Vegetal egg cytoplasm promotes gastrulation and is responsible for specification of vegetal blastomeres in embryos of the ascidian Halocynthia roretzi

Development ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1271-1279 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Radial Symmetry ◽  
Second Phase ◽  
Equatorial Position ◽  
Animal Cells ◽  
Halocynthia Roretzi ◽  
Animal Pole ◽  
Vegetal Pole ◽  
Ooplasmic Segregation ◽  
Derivatives Of ◽  
Egg Cortex

An animal-vegetal axis exists in the unfertilized eggs of the ascidian Halocynthia roretzi. The first phase of ooplasmic segregation brings the egg cortex to the vegetal pole very soon after fertilization. In the present study, when 5–8% of the egg cytoplasm in the vegetal pole region was removed between the first and second phase of segregation, most embryos exhibited failure of gastrulation, as reported previously in Styela by Bates and Jeffery (Dev. Biol, 124, 65–76, 1987). The embryos that were deficient in vegetal pole cytoplasm (VC-deficient embryos) developed into permanent blastulae. They consisted for the most part of epidermal cells and most lacked the derivatives of vegetal blastomeres, such as endoderm, muscle and notochord. Removal of cytoplasm from other regions did not affect embryogenesis. The cleavage of the VC-deficient embryos not only exhibited radial symmetry along the animal-vegetal axis but the pattern of the cleavage was also identical in the animal and vegetal hemispheres. Examination of the developmental fates of early blastomeres of VC-deficient embryos revealed that the vegetal blastomeres had assumed the fate of animal cells. These results suggested that the VC-deficient embryos had been totally animalized. When vegetal pole cytoplasm was transplanted to the animal pole or equatorial position of VC-deficient eggs, gastrulation occurred, starting at the site of the transplantation and tissues derived from vegetal blastomeres formed. Therefore, it appears that vegetal pole cytoplasm specifies the site of gastrulation and the cytoplasm is responsible for the specification of vegetal blastomeres. It is suggested that during the second phase of ooplasmic segregation, cytoplasmic factors responsible for gastrulation spread throughout the entire vegetal hemisphere.

Fertilization and ooplasmic movements in the ascidian egg

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 237-249 ◽  
Author(s):  
C. Sardet ◽  
J. Speksnijder ◽  
S. Inoue ◽  
L. Jaffe
Keyword(s):  
Polar Body ◽  
Surface Velocity ◽  
Second Phase ◽  
Animal Pole ◽  
Male Pronucleus ◽  
Vegetal Pole ◽  
Female Pronucleus ◽  
Ooplasmic Segregation ◽  
Second Polar Body ◽  
Sperm Aster

Using light microscopy techniques, we have studied the movements that follow fertilization in the denuded egg of the ascidian Phallusia mammillata. In particular, our observations show that, as a result of a series of movements described below, the mitochondria-rich subcortical myoplasm is split in two parts during the second phase of ooplasmic segregation. This offers a potential explanation for the origin of larval muscle cells from both posterior and anterior blastomeres. The first visible event at fertilization is a bulging at the animal pole of the egg, which is immediately followed by a wave of contraction, travelling towards the vegetal pole with a surface velocity of 1.4 microns s-1. This wave accompanies the first phase of ooplasmic segregation of the mitochondria-rich subcortical myoplasm. After this contraction wave has reached the vegetal pole after about 2 min, a transient cytoplasmic lobe remains there until 6 min after fertilization. Several new features of the morphogenetic movements were then observed: between the extrusion of the first and second polar body (at 5 and 24–29 min, respectively), a series of transient animal protrusions form at regular intervals. Each animal protrusion involves a flow of the centrally located cytoplasm in the animal direction. Shortly before the second polar body is extruded, a second transient vegetal lobe (‘the vegetal button’) forms, which, like the first, resembles a protostome polar lobe. Immediately after the second polar body is extruded, three events occur almost simultaneously: first, the sperm aster moves from the vegetal hemisphere to the equator. Second, the bulk of the vegetally located myoplasm moves with the sperm aster towards the future posterior pole, but interestingly about 20% remains behind at the anterior side of the embryo. This second phase of myoplasmic movement shows two distinct subphases: a first, oscillatory subphase with an average velocity of about 6 microns min-1, and a second steady subphase with a velocity of about 26 microns min-1. The myoplasm reaches its final position as the male pronucleus with its surrounding aster moves towards the centre of the egg. Third, the female pronucleus moves towards the centre of the egg to meet with the male pronucleus. Like the myoplasm, the migrations of both the sperm aster and the female pronucleus shows two subphases with distinctly different velocities. Finally, the pronuclear membranes dissolve, a small mitotic spindle is formed with very large asters, and at about 60–65 min after fertilization, the egg cleaves.

