Mesenchyme Blastula Research Articles

Alkaline phosphatase of sea urchin embryos: Chromatographic and electrophoretic characterization

A change in the molecular form of alkaline phosphatase in sea urchin embryos accompanies the marked increase in activity that occurs at gastrulation. On the basis of chromatographic and electrophoretic analyses, two major classes of alkaline phosphatase are identified: early enzyme, from unfertilized eggs to mesenchyme blastula, characterized by a major peak of activity, with a K av of 0.123 on Sephadex G-200 columns, elution from DEAE-Sephadex columns by 0.5 M NaCl, and a migration value of 0.51 (relative to bromophenol blue) after electrophoresis in 7.5% polyacrylamide gels; late enzyme, from gastrula to plutei, characterized by a K av of 0.137, elution from DEAE-Sephadex by 0.55–0.75 M NaCl, and a migration value of 0.56. By chromatographic and electrophoretic criteria the early enzyme appears to have a slightly greater molecular volume, lower net negative charge, and more heterogeneous composition than the late enzyme. Both enzyme preparations were maximally active at a pH 9.4–9.5. Enzyme from all stages appears to be predominantly associated with cell membranes. Extracting the enzyme by treatment with n-butanol, precipitating the enzyme from the dialyzed aqueous phase with ethyl alcohol, and chromatographing the alcohol preparation on columns of sieving and anion-exchanging media resulted in a substantial purification of the enzyme from all stages.

Poly(A)-containing polyribosomal RNA in sea urchin embryos: Changes in proportion during development

Evidence is described which suggests that a large fraction of sea urchin embryo polyribosomal RNA is not polyadenylated, and that the proportion of poly(A)-containing RNA increases during embryological development. Cleavage, early blastula, and mesenchyme blastula stage embryos were treated with cordycepin, and the effects of the drug on polyribosome appearance were determined. Polyribosomes in cleaving embryos and early blastulae were unaffected by the drug. However, there was a significant reduction in the amount of polyribosomes present and in the amount of newly synthesized RNA entering polyribosomes in mesenchyme blastulae after cordycepin treatment. Polyribosomal RNA was isolated from cleavage, early blastula, and mesenchyme blastula stage embryos, and the RNA was characterized by sucrose gradient centrifugation. Cleaving embryo polyribosomal RNA has a prominent 9 S peak and a smaller 20 S peak, and the larger RNA is heterodisperse. There is no poly(A) associated with the 9 S and 20 S material, but there is poly(A) associated with the RNA sedimenting faster than 20 S. Early blastula and mesenchyme blastula polyribosomal RNA have 22 S and 26 S peaks, respectively. This later stage RNA is quite heterodisperse and poly(A) is associated with RNA in all size classes larger than 9 S. Finally, the proportion of polyadenylated light and heavy polyribosome RNA was determined for cleavage, early blastula, and mesenchyme blastula stage embryos. Approximately 20–25% of newly synthesized light polyribosome RNA is polyadenylated and this amount is relatively constant during early development. However, the proportion of newly synthesized RNA in heavy polyribosomes that is polyadenylated increases during development: 25% at cleavage, 33% at early blastula, and 48% at mesenchyme blastula stages.

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

The relationship between 35SO 4 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 35SO 4 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.

RNA synthesis in isolated mitochondria from sea urchin embryos

RNA synthesis in vitro by mitochondria of sea urchin has been characterized using embryos isolated at the mesenchyme blastula stage. This synthesis is supported by a specific mitochondrial RNA polymerase which possesses peculiar properties similar to that of the same enzyme found in other organisms. Synthesis of RNA in sea urchin mitochondria is inhibited by rifampicin, acriflavin and actinomycin D and unaffected by α-amanitin. Measurements of the reaction at different developmental stages demonstrate that RNA synthesis already increases significantly a few minutes after fertilization.

The Enrichment of Egg-type RNA in Cleavage Stage Embryos of the Sand Dollar Dendraster excentricus

RNA populations synthesized during the first four cleavages of the sand dollar Dendraster excentricus were almost entirely egg-type as determined by RNA-DNA hybridization under conditions which measure repetitive DNA sequences. The relative concentrations of these egg-type RNA populations increased progressively up to the late blastula stage, then decreased, beginning at mesenchyme blastula, to a low level at the early prism stage. The egg-type RNA populations were transcripts of nuclear DNA, were heterogeneous in size and were species specific. RNA-DNA duplexes containing this RNA were heterogeneous with respect to thermal stability.

