a vase-shaped structure beneath the blastoderm composed of white yolk. The bulbous portion in the center of the yolk is termed the body of the latebra. The thinner portion extending toward the blastoderm is referred to as the neck of the latebra. As the neck of the latebra approaches the blastoderm, it flares out to become the nucleus of Pander. The remainder of the yolk often features alternating concentric layers of white and yellow yolk. These layers, which appear as rings in sections, are thought to represent the daily accumulation of yolk during oogenesis. In this study eggs were imaged with a single slice spin echo sequence using MRI parameters that maximized the visualization of the latebra and concentric rings in the egg yolk. Some experiments were conducted for 2 to 3 day periods with eggs kept in the bore of the magnet using a small incubator that was constructed using a temperature-controlled water pump. The concentric rings of the yolk and the body of the latebra flatten and become more elliptical during development. The neck of the latebra becomes shorter and disappears around the 7th day of incubation. The body of the latebra starts to become incorporated into the embryo at about the 7th day of incubation and usually disappears by the 13th day. The concentric rings are no longer visible as distinct entities at this time. Histochemical procedures carried out as a result of MRI findings indicate that the latebra is an iron-rich structure.     

Early development of the kelp fly,Coelopa frigida (Diptera). II. Morphology of cleavage and blastoderm formation

Cleavage and blastoderm formation in Coelopa frigida are extremely rapid developmental processes. In short (6–7 minutes) successive cell cycles, nuclei multiply and spread out through the egg. The movement seems to be aided by endoplasmic vesicles and cisternae which are in direct contact with the nuclear membrane. The first cells to separate from the egg plasmodium in early superficial cleavage stages are the pole cells. Precursor material from multivesicular bodies forms the pole cell membranes. The primary nuclei from the posterior pole region are removed from the blastoderm by the pole cell segregation. Blastoderm nuclei from the regions adjacent to the posterior pole migrate into the residual periplasm after pole cell segregation has been completed and constitute the blastoderm nuclei in that region of the egg. Nucleoli are not revealed during internal cleavage. They appear in pole cells shortly after their segregation. The generation time of the blastoderm nuclei increases after the twelfth cleavage. Concurrently, nucleoli form in the blastoderm nuclei and permanent cell membranes separate individual blastoderm cells. After blastoderm cells have been separated from each other, they remain in contact with the interior yolk sac by means of cytoplasmic canals. This contact is maintained at least during the early phases of blastokinesis. Observations on nuclear migration and rapid membrane formation are discussed as examples of protein assembly from subunits as an alternative to de novo protein synthesis in early stages of development.     

Blastoderm degeneration, an early embryonic failure in dwarf Single Comb White Leghorn chickens.

Blastoderm degeneration is an early embryonic lethal condition observed in selected paired matings within a line of dwarf Single Comb White Leghorn chickens that results in a 25% reduction of the hatch of fertilized eggs. The disorder is macroscopically evident at 32 h of incubation by the presence of a small localized indentation on the outer periphery of the expanding blastoderm. The affected blastoderms undergo a series of rapid macroscopic degenerative changes that conclude at about 120 h characterized by the presence of dispersed blastoderm fragments on the surface of the egg's yolk. Microscopically, this embryonic failure appears to manifest itself between Hamburger-Hamilton stages 8 and 9 of development and is characterized by a series of retarded developmental processes: closure of the anterior neuropore, brain vesicle differentiation, somite formation, and cardiac development. The disorder is inherited as an autosomal recessive trait. Attempts to identify factors that influence the disorder have thus far been unsuccessful. The symbol bld is proposed for this recessive gene.     