Regionality of egg cytoplasm that promotes muscle differentiation in embryo of the ascidian, Halocynthia roretzi

Development ◽  
1992 ◽  
Vol 116 (3) ◽  
pp. 521-529 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Muscle Cells ◽  
Muscle Differentiation ◽  
Posterior Region ◽  
Second Phase ◽  
Halocynthia Roretzi ◽  
Early Embryos ◽  
Vegetal Pole ◽  
Restricted Region ◽  
Ooplasmic Segregation ◽  
Cytoplasmic Determinants

Development of ascidians occurs in typical mosaic fashion: blastomeres isolated from early embryos differentiate into tissues according to their normal fates, an indication that cytoplasmic determinants exist in early blastomeres. To provide direct evidence for such cytoplasmic determinants, we have devised methods for fusing blastomeres and cytoplasmic fragments from various regions. (1) Presumptive-epidermis blastomeres were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi and development of muscle cells was monitored by an antibody to ascidian myosin. Muscle differentiation was observed only when presumptive-epidermis blastomeres were fused with fragments from the posterior region of B4.1 (posterior-vegetal) blastomeres, the normal progenitor of muscle cells. The results indicate that muscle determinants are present and localized in the cytoplasm that enters muscle-lineage cells. (2) To investigate the presence and localization of muscle determinants in the egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the presumptive- epidermis blastomeres, and formation of muscle cells was assessed by monitoring myosin, actin and acetylcholinesterase expression. These proteins were expressed only when cytoplasm from a restricted region of the eggs, i.e. the vegetal region, after the first phase of ooplasmic segregation, and posterior region, after the second phase of segregation, were fused. Based on these experiments, it is suggested that muscle determinants are segregated by ooplasmic movements after fertilization. They move initially to the vegetal pole of the egg and, prior to first cleavage, to the posterior region from whence future muscle-lineage blastomeres are formed. The inferred movements of muscle determinants correspond to those of the myoplasm, a microscopically visible portion of the egg cytoplasm.

Localization of determinants for formation of the anterior-posterior axis in eggs of the ascidian Halocynthia roretzi

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3093-3104 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Muscle Cells ◽  
Bilateral Symmetry ◽  
Radial Symmetry ◽  
Second Phase ◽  
Cleavage Pattern ◽  
Halocynthia Roretzi ◽  
Ooplasmic Segregation ◽  
Anterior Posterior ◽  
Anterior Posterior Axis

Unfertilized eggs of the ascidian Halocynthia roretzi are radially symmetrical along the animal- vegetal axis. After fertilization, ooplasmic segregation results in formation of an anterior-posterior axis horizontally, and eggs become bilaterally symmetrical. When 8–15% of the cytoplasm of the posterior- vegetal region of the egg was removed after the second phase of ooplasmic segregation, most of the embryos completed gastrulation but developed into radialized larvae along the animal-vegetal axis with no apparent anterior-posterior axis. Removal of cytoplasm from other regions did not affect formation of this latter axis. The cleavage pattern of the embryos that were deficient in posterior- vegetal cytoplasm (PVC) exhibited radial symmetry instead of the complicated bilateral symmetry of normal embryos. Detailed comparisons of cleavage patterns revealed the duplication of the anterior cleavage pattern in the originally posterior halves of the PVC-deficient embryos. The PVC-deficent larvae lacked muscle cells, which are normally derived from the posterior blastomeres. Examination of the developmental fates of the early blastomeres of the PVC-deficient embryos revealed that all of the vegetal blastomeres had assumed anterior fates. These results suggest that the PVC-deficient embryos are totally anteriorized. When posterior-vegetal cytoplasm was transplanted to the anterior-vegetal position of PVC-deficient eggs, the axial deficiency was overcome, and reversal of the anterior-posterior axis was observed. The results of transplantation of posterior-vegetal cytoplasm to the anterior-vegetal position in normal eggs demonstrated that formation of the anterior structure is suppressed by posterior-vegetal cytoplasm. These results suggest that posterior fate is specified by the presence of posterior-vegetal cytoplasm, while anterior fate is specified by the absence of posterior-vegetal cytoplasm. Thus, posterior-vegetal cytoplasm determines the anterior-posterior axis by generating the posterior cleavage pattern and conferring posterior fates on cells, as well as by inhibiting anterior fates that would otherwise occur by default.