Nuclear basic protein acetylation during early sea urchin development

The acetylation of nuclear basic proteins was studied during early Strongylocentrotus purpuratus development (32–64 cell stage to pluteus formation). 1. 1. There are basic nuclear proteins present at the 32–64 cell stage that coelectrophorese with all of the histone fractions of a more advanced stage. The most highly acetylated proteins at this early cleavage stage are those that migrate in position of the F3 and F2a1 arginine-rich histones. 2. 2. At the early blastula, mesenchyme blastula, gastrula, prism and pluteus stages, the lysine-rich histone F1 is minimally acetylated. The extent of acetylation of the F2b-F2a2 slightly lysine-rich histones varies somewhat during development and is highest at the mesenchyme blastula and gastrula stages. F2a1 acetylation is slightly lower at prism stage. 3. 3. 10 −3 M puromycin inhibits prism stage protein synthesis completely, but total protein acetylation continues at grater than two-thirds the normal rate. 4. 4. Late gastrula stage embryos were pulse-labeled for 1 h with [ 14C]acetate and [ 3H]thymidine. During a 1-h cold chase period, the [ 14C]acetyl groups of fractions F2a1 and F2b-F2a2 decayed twice as fast as the labeled DNA, indicating rapid turnover of the histone acetyl groups. The above results suggest that the acetylation of invertebrate histone fractions is very similar to mammalian histone acetylation.

Investigation of the mode of nuclear control over protein synthesis in early development of loach and sea urchin

ABSTRACT It is shown that in loach embryos the incorporation of precursors into protein takes place in the blastoderm cells only. The change of the rate of incorporation of labelled amino acids into protein of the blastoderm separated from the yolk at successive developmental stages reflects the changes in the level of protein synthesis in intact embryos of the same developmental stages. Typical periodic changes of the intensity of protein synthesis in early embryogenesis of the loach are detected: low incorporation of amino acids at blastula stages is followed by an increase of synthesis during gastrulation and by a decrease with the onset of organogenesis. To study the genetic control over protein synthesis at various developmental stages of loach and sea-urchin embryos the effects of ionizing radiation and long-term treatment with actinomycin D have been examined. X-Irradiation doses produce an insignificant direct effect on protein synthesis, while radiation damage of the nuclear apparatus results in a gradual but ever increasing inhibition of protein synthesis. The inhibition of RNA synthesis with actinomycin or ionizing radiation damage of the nuclei produce essentially the same effect on the intensity of protein synthesis. Protein synthesis in androgenetic haploid hybrid embryos (loach ♀ ×goldfish ♂) and in loach androgenetic haploid embryos after producing a partial elimination of chromosomes does not differ from ‘enucleated’ loach embryos completely deprived of chromosomes. These data suggest that high-polymeric RNA formed after the elimination of some chromsomes is unable to provide a normal level of protein synthesis. Protein synthesis is not controlled by the nuclei up to the stages of early blastula (sea urchin) and of late blastula (loach), being evidently programmed in oogenesis. To provide a rapid activation of protein synthesis in the course of gastrulation in the loach the function of the nuclei has to be realized during mid-blastula stages. An increase of the rate of the incorporation of amino acids at the stages of mesenchyme blastula in the sea urchin depends on the synthesis of RNA at the early blastula. At the same time protein synthesis during gastrulation in the loach and at mesenchyme blastula in the sea urchin is much less dependent on the simultaneous RNA synthesis. Protein synthesis at these stages seems to be provided by the long-living templates and controlled by non-gene mechanisms of the regulation of translation. Thus early embryonic differentiation in the loach and sea-urchin development is related to the activation of protein synthesis. The latter is provided by the preceding morphogenetic nuclear function, which makes protein synthesis relatively independent of simultaneous synthesis of templates that ensures subsequent development stages.

De novo Synthesis of Transfer and 55 RNA1f in Cleaving Sea Urchin Embryos

SIGNIFICANT changes in RNA metabolism have been described during early sea urchin development. Until recently the only detectable class of RNA synthesized during cleavage stages was that with a low G + C base composition and heterogeneous sedimentation properties (DNA-Jike RNA)1. The genes for nucleolar ribosomal RNA (26S and 18S) were believed to become active only following gastrulation2–4 and the products of nuclear transfer RNA (4S) genes were first detected at the mesenchyme blastula stage5. Any label in the 4S region of sucrose gradients of RNA from the cleavage stages of embryogenesis was interpreted as reflecting the turnover of the pCpCpA region of pre-existing transfer RNA (tRNA) molecules.

Isolation of homologous nuclear DNAs from sea-urchin embryos.

ABSTRACT Studies are presented on the ability of low-molecular-weight nuclear DNA fractions to hybridize to higher-molecular-weight nuclear DNAs taken from different stages of early developing sea-urchin embryos, Strongylocentrotus purpuratus. Using preparative DNA-DNA hybridization, a fraction of 60-s mid-blastula DNA was isolated for its ability to anneal to 10-s morula DNA. Approximately 80 regions on each 60-s molecule were found to be homologous to 10-s DNA. High-molecular-weight (> 240-s) nuclear DNA from mesenchyme blastula stage and later stages (compared with DNA from pre-blastula nuclei) shows an increase in the number of regions homologous to fractions of 10-and 60-s DNAs.

