Ultraviolet-sensitive ooplasmic factors are responsible for the development of an endoderm-specific alkaline phosphatase in the ascidian embryo

The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue-specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m²) of ultraviolet (UV) light did not eliminate the AP-promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle-marker. A higher dose (4500 J/m²) of UV light markedly reduced the AP-promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants.     

Development of myoplasm-enriched ascidian embryos

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40-50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10-30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40-50% of the total egg volume, is that ascidian embryos are capable of regulative development.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2-3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A)+ RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.     

Ultraviolet irradiation during ooplasmic segregation prevents gastrulation, sensory cell induction, and axis formation in the ascidian embryo

The effect of ultraviolet (uv) light on embryonic development was examined in the ascidian Styela clava. uv irradiation (3.0 x 10(-3) J mm-2) of the entire surface of fertilized eggs during ooplasmic segregation prevented gastrulation, sensory cell induction, and embryonic axis formation. The uv-irradiated embryos completed ooplasmic segregation and cleaved normally, but vegetal blastomeres did not invaginate at the beginning of gastrulation, sensory cells in the larval brain did not develop tyrosinase or melanin pigment, and the larval tail did not develop. Endoderm, epidermis, and muscle cells differentiated in the uv-irradiated embryos, however, as evidenced by expression of endodermal alkaline phosphatase (AP), an epidermal-specific antigen, and alpha-actin, myosin heavy chain, and acetylcholinesterase (AChE) in muscle cells. Higher doses of uv light (6.0-9.0 x 10(-3) J mm-2) suppressed expression of the epidermal antigen and muscle cell markers, whereas the development of endodermal AP was insensitive. Irradiation at various times between fertilization and the 16-cell stage revealed that gastrulation, sensory cell differentiation, and axis formation are sensitive to uv light only during ooplasmic segregation. Irradiation of restricted regions of the zygote during ooplasmic segregation showed that the uv-sensitive components are localized in the vegetal hemisphere. The absorption characteristics of the uv-sensitive components suggest that they are nucleic acids. The results show that uv-sensitive components that specify gastrulation, sensory cell induction, and embryonic axis formation are localized in the vegetal hemisphere of Styela eggs.     

Survival of maternal mRNA in anucleate and unfertilized mouse eggs

Unfertilized mouse eggs were parthenogenetically activated in vitro and then bisected. Anucleate fragments were aged in vitro, and their protein synthesis was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proteins were compared with those which were synthesized by aging unfertilized eggs and those which were translated in vitro from mRNA extracted from the unfertilized eggs. Normally cleaving parthenogenetic eggs served as controls. Cytoplasts and unfertilized eggs synthesized considerable quantities of protein after 2 days in culture. The protein patterns of cytoplasts and unfertilized eggs shifted in this time mainly within a group of proteins with a molecular mass of about 35 kDa. This shift was also seen in controls between day 1 and 2 but was delayed in unfertilized eggs. There was no clear appearance of new proteins in aging cytoplasts, which might have indicated a selective activation of maternal mRNA at a certain time after the activation stimulus, nor was such a change apparent in unfertilized eggs. The survival of maternal allozymes of glucose phosphate isomerase was tested in cytoplasts derived from fertilized eggs. The allozymes remained active during 4 days of aging and did not change their quantitative correlation.     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

Muscle determinants in the ascidian egg are inactivated by UV irradiation and the inactivation is partially rescued by injection of maternal mRNAs

The ascidian egg contains cytoplasmic determinants that specify the fate of larval muscle cells. In a previous study, we developed an experimental system to identify the molecular nature of muscle determinants, in which unfertilized Ciona savignyi eggs were fragmented into four pieces by centrifugation. When inseminated, only nucleated fragments (red fragments) develop into partial embryos that only show differentiation of epidermal cells. One type of enucleated fragment (black fragment) has the remarkable ability to promote muscle differentiation when fused with red fragments. In the present study, using this experimental system, we investigated the molecular nature of muscle determinants. UV irradiation of black fragments suppressed the ability to promote expression of the muscle-specific protein, myosin heavy chain. The wavelength of UV light responsible for the inactivation (250–275 nm) suggested that UV-sensitive targets are nucleic acids. Injection of poly(A)⁺ RNA isolated from an un-irradiated black-fragment-rich fraction into UV-irradiated black fragments partially recovered the ability to promote the expression of myosin heavy chain protein. Poly(A)⁺ RNA from a red-fragment-rich fraction did not rescue the suppression of UV-irradiated black fragments. These results suggest that maternal mRNAs enriched in black fragments are closely associated with muscle determinants in the ascidian egg.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Contribution of maternal mRNA for maintenance of Ca2+-dependent reaggregating activity in dissociated cells of Xenopus laevis embryos