Localization of egg cytoplasm that promotes differentiation to epidermis in embryos of the ascidian Halocynthia roretzi

Development ◽  
1994 ◽  
Vol 120 (2) ◽  
pp. 235-243 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Specific Antigen ◽  
Equatorial Region ◽  
Epidermal Cells ◽  
Second Phase ◽  
Halocynthia Roretzi ◽  
Cytoplasmic Factors ◽  
Early Embryos ◽  
Ooplasmic Segregation ◽  
Anucleate Cell ◽  
Cell Fragments

Embryogenesis in ascidians is of the mosaic type. This property suggests the presence of cytoplasmic factors in the egg that are responsible for specification of the developmental fates of early blastomeres. The epidermal cells that surround the entire tadpole larva originate exclusively from blastomeres of the animal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of epidermis fate, we carried out cytoplasmic transfer experiments by fusing blastomeres and anucleate cell fragments from various regions of eggs and embryos. Initially, presumptive non-epidermis blastomeres (blastomeres from the vegetal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of epidermal cells was monitored by following the expression of an epidermis- specific antigen, as well as by observations of morphology and the secretion of larval tunic materials. Formation of epidermis was observed when vegetal blastomeres were fused with cytoplasmic fragments from the presumptive epidermis blastomeres. The results suggested that cytoplasmic factors that promoted epidermis differentiation (epidermis determinants) were present in epidermis progenitors. Vegetal blastomeres only manifested this change in fate when fused with cytoplasmic fragments of roughly equal or larger size. Next, to examine the presence and localization of epidermis determinants in the uncleaved egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the vegetal blastomeres. The results suggested that epidermis determinants were already present in unfertilized eggs and that they were segregated by movements of the ooplasm after fertilization. After the first phase of ooplasmic segregation, these determinants were widely distributed, with the highest activity being located in the equatorial region. There were no indications of regional differences in the activity within the equatorial region of eggs at this stage. After the second phase of ooplasmic segregation, prior to the first cleavage, the activity moved in the animal direction, namely, to the animal hemisphere, from which future epidermis-lineage blastomeres are normally formed.

A gastrulation center in the ascidian egg

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 53-63 ◽  
Author(s):  
William R. Jeffery
Keyword(s):  
Nervous System ◽  
Uv Irradiation ◽  
Second Phase ◽  
Uv Sensitivity ◽  
Maternal Mrna ◽  
Free Translation ◽  
Vegetal Pole ◽  
Cell Embryo ◽  
Ooplasmic Segregation ◽  
Uv Dose

A gastrulation center is described in ascidian eggs. Extensive cytoplasmic rearrangements occur in ascidian eggs between fertilization and first cleavage. During ooplasmic segregation, a specific cytoskeletal domain (the myoplasm) is translocated first to the vegetal pole (VP) and then to the posterior region of the zygote. A few hours later, gastrulation is initiated by invagination of endoderm cells in the VP region of the 110-cell embryo. After the completion of gastrulation, the embryonic axis is formed, which includes induction of the nervous system, morphogenesis of the larval tail and differentiation of tail muscle cells. Microsurgical deletion or ultraviolet (UV) irradiation of the VP region during the first phase of myoplasmic segregation prevents gastrulation, nervous system induction and tail formation, without affecting muscle cell differentiation. Similar manipulations of unfertilized eggs or uncleaved zygotes after the second phase of segregation have no effect on development, suggesting that a gastrulation center is established by transient localization of myoplasm in the VP region. The function of the gastrulation center was investigated by comparing protein synthesis in normal and UV-irradiated embryos. About 5% of 433 labelled polypeptides detected in 2D gels were affected by UV irradiation. The most prominent protein is a 30 kDa cytoskeletal component (p30), whose synthesis is abolished by UV irradiation. p30 synthesis peaks during gastrulation, is affected by the same UV dose and has the same UV-sensitivity period as gastrulation. However, p30 is not a UV-sensitive target because it is absent during ooplasmic segregation, the UV-sensitivity period. Moreover, the UV target has the absorption maximum of a nucleic acid rather than a protein. Cell-free translation studies indicate that p30 is encoded by a maternal mRNA. UV irradiation inhibits the ability of this transcript to direct p30 synthesis, indicating that p30 mRNA is a UV-sensitive target The gastrulation center may function by sequestration or activation of maternal mRNAs encoding proteins that function during embryogenesis.

