Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8?C16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

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.     

Programmed Endocytosis During Epiboly of Fundulus heteroclitus

A major question in the analysis of teleost epiboly is the fateof the yolk cytoplasmic layer. It diminishes during epibolyand eventually disappears at the completion of epiboly. Thispaper is concerned with the fate of the surface of the yolkcytoplasmic layer during epiboly. When gastrulae during epibolyare bathed in lucifer yellow (CH) and then observed with fluorescentmicroscopy or bathed in ferritin and then fixed and observedwith TEM, a thin circumferential ring of endocytic vesiclesis observed, confined to the external yolk syncytial layer justperipheral to the advancing margin of the blastoderm. Even thoughthe entire egg is immersed in the marker, endocytosis is confinedto this limited region. More precisely, this endocytosis occursonly within the region of the external yolk syncytial layer,where the surface is most folded. The endocytic vesicles thusformed move downward and settle on the surface of the membraneseparating the yolk from the cytoplasm in the yolk syncytiallayer. They do not join the surface of the internal yolk syncytiallayer; hence they do not contribute to its expansion. Priorto the onset of epiboly there is no such endocytosis at thesurface of the egg. Since this endocytosis occurs only duringepiboly and only at the surface of the external yolk syncytiallayer just peripheral to the advancing margin of the blastoderm,and in the absence of large molecules in the medium, we concludethat it is programmed. We, therefore, present this as a caseof programmed internalization of cell surface serving as themorphogenetic mechanism responsible for the disappearance ofthe surface of the yolk cytoplasmic layer during gastrulationof the teleost Fundulus heteroclitus     

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25°C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12–24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.     

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.     

Scanning electron microscopy of Drosophila embryogenesis: 1. The structure of the egg envelopes and the formation of the cellular blastoderm

As part of a series of detailed observations on embryogenesis in Drosophila, the protective coverings of the egg and surface changes in the embryo prior to gastrulation have been studied with the SEM. Four specializations of the chorion are described: the plastron, micropylar cone, operculum, and the posterior thickening. After removal of the protective coverings the surface changes during development can be observed. During the first eight synchronous nuclear divisions a dense array of thin microprojections covers the whole embryo. After the ninth division between 373 and 408 nuclei reach the surface and become located in cytoplasmic projections. From counts of the number of surface bulges during the syncytial blastema stages, it was established that 13 synchronous divisions take place producing between 5600 and 6500 surface nuclei. During formation of the cellular blastoderm, the location of the prospective cells becomes obscured by a dense pattern of microprojections from each cell. However, with the completion of the blastoderm, the surfaces of the cells become smooth and the cell outlines distinct. The usefulness of the SEM in developmental studies is discussed.     

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.     

Microtubules During Blastoderm Formation in the Egg of the Silkworm, Bombyx mori L.

Microtubules in the silkworm egg, Bombyx mori , were observed by electron microscopy, in order to investigate the relationship between cytoskeletal organelles and the migration of energids, the cleavage nuclei accompanied by the associated cytoplasm, near the egg surface or during blastoderm formation. Numerous microtubules were observed in the associated cytoplasm of an energid even in the interphase of mitosis.
At about 8.5 hr after oviposition, when many energids had already cleft and distributed near the peripheral yolk granule region, long microtubules distributed radially from the perinuclear region to the periphery in the associated cytoplasm. When an energid was protruding, the microtubules above the nucleus distributed at a more acute angle than those under the nucleus. When a blastoderm cell had just been formed, the microtubules were observed only under the nucleus.
Colchicine, an inhibitor of microtubules, stopped the migration of energids and inhibited the formation of blastoderm cells even after many energids had already distributed at the peripheral yolk granule region. The relationship between the microtubules and the migration of energids near the egg surface or during blastoderm formation was discussed.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Blastoderm development in honey bee embryogenesis as seen in the scanning electron microscope

