Expression of an antigen specific for trunk lateral cells in quarter embryos of the ascidian, Halocynthia roretzi

Cell-lineage analysis has demonstrated that a pair of the right and left A7.6 cells of a 64-cell embryo of the ascidian Halocynthia roretzi, descendants of A4.1 cells of an 8-cell embryo, give rise to trunk lateral cells (TLCs). In this study, in order to investigate cellular mechanisms involved in the specification of TLCs, we have examined the expression of a TLC-specific antigen in cleavage-arrested embryos and in quarter partial embryos. Although cleavage arrest of embryos by treatment with cytochalasin B at early stages, prior to and including the 16-cell stage, inhibited expression of the TLC-specific antigen, embryos arrested at the 32-cell stage and at later stages developed the antigen. The only blastomeres exhibiting expression of the antigen were the presumptive TLCs, as predicted by cell-lineage assignments. When the developmental potential of quarter embryos that originated from four isolated blastomere-pairs (a4.2, b4.2, A4.1, and B4.1 pairs) of an 8-cell embryo was examined, the A4.1 quarter embryos, which are developmentally fated to give rise to TLCs, rarely showed evidence of expression of the antigen. Expression of the antigen was not observed in a4.2 and b4.2 quarter embryos, which are not associated with the TLC fate. By contrast, expression of the antigen was detected in about a half of the B4.1 quarter embryos which are also not associated with the TLC fate. These results are discussed with reference to the relationship between TLCs and mesenchyme cells.     

Determinative properties of muscle lineages in ascidian embryos

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated muscle expression in isolated partial embryos of A4.1-derived cells requires an association with cells from other lineages whereas muscle from B4.1 blastomeres develops autonomously. Clear differences also occurred in the time acetylcholinesterase activity was first detected in partial embryos from these two sources. Isolated b4.2 cells failed to show any muscle development even in combination with anterior-animal cells (a4.2) and are presumably even more dependent on normal cell interactions and associations. Others have noted an additional distinction between the different sources of muscle: muscle cells from non-B4.1 lineages occur exclusively in the distal part of the tail, while the B4.1 descendants contribute those cells in the proximal and middle regions. During the course of ascidian larval evolution tail muscle probably had two origins: the primary lineage (B4.1) whose fate was set rigidly at early cleavage stages and secondarily evolved lineages which arose later by recruitment of cells from other tissues resulting in increased tail length. In contrast to the B4.1 lineage, muscle development in the secondary lineages is controlled less rigidly by processes that depend on cell interactions.     

Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.     

Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Requirement of cell division for muscle actin expression in the primary muscle cell lineage of ascidian embryos

The role of cell division in the expression of muscle actin and its relationship to acetylcholinesterase (AChE) development was examined in cleavage-arrested embryos of the ascidian Styela. Muscle actin expression was detected by two-dimensional gel electrophoresis of radioactively labelled proteins and by in situ hybridization with a cDNA probe, whereas AChE activity was assayed by enzyme histochemistry. In the majority of cases, muscle actin expression was first detected in embryos arrested after the 16-cell stage. Some embryos showed muscle actin expression after arrest at the 8-cell stage, however, muscle actin mRNA did not accumulate in embryos arrested at earlier cleavages. The cells that expressed muscle actin in 8- to 64-cell cleavage-arrested embryos belonged to the primary muscle lineage; secondary muscle cell precursors did not express muscle actin. Zygotic muscle actin mRNA appeared to accumulate with myoplasmic pigment granules in the perinuclear region of cleavage-arrested embryos, suggesting that the myoplasm may have a role in the organization of muscle cells. In contrast to muscle actin, AChE was detected in a small proportion of embryos treated with cytochalasin as early as the 1- or 2-cell stage, and most embryos treated with cytochalasin at later cleavages expressed this enzyme in some of their cells. Most primary muscle lineage cells expressed both muscle actin mRNA and AChE, however, some cells expressed only muscle actin mRNA or AChE. The results suggest that at least three cleavages are required for muscle actin expression and that muscle actin and AChE expression can be uncoupled in cleavage-arrested embryos.     

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35^S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Differentiation Expression in Blastomeres of Cleavage-Arrested Embryos of the Ascidian Halocynthia roretzi

The question whether two or more different genetic programs are expressed in the common cytoplasm of single blastomeres or the expression of one genetic program somehow excludes expression of the other, was analyzed by assessing the occurrence of a muscle-specific and two epidermis-specific antigens in cleavage-arrested blastomeres in early embryos of the ascidian Halocynthia roretzi . Blastomeres which had been arrested in 1- to 4-cell stages expressed only the epidermis markers. Arrested 8-cell to 32-cell embryos produced both epidermis and muscle markers, but each cell expressed only one program of differentiation, even though some possessed the potential to express both. The differentiation expressions followed their cell lineages. These results indicate that at least in this experimental system differentiation markers of the two different cell-types are expressed exclusively.
A distinct order was noticed in expression of the two epidermis markers in a single blastomeres; a marker is always superiorly expressed, whereas the other appears only when the superior marker is expressed.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo

We describe a fluorimetric method for measuring the level of H2O2 in individual mouse oocytes and early embryos. Levels of H2O2 are low but detectable in unfertilized oocytes recovered freshly from the female reproductive tract. The levels in early cleaving embryos (1-cell to 8-cell stages) immediately after recovery from the female tract seem to be slightly higher the later the stage examined. However, when embryos are cultured in vitro from the 1-cell or early 2-cell stage, H2O2 levels rise when the embryos reach the mid-2-cell stage and remain elevated until they enter the early 4-cell stage. No equivalent elevation of H2O2 is seen during the transition from 1-cell to 2-cell or from 4-cell to 8-cell stages. Embryos that are able to develop successfully in vitro, as well as those that show a developmental block at the 2-cell stage on culture in vitro, both show this rise in H2O2 levels after in vitro culture. The relationship between the rise in H2O2 and the '2-cell block' to development is discussed.     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.