Memoirs: The Direct Development of a New Zealand Ophiuroid

1941 ◽  
Vol s2-82 (327) ◽  
pp. 377-440
Author(s):  
H. BARRACLOUGH FELL
Keyword(s):  
Bilateral Symmetry ◽  
Ventral Surface ◽  
Radial Symmetry ◽  
Early Ontogeny ◽  
Body Cavity ◽  
Cell Stage ◽  
Animal Pole ◽  
Early Stages ◽  
Vegetal Pole ◽  
Primitive Structure

1. The first cleavage may be either equal, or markedly unequal; when it is equal the next segmentation affects both blastomeres; when it is unequal the larger blastomere is believed to give rise to three cells, and the smaller remains undivided till the next cleavage. 2. At the eight-cell stage there are two quartets of blastomeres. The upper quartet, micromeres, occupy the animal pole. The lower quartet, macromeres, occupy the vegetal pole. 3. The blastula comprises micromeres and macromeres, and the blastocoel is small and becomes eccentric. No cilia are developed. 4. The gastrula is formed by the shallow imagination of the macromeres, accompanied by an extensive process of epiboly affecting the micromeres. More marked epiboly of cells on two sides of the blastomere produces in the early stages two crests which later disappear. These may indicate a trace of bilateral symmetry. Epiblast comes to lie on solid mes-hypoblast. The archenteron is transient, and gives rise to no structures. The blastopore occupies the position of the definitive mouth. 5. No larva ever forms, nor is there any vestige of a larval stage. 6. The solid gastrula is converted into the adult by assuming a radial symmetry directly, with no intermediate bilaterally symmetrical form, unless the two epibolic crests are regarded as vestiges of larval symmetry. 7. The podia appear as solid outgrowths, in which the hydrocoelic cavity develops by splitting. 8. The definitive enteron appears as a split extending upward from the ventral surface through the solid hypoblast. 9. The young ophiuroid leaves the egg before the appearance of the general body cavity, and moves about, but does not at first take food. 10. The general coelomic body cavity and the perihaemal cavity develop by splitting in a mass of mesenchyme derived from the outer layers of mes-hypoblast. 11. The formation of the skeletal system is delayed till the stage of between two and three arm-segments. 12. The development of the skeleton follows closely that described for Amphiura squamata. 13. The tooth is shown to originate independently of the torus angularis; its rudiments comprise nine symmetrically disposed spicules. 14. The terminal plate arises later than the radials, and has a distinctive ‘primitive structure’. 15. The spine is shown to have a different development to that of the tooth, and therefore would seem to have no connexion with the latter in phylogeny or ontogeny. 16. It is suggested that the aberrant early stages are to be correlated with the retarding effect of the yolk mass present in the egg during ontogeny. The aberrant features may have had a different origin in phylogeny. 17. It is suggested that the simultaneous appearance in ontogeny of homologous organs situated at equal radial distances from the centre is to be explained in terms of hormonic activity. 18. It is concluded that evolution has considerably affected the early ontogeny without leaving its mark on phylogeny. The adult thus conforms to its class, the young form does not.

Studies of the cleavage in the frog egg

Development ◽  
1969 ◽  
Vol 21 (1) ◽  
pp. 119-129
Author(s):  
T. Kubota
Keyword(s):  
Sea Urchin ◽  
Sea Urchins ◽  
Cleavage Furrow ◽  
Mitotic Apparatus ◽  
Animal Pole ◽  
Sea Urchin Eggs ◽  
Rana Nigromaculata ◽  
Vegetal Pole ◽  
Egg Cortex

In sea-urchin eggs, once karyokinesis reaches metaphase or anaphase, the cleavage furrow can be formed even if the mitotic apparatus is destroyed (Swann & Mitchison, 1953) or removed (Hiramoto, 1956). A similar result was obtained in frog eggs (Kubota, 1966). In amphibian eggs a much longer time is available for performing experiments than in sea urchins as the furrow first appears at the animal pole and slowly travels toward the vegetal pole. Taking advantage of this situation, Waddington (1952) and Dan & Kuno-Kojima (1963) performed various kinds of operations to elucidate the roles of the egg cortex and the inner cytoplasm in furrow formation, and Selman & Waddington (1955) also made cytological observations of the process. In the present paper a shift of the inner cytoplasm relative to the cortex and its influence on the course of the furrow was analysed for eggs of the frog Rana nigromaculata.

scholarly journals The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation.

