The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

Qualitative and quantitative changes in protein synthesis occur at the 8-16-cell stage of embryogenesis in the cow

Cow oocytes and preimplantation embryos were cultured in medium containing radiolabelled methionine and the proteins synthesized were analysed by one-dimensional electrophoresis and fluorography. Marked changes in the pattern of synthesis were observed at the 8-16-cell stage of development. Quantitatively, a gradual decrease in the rate of protein synthesis occurred between the zygote and 8-cell stage and then the rate increased progressively to the blastocyst stage. Incorporation of radiolabelled uridine into RNA was first detected at the 16-cell stage. Taken together, these results suggest that protein synthesis is programmed by maternal mRNA up to the 8-cell stage but switches to mRNA derived from the zygote genome between the 8- and 16-cell stage.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Stimulation of Protein Synthesis in the Mitochondria of Sea Urchin Embryos before Gastrulation

A transient increase in protein synthesis was observed in mitochondria at the mesenchyme blastula stage of sea urchin ( Hemicentrotus pulcherrimus ) embryos. This stimulated activity was inhibited by chloramphenicol but not by cycloheximide. Reconstituting experiments in which poly U-dependent protein synthesis was carried out showed the mitochondrial peptide elongation factor to be essential for increasing the protein synthetic activity in mesenchyme blastula, but aminoacyl tRNA synthetase and ribosome fraction containing initiation factor not to be involved in this increase. These findings are discussed in relation to the differentiation of embryos at the gastrulation stage.     

CHANGES IN CELL SURFACE MORPHOLOGY DURING DIFFERENTIATION OF MICROMERE- AND MESOMERE-DERIVED CELLS OF THE SEA URCHIN EMBRYO

Micromeres and mesomeres isolated from 16-cell embryos of the sea urchin, Strongylocentrotus intermedius , were cultured in vitro , and changes in the cells surface architecture during the differentiation of the micromere- and mesomere-derived cells were observed using scanning electron microscopy. Two types of the distribution of the surface microvilli were observed in both blastomere-derived cell masses. One type showed a uniform distribution of the microvilli and the other type showed an uneven one. Though many microvilli were observed in most of both mesomere and micromere-derived cells at the 64-cell stage and the early blastula stage (16 hr after the 16-cell stage at 6°C) respectively, the microvilli decreased in number at the later stages in both blastomere-derived cell masses as compared with the 64-cell stage and the early blastula stage respectively. Rapid disappearance of the surface microvilli was observed in the micromere-derived cells in contrast with the mesomere-derived cells which still had many microvilli even at the midmesenchyme stage.     

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation.

When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearance of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. The active fraction does not readily filter through Amicon XM-50 and is retarded by column chromatography on Bio-Gel P-60.     

Change in the Activity of Na1, K+-ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development

The activity of ouabain-sensitive Na⁺, K⁺-ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo-wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre-gastrula stage and ectodermal cells at the late gastrula stage, the Na⁺, K⁺-ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells. Cycloheximide did not only block the activity increase in this period but also caused evident decrease in the activity in embryos at all examined stages. The activity increase in this period was strongly blocked by the treatment with actinomycin D, starting before the mesenchyme blastula stage, and was not seriously inhibited by the treatment starting at the mesenchyme blastula stage. The treatment starting at the initiation of gastrulation only slightly blocked further increase in the activity. Probably, an accumulation of mRNA encoding Na⁺, K⁺-ATPase occurs mainly in ectodermal cells and is completed up to the early gastrula stage.     

Cloning,expression, and localization of a new member of a Paracentrotus lividus cell surface multigene family

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed. © 1996 Wiley-Liss, Inc.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) activates the maternal program of apoptosis shortly after MBT in Xenopus embryos

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.     

Changes in cell surface charges during differentiation of isolated micromeres and mesomeres from sea urchin embryos

Changes in the negative surface charge were observed by cell electrophoresis during the differentiation of micromeres and mesomeres isolated from 16-cell-stage sea urchin embryos. Micromeres and mesomeres were separated by a sucrose density gradient column and were cultured in normal seawater. An isolated micromere developed to a cell aggregate, and, at the mesenchyme-blastula stage of control, the aggregate began to scatter into single cells. These processes are quite similar to those of the primary mesenchyme cells in situ. An isolated mesomere, on the other hand, developed into an ectodermal vesicle. At desired stages of development, the cell aggregates which derived from single blastomeres were dissociated into single cells, and their electrophoretic mobilities were measured. It was found that the electrophoretic mobility of the micromere- and mesomere-derived cells concomitantly increased from the early blastula stage up to the early mesenchyme stage. In contrast with the mesomere-derived cells, however, the micromere-derived cells showed another increase in electrophoretic mobility when the cells began to migrate as primary mesenchyme cells. These results show that a correlation exists between the increase in cell surface negative charge and the migration of the primary mesenchyme cells.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

Temporal-spatial expression of two Paracentrotus lividus cell surface proteins

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.     

Synthesis of Proteins Enriched in Li+- Induced Vegetalized Embryos of Sea Urchin during Early Development

Sea urchin embryos were vegetalized by a pulse treatment with 60 mM Li⁺ between 2.5 hr and 6 hr after fertilization at 20°C. Normal and Li⁺ -treated embryos were exposed to [³⁵S]-methionine for 2 hr at various stages and [³⁵S]-labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). On fluorograph of 2D-PAGE at the pre-hatching blastula stage, significant difference of labeled proteins between normal and vegetalized embryos was not observed in the range from neutral to acidic pH, but pls of several proteins were found to be shifted toward alkaline pH. At the mesechyme blastula stage, five major proteins [M.W. 36 K, 43 K (two species), 71 K and 150 K] were enriched in Li⁺-treated embryos among a few hundreds of synthesized proteins. At the late gastrula stage, the labeling intensities of these proteins except for one of 43 K proteins increased remarkably in Li⁺-treated embryos. Furthermore, two proteins (M.W. 105 K and 135 K) were also enriched in Li⁺-treated embryos at this stage. At the prism stage, these proteins enriched in Li⁺-treated embryos became hardly detectable, and the synthesis of at least four proteins (M.W. about 20 K, 41 K, 43 K and 200 K<) appeared to increase in normal embryos, but not in Li⁺-treated embryos. Synthesis of proteins eniched in Li⁺-treated embryos probably support endodermal cell differentiation.