Regularities of early embryogenesis in teleosts as revealed by studies of the temporal pattern of development

Summary The absolute durations in minutes of the periods from insemination till the onset of gastrulation, the end of epiboly and appearance of the 10th pair of somites, as well as the intervals between these stages, have been determined in 7 species of Teleostei. Their relative duration, in terms of mitotic cycle time units {ie313-1} has also been determined. The biological age (expressed in number of {ie313-2} units) at which the isolated blastoderm becomes capable of differentiating in vitro was also determined in 4 of the species.The relative duration of the periods studied in all the species was shown to be stable at optimal temperatures. The corresponding periods of development have similar duration in closely related teleost species (i.e. belonging to the same genus).The relative duration of the cleavage period varies among the species belonging to different families. These variations are correlated with differences in the amount of yolk in the egg and the degree of psychrophily of the species compared. By contrast, the relative duration of the period between the onset of gastrulation and the stage of 10 pairs of somites was found to be very similar in species which belong to different orders and differ with respect to the amount of yolk in the egg and the temperature ranges of development. The rate of movement of blastoderm cells during epiboly is higher, the larger the eggs in the species under study. The variations in the age at which the isolated blastoderm acquires the ability to differentiate in vitro are not always correlated with the amount of yolk in the egg.The hypothesis is proposed that an interaction between the yolk and active cytoplasm, which determines time of the onset of ribosomal RNA synthesis, is of importance in controlling the temporal relationships in early embryogenesis.     

The specificity of intracellular changes in chick embryo blastoderm during the latent period of embryogenesis]

Yolk inclusions, lipids and polysaccharides found in the chicken embryo blastoderm cells are utilized during the latent period of embryogenesis. The yolk outside the blastoderm is not utilized. A delay in the development of the embryo of first days of incubation is related to a switching over the metabolism from utilizaiton of intracellular nutrient material to assimilation of the extracellular yolk. In the course of morphogenetical movements of the embryo, in the process of gastrulation, took place an increased biosynthesis in the blastoderm cell membranes.     

Behavior of silkworm yolk protein on phospholipid membranes

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of −70 mV for yolk and −198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.     

Early embryonic development,including germ-disk stage,in the theridiid spider Achaearanea japonica (Bös. et Str.)

Early embryonic development, from the first cleavage to the germ-disk stage, in the theridiid spider Achaearanea japonica was examined by light and electron microscopy. The eggs are syncytial during the first four cleavages, and then invaginations of cell membranes fuse to generate the blastomeres at the sixteen-nucleus stage. The cleavage pattern is a modified type of total cleavage. It appears that radial bundles of microtubules that radiate from the perinuclear cytoplasm may participate in the migration of cleavage nuclei for the formation of the blastoderm. The large yolk granules are sequestered by cell membranes from the blastomeres or blastoderm cells into the interior of the embryo together with various organelles and glycogen granules. Most of the blastoderm cells converge in the upper hemisphere to form the germ disk, whereas a few cells remain in the lower hemisphere. The embryo at the germ-disk stage contains many spherical germ-disk cells. Almost no large yolk granules are found in these cells, but the flat remaining cells each contain several large yolk granules. These remaining cells may preserve a flat shape to cover the surface of the embryo that does not include the germ disk. © 1995 Wiley-Liss, Inc.     

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Hepten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo

The early chick blastoderm expresses two endogenous galactose-bindinglectins of 14 kDa and 16 kDa. We have studied the effect thelectin hapten inhibitors thiodigalactoside and the syntheticneoglycoprotein lactosyl-bovine serum albumin as well as polyclonalanti-lectin antibodies on the development of early chick embryoscultured in a defined medium. Controls consisted of maltose,maltosyl bovine serum albumin and rabbit IgG. Embryos treatedat the onset of cell migration during early gastrulation underwentblastoderm retraction with decrease in surface area. In addition,they exhibited a lack of demarcation between the presumptiveembryonic area (area pellucida) and the presumptive extraembryonicarea (area opaca). These blastoderms also lacked a primitivestreak, that is, the structure that forms in the area pellucidaduring gastrulation as cell migrate to form the endodermal andmesodermal layers of the embryo. Embryos treated at later stagesof gastrulation showed development similar to that of controlsin that they were able to undergo early organogenesis. The resultssuggest that lectin mediated mechanisms are essential for themigratory movements of early gastrulation and that, at lategastrulation, other mechanisms exist in the embryo to compensatefor lectin function. blastoderm chick embryo galectin