1990 ◽  
Vol 110 (5) ◽  
pp. 1589-1598 ◽  
Author(s):  
J E Speksnijder ◽  
C Sardet ◽  
L F Jaffe
Keyword(s):  
Imaging System ◽  
Sperm Nucleus ◽  
Calcium Wave ◽  
Free Calcium ◽  
S Wave ◽  
Animal Pole ◽  
Calcium Dependent ◽  
Sperm Entry ◽  
Vegetal Pole ◽  
Ooplasmic Segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.

The development of animal cap cells in Xenopus: the effects of environment on the differentiation and the migration of grafted ectodermal cells

Development ◽  
1987 ◽  
Vol 101 (1) ◽  
pp. 23-32 ◽  
Author(s):  
E.A. Jones ◽  
H.R. Woodland
Keyword(s):  
Striated Muscle ◽  
Animal Cells ◽  
Animal Pole ◽  
Mesoderm Development ◽  
Dorsal Axis ◽  
Dorsal Mesoderm ◽  
Vegetal Pole ◽  
Pole Cells ◽  
Inductive Signal ◽  
Host Tissues

We have used blastocoel and vegetal pole grafts to investigate the effect of environment on differentiation and movement of animal pole cells of Xenopus. In the blastocoel of embryos earlier than stage 10, fragments of animal pole primarily form mesoderm. The cells are either integrated into normal host tissues or they organize a secondary posterior dorsal axis. If either host or graft is later than stage 9 the graft forms ectoderm and its cells all migrate into the host ectoderm. Inner layer animal cells form sensorial layer; outer cells move to the epidermis. Thus considerable powers of appropriate movement are seen. In the vegetal pole no movement occurs. If the graft is stage 9 or earlier, or the host is stage 101/2 or earlier, the graft forms mesoderm, including striated muscle in the gut. This shows that muscle can develop in wholly the wrong environment, it suggests that the dorsal inductive signal from mesoderm is rather general in the vegetal mass and suggests that dorsal mesoderm development involves little subsequent adjustability. If the host is stage 11 or later, or the graft later than stage 9, the graft forms epidermis in the gut. This shows that the epidermal pathway of development is also insensitive to environment.

Localized regions of egg cytoplasm that promote expression of endoderm-specific alkaline phosphatase in embryos of the ascidian Halocynthia roretzi

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 1-7 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Alkaline Phosphatase ◽  
Histochemical Staining ◽  
Halocynthia Roretzi ◽  
Endoderm Differentiation ◽  
Initial Event ◽  
Cytoplasmic Factors ◽  
Early Embryos ◽  
Vegetal Pole ◽  
Cytoplasmic Determinants ◽  
Cytoplasmic Transfer

Embryogenesis in ascidians is known to be of the mosaic type, a property that suggests the presence of cytoplasmic factors in the egg which are responsible for specification of the developmental fates of early blastomeres. Endoderm cells are present in the trunk region of tadpole larvae, and these cells specifically express alkaline phosphatase (AP). Endoderm cells originate exclusively from blastomeres of the vegetal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of endoderm specification, we carried out cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various regions. Initially, presumptive-epidermis blastomeres (blastomeres from the animal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of endoderm cells was monitored by histochemical staining for AP. AP activity was observed only when presumptive-epidermis blastomeres were fused with cytoplasmic fragments from the presumptive-endoderm blastomeres. The results suggest that cytoplasmic factors that promote the initial event of endoderm differentiation (endoderm determinants) are present in endoderm-lineage blastomeres. Next, to examine the presence and localization of endoderm determinants in the egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the presumptive-epidermis blastomeres. The results suggest that endoderm determinants are already present in unfertilized eggs, and that they are segregated by movements of the ooplasm after fertilization. Initially, these determinants move to the vegetal pole of the egg. Then, prior to the first cleavage, their distribution extends in the equatorial direction, namely, to the entire vegetal hemisphere from which future endoderm-lineage blastomeres are formed.

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