Summary

Based on observations in the scanning electron microscope, we describe the successive changes at the cell surface of fertilized honey bee eggs which, over a period of 20 h, lead to blastoderm formation. Each change starts in the differentiation center located in the anterior egg half and from there spreads as a wave towards both poles. However, one of the waves is heterogeneous: the protrusions or caps of oolemma which start enveloping the preblastoderm nuclei arise in different ways in the anterior third and in the more posterior regions of the egg cell. After the formation of these protrusions, three mitotic waves pass over the peripheral nuclei. The last of these blastemal mitoses results in a two-layered arrangement of peripheral nuclei, each surrounded by an outpocketing of the egg cell basally confluent with the central yolky part. This two-layered state after some time transforms into a single layer of columnar outpocketings which increase in height by extending their borders centripetally into the secondary periplasm (inner ‘Keimhautblastem’). The outpocketings disconnect from the central yolk mass as separate blastoderm cells only a short time before gastrulation. The events described here consume nearly 40% of the time period between oviposition and hatching; thus, and by comparison with most other insects, blastoderm formation in the honey bee is a very lengthy process.     

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.     

Embryonic development of the jumping bristletail Pedetonutus unimaculatus Machida,with special reference to embryonic membranes (Hexapoda: Microcoryphia,Machilidae)

In the machilid Pedetonutus unimaculatus, a germ disc is formed by the aggregation and proliferation of cells within a broadly defined embryonic area. Cells adjacent to the embryonic area form the serosal fold that grows beneath the embryo. Then the embryonic margin is extended to form a cell layer or amnion that lies between the embryo and serosal fold. Thus, an amnioserosal fold is formed by the addition of the amnion to the serosal fold. Serosal cells cover the entire surface of the egg and begin to secrete a serosal cuticle. Soon the amnioserosal fold is withdrawn, and the embryo is exposed to the egg surface. The spreading amnion replaces the serosal cells that finally degenerate through the formation of a secondary dorsal organ. In the areas of amnion anterior and lateral to the embryo, yolk folds form and encompass the embryo. The amnion is a provisional dorsal closure and never participates in the formation of the definitive one. The amnioserosal fold of the Microcoryphia appears to have the functional role of secreting a serosal cuticle beneath the embryo. This fold of the Microcoryphia may be regarded as an ancestral form of the amnioserosal folds of the Thysanura-Pterygota. the yolk folds may appear to be passive transformation of the yolk mass linked to positioning of the growing embryo within the egg. There is no evidence that the yolk folds and the cavity appearing between them in the Microcoryphia are homologous to the amnioserosal fold and amniotic cavity in the Thysanura-Pterygota. The yolk folds appear to be one of the embryological autapomorphies in the Microcoryphia. © 1994 Wiley-Liss, Inc.     

胡子鲶的胚胎和幼鱼发育的研究

胡子鲶(Clarias fuscus)的胚胎和幼鱼的发育过程与蟾胡子鲶(C.batrachus)类似,器官分化时间和大小比例则有一定的差异。胡子鲶卵呈球形,富含卵黄,卵径1.7—1.9毫米;出膜仔鱼全长4.8—5.1毫米,比蟾胡子鲶约大1/3;幼鱼卵黄囊被吸收消失的时间比蟾胡子鲶迟1—2天。心脏的出现和搏动与开始出现血液循环的间隔时间只有3—5小时;耳石的出现几乎和心脏的出现处在同一时间内,与白鲢有较大的差异。在仔鱼前期居维氏管明显,但出现的时间比蟾胡子鲶稍迟些。仔鱼出膜形式与蟾胡子鲶不同,也与一般鱼类出膜形式不同。仔鱼是以腹部卵黄囊顶破卵膜,以卵黄囊先出膜,一般鱼类多数是以头部或尾部先出膜的。在水温28.5—31℃的条件下,从受精到孵化出膜的时间为28小时25分;幼鱼期共历时12—15天。       

Scanning electron microscopy of the post-embryonic stages of the climbing perch,Anabas testudineus

Different developmental stages, fertilized eggs through hatchlings, of the climbing perch,Anabas testudineus, have been studied by scanning electron microscopy. The surface specialization of eggs and hatchlings reveals that while the egg surface is reticulate in appearance, the hatchlings are covered with microridges. Vitelline arteries are seen at the pharyngeal and abdominal regions. They supply nutrients directly from the yolk sac to the developing embryo. Three pairs of such arteries are distinctly seen in the pharyngeal region. Mucous glands are discernible at places over the entire body surface of the embryo before the formation of scales. The skin seems to be helpful in gaseous exchange till the gills and accessory respiratory organs develop and become functional.     