Dissociated Xenopus laevis blastula cells, where reaggregation was inhibited in Ca2+-free medium, reaggregated immediately after the addition of Ca2+. This reaggregation was not inhibited by cordycepin or actinomycin D treatment during culture, although cycloheximide and puromycin were inhibitory. The reaggregation was not inhibited even when fertilized eggs were microinjected with cordycepin and their RNA synthesis was continuously inhibited through cleavage to blastula stages. In neurula cells, cordycepin treatment induced significant reduction in sizes of aggregates formed. These results suggest that the Ca2+-dependent reaggregating activity of blastula cells is maintained by the translation of maternal, rather than newly synthesized, mRNA.     

Polyadenylic acid-containing of rna in unfertilized and fertilized eggs of the rabbit.

Molecular hybridization between ³H-polyuridylic acid and unlabeled RNA prepared from unfertilized rabbit eggs and 10-h postfertilization stage rabbit embryos has been used to measure the amount and subcellular localization of adenylated maternal RNA. The results reported indicate that there is poly (A)-containing RNA (putative messenger RNA) in unfertilized rabbit eggs. The amount of poly (A) in the RNA in rabbit eggs does not increase immediately after fertilization and is located primarily in the ribosomal fraction of the cell. The rate of protein synthesis in fertilized eggs is insensitive to α-amanitin at concentrations which inhibit RNA synthesis. These results suggest that maternal mRNA makes an important contribution to protein synthesis in early stages of cleavage in the rabbit embryo.     

Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Summary Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians. Offprint requests to: W.R. Bates     

cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides

A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper.     

A direct effect of some rifamycin derivatives on the morphology of mammalian mitochondria

Protein synthesis has been investigated in cell-free preparations from mature ovarian oocytes, unfertilized and fertilized eggs, and early embryos of Drosophila melanogaster. Preparations from unfertilized eggs have a specific activity that is 5- to 6-fold higher than the activity of fractions from ovarian oocytes. There is an additional small increase in activity of preparations from fertilized eggs. The specific activity that is rapidly attained in the fertilized egg remains essentially constant for 2 to 2.5 h after fertilization, decreases sharply during blastoderm formation, and again increases during gastrulation. The activities of unfertilized eggs decline slightly during the first 2 h after oviposition, and then decrease more sharply. About 35 % of the ribosomes in preparations from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients, whereas no polyribosomes could be detected in preparations from ovarian oocytes. In both ovarian oocytes and fertilized eggs, less than 1 % of the ribosome populations were present as subunits. Additional ribonucleoprotein material of buoyant densities different from those of ribosomal subunits or ribosomes was found throughout the sucrose gradients. About 3.5 % of the ribosomes were found to be membrane-bound in preparations from both unfertilized and fertilized eggs.     

The change in osmotically inactive fraction produced by cell activation

1. Resting and activated eggs of the sea urchin Arbacia punctulata were swollen in hypotonic sea water (60, 70, 80, and 90 per cent), and allowed to attain equilibrium volumes (Figs. 1 and 2). 2. Both fertilized and unfertilized eggs obey the Boyle-van't-Hoff law, but the value for b, the "osmotically inactive fraction" or non-swellable volume, was different for the two, averaging in the cases studied 7.3 per cent for unfertilized and 27.4 per cent for fertilized. 3. On activation, the eggs of the sea urchin undergo a definite increase in total cell volume, of approximately 2.7 per cent. 4. Some evidence is adduced for the possibility that the alteration in cell volume and in o.i.f. may depend upon the species in question. 5. A parallelism between change in b and alteration of respiratory metabolism in Arbacia, Chaetopterus, and Arbacia fragments is pointed out. This requires further investigation in other species to establish generality. 6. Equations for the calculation of the point at which osmotic pressures and cell volumes are identical for unfertilized and fertilized eggs are included. 7. A mechanical analogue of the phenomena is introduced (Fig. 3).