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.

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.

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.

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.

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.

Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 551-560 ◽  
Author(s):  
N.D. Hopwood ◽  
A. Pluck ◽  
J.B. Gurdon
Keyword(s):  
Ectopic Expression ◽  
Muscle Cells ◽  
Muscle Differentiation ◽  
Cardiac Actin ◽  
Early Embryos ◽  
Muscle Genes ◽  
Myogenic Factor ◽  
Somitic Mesoderm

We have cloned a Xenopus cDNA that encodes a homologue of the human myogenic factor, Myf-5. Xenopus Myf-5 (XMyf5) transcripts first accumulate in the prospective somite region of early gastrulae. The pattern of XMyf5 expression is similar to that of the Xenopus MyoD (XMyoD) gene, except that XMyf5 transcripts are largely restricted to posterior somitic mesoderm even before any somites have formed. Transient ectopic expression of XMyf5 activates cardiac actin and XMyoD genes in animal cap cells, but does not cause full myogenesis, even in combination with XMyoD. These results suggest that XMyf5 acts together with XMyoD as one of the set of genes regulating the earliest events of myogenesis, additional factors being required for complete muscle differentiation.

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.

scholarly journals CENTRIOLES AND THE FORMATION OF RUDIMENTARY CILIA BY FIBROBLASTS AND SMOOTH MUSCLE CELLS

1962 ◽  
Vol 15 (2) ◽  
pp. 363-377 ◽  
Author(s):  
Sergei Sorokin
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Sensory Function ◽  
Second Phase ◽  
Electron Microscopic ◽  
Internal Development ◽  
Whole Process ◽  
Third Phase ◽  
Mammalian Tissues

Cells from a variety of sources, principally differentiating fibroblasts and smooth muscle cells from neonatal chicken and mammalian tissues and from organ cultures of chicken duodenum, were used as materials for an electron microscopic study on the formation of rudimentary cilia. Among the differentiating tissues many cells possessed a short, solitary cilium, which projected from one of the cell's pair of centrioles. Many stages evidently intermediate in the fashioning of cilium from centriole were encountered and furnished the evidence from which a reconstruction of ciliogenesis was attempted. The whole process may be divided into three phases. At first a solitary vesicle appears at one end of a centriole. The ciliary bud grows out from the same end of the centriole and invaginates the sac, which then becomes the temporary ciliary sheath. During the second phase the bud lengthens into a shaft, while the sheath enlarges to contain it. Enlargement of the sheath is effected by the repeated appearance of secondary vesicles nearby and their fusion with the sheath. Shaft and sheath reach the surface of the cell, where the sheath fuses with the plasma membrane during the third phase. Up to this point, formation of cilia follows the classical descriptions in outline. Subsequently, internal development of the shaft makes the rudimentary cilia of the investigated material more like certain non-motile centriolar derivatives than motile cilia. The pertinent literature is examined, and the cilia are tentatively assigned a non-motile status and a sensory function.

Development of Transient Outward Currents Coupled With Ca2+-Induced Ca2+ Release Mediates Oscillatory Membrane Potential in Ascidian Muscle Cells

2004 ◽  
Vol 92 (2) ◽  
pp. 1056-1066 ◽  
Author(s):  
Koichi Nakajo ◽  
Yasushi Okamura
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Muscle Cell ◽  
Single Channel ◽  
Muscle Cells ◽  
Developmental Expression ◽  
Dependent Manner ◽  
Membrane Excitability ◽  
Halocynthia Roretzi ◽  
Outward Currents

Isolated ascidian Halocynthia roretzi blastomeres of the muscle lineage exhibit muscle cell-like excitability on differentiation despite the arrest of cell cleavage early in development. This characteristic provides a unique opportunity to track changes in ion channel expression during muscle cell differentiation. Here, we show that the intrinsic membrane property of ascidian cleavage-arrested muscle-type cells becomes oscillatory by expressing transient outward currents ( Ito) activated by Ca2+-induced Ca2+ release (CICR) in a maturation-dependent manner. In current-clamp mode, most day 4 (72 h after fertilization) cleavage-arrested muscle cells exhibited an oscillatory membrane potential of –20 mV at 15 Hz, whereas most day 3 (48 h after fertilization) cells exhibited a spiking pattern. In voltage-clamp mode, the day 4 cells exhibited prominent transient outward currents that were not present in day 3 cells. Ito was abolished by the application of 10 mM caffeine, implying that CICR was involved in Ito activation. Ito was based on K+ efflux and sensitive to tetraethylammonium and some Ca2+-activated K+ channel inhibitors. We found a 60-pS single channel conductance that was activated by local Ca2+ release in ascidian muscle cell. Voltage-clamp recording with an oscillatory waveform as a command pulse showed that CICR-activated K+ currents were activated during the falling phase of the membrane potential oscillation. These results suggest that developmental expression of CICR-activated K+ current plays a role in the maturation of larval locomotion by modifying the intrinsic membrane excitability of muscle cells.

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