Early embryonic development of the mayfly Ephemera japonica McLachlan (Insecta: Ephemeroptera,Ephemeridae)

In the newly laid egg of the mayfly Ephemera japonica, an egg nucleus (oocyte nucleus) at metaphase of the first maturation division is in the polar plasm at the mid-ventral side of the egg, and a male pronucleus lies in the periplasm beneath a micropyle situated just opposite the polar plasm or at the mid-dorsal side of egg. The maturation divisions are typical. An extensive and circuitous migration of the male pronucleus is involved in the fertilization process: it first moves anteriad in the periplasm from beneath the micropyle to the anterior pole of the egg and then turns posteriad in the yolk along the egg's long axis to the site of syngamy, near the center of the egg. Cleavage is superficial. The successive eight cleavages, of which the first five are synchronized, result in the formation of the blastoderm, and about ten primary yolk cells remain behind in the yolk. Even in the newly formed blastoderm, the thick embryonic posterior half and the thin extraembryonic anterior half areas are distinguished: the former cells are concentrated at the posterior pole of the egg to form the germ disc, and the latter cells become more flattened, forming serosa. Time-lapse VTR observations reveal a yolk stream that is in accord with the migration of the male pronucleus in time and direction. The yolk stream is also generated in activated unfertilized eggs, and it is probable that the migration of the male pronucleus in association with the fertilization may be directed by the yolk stream. J. Morphol. 238:327–335, 1998. © 1998 Wiley-Liss, Inc.     

Mechanism of Fundulus Epiboly--A Current View

This paper summarizes evidence for the following picture ofFundulus epiboly, with an eye toward laying groundwork for futureinvestigation. The major force in epiboly is the yolk syncytiallayer (YSL). Prior to epiboly, it spreads well beyond the borderof the blastoderm to form the wide external YSL (E-YSL). Thishas contractile properties, which, however, are restrained priorto epiboly by the attached enveloping layer (EVL) of the blastoderm.Epiboly begins when the E-YSL contracts and narrows, throwingits surface into folds and pulling the internal YSL (I-YSL)and the attached EVL vegetally. When the narrowing of the E-YSLhas ceased, it is postulated that its contractility continuesas a circumferential wave of vegetally directed contractionthat moves over the yolk toward the vegetal pole, dragging theI-YSL and the attached EVL (and blastoderm) with it. The mostobvious visible manifestation of this wave is a marked marginalconstriction, where the YSL joins the yolk cytoplasmic layer(YCL). As this contractile wave passes over the yolk, cytoplasmfrom the YCL mingles with that of the advancing E-YSL, and YCLsurface adds to the already highly convoluted surface of theE-YSL. This folded surface is the site of a thin, highly localizedband of rapid endocytosis that encircles the egg and passesover it with the E-YSL in a wave throughout epiboly. This internalization,which is receptor independent and therefore somehow programmed,accompanies the putative contractile wave, and accounts forthe disappearance of the surface of the YCL. Since the YCL surfacestands in the way of the advancing YSL, its internalizationis part of the mechanism of epiboly. As the I-YSL expands inresponse to this marginal pull, its abundant microvilli graduallydisappear, providing surface for its epiboly. The firmly attachedEVL likewise expands toward the vegetal pole in response tothe pull of the autonomously expanding YSL. As epiboly of theEVL progresses, it adjusts to the geometric problems posed bya sheet expanding over a sphere by active cell rearrangementwithin the cell monolayer. Thus, epiboly of the EVL has an activeas well as a passive component. Deep cells are not causallyinvolved in epiboly, but move about in coordinated ways in theconstantly increasing space between the I-YSL and the EVL providedby epiboly and form the germ ring and the embryonic shield andeventually the embryo proper. An attempt is made to pull allof this together, and more, in order to achieve as comprehensivean understanding of epiboly as present evidence will allow.     

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.     

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.