Properties of glycoprotein particles and their changes during development of sea urchin eggs and embryos

1. 1. A singular proteinous component was isolated from the 105 000 g supernatant of morulae of Anthocidaris crassispina and Hemicentrotus pulcherrimus. The component was considered to consist of particles of 26S of S 20,w from its sedimentation patterns at ultracentrifugation. The 26S particles contain carbohydrates such as hexoses and hexosamines at 8.8 and 3.7, respectively, as values relative to a protein content of 100. 2. 2. The 26S glycoprotein particles were shown to be composed of several heterogeneous subunits, dissociated by 8 M urea treatment and separated by electrophoresis through a polyacrylamide gel column containing 6 M urea. None of the subunit bands of the 26S particles in electrophoresis corresponded to any of the bands formed by electrophoresis of yolk granules treated in the same way. 3. 3. Sedimentation properties of the 26S particles were studied during embryonic and larval development. The results indicated that the particles became lighter stepwise by about 1.5S, first at mesenchymal blastula and then at pluteus stage, as estimated by density gradient centrifugation. By analytical centrifugation, the 26S particles were estimated to become about 22S at pluteus stage. 4. 4. Quantitative changes of the 26S particles were also observed to occur in the course of development in parallel with the changes in sedimentation constant. 5. 5. In keeping with the changes in their sedimentation properties, changing patterns of subcomponent composition of the 26S particles were revealed by gel electrophoresis. Changes in the electrophoretic pattern were also observed to occur simultaneously at mesenchyme blastula and pluteus stages. A tentative idea is presented for the molecular events causing the developmental changes in characteristics of the particles, as seen from the electrophoretic patterns of the 26S particles treated in these ways.

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos ( Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens. As previously assessed by means of immunodiffusion, one of the antisera used herein contained antibodies to two soluble antigens present in the unfertilized egg but decreasing in amount during early development and undetectable after gastrulation. This antiserum gave parchy staining in the cytoplasm of the oocytes. However, in the mature egg, the staining was localized to the margin of the cells. After fertilization, the fluorescence was seen in the fertilization membrane and possibly also in the layer underneath. Another antiserum contained antibodies to a third antigen present in the egg at low concentration but increasing during development as assessed by immunodiffusion. By immunofluorescence, this antigen could not be traced before hatching. At the mesenchyme blastula stage, however, it occurred in the mesenchyme cells. This localization persisted throughout gastrulation. In contrast to these specific staining patterns, antisera to whole homogenates of either eggs or developmental stages gave a widely scattered cytoplasmic staining of both oocytes, unfertilized eggs and developmental stages.

The occurrence of stage specific antigens during early sea urchin development

AbstractRabbit antisera to homogenates of unfertilized sea urchin eggs were absorbed with pluteus extract. In the same way, antisera to gastrulae or plutei were also made “stage specific” by absorption with extracts of eggs. When tested by means of immunoelectrophoresis the absorbed sera formed precipitates with antigens in extracts from homologous developmental stages. Precipitates of such stage specific antigens were removed from the agar plates and reinjected into rabbits. In this way, monospecific antisera to four stage specific antigens were obtained. Upon closer analysis, the egg specific antigen was found to be heterogeneous, consisting of two components. Both components decreased in concentration and had disappeared by the time of gastrulation. The two pluteus specific antigens seemed to be absent from eggs and earlier stages. By means of isotope incorporation experiments and autoradiography of immunodiffusion plates, it was found that one of these antigens became detectable at the mesenchyme blastula stage while the other was first detected at the prism stage. The fourth antigen, which was clearly detectable from gastrulation onwards, was found to be already present in the unfertilized eggs but at much lower concentrations than in late developmental stages.

Control of enzyme synthesis in early sea urchin development: Aryl sulfatase activity in normal and hybrid embryos

Aryl sulfatase activity begins to increase at the mesenchyme blastula stage and continues to increase through the pluteus stage. Total activity increases 30-fold in Strongylocentrotus purpuratus embryos and 10-fold in Allocentrotus fragilis embryos. The bulk of new sulfatase activity is localized on or near the cell surface; thus, unlike the unfertilized egg or early cleavage stages, up to 75% of the sulfatase activity of larvae can be extracted with isotonic Na:K, and intact larvae can hydrolyze exogenous substrate. Mixing experiments do not reveal any soluble activators, but do indicate the presence of a heat-stable, dialyzable inhibitor in eggs and embryos. Developmental changes in this inhibitor, however, are not sufficient to account for the observed change in enzyme activity. Studies with viable hybrids between S. purpuratus and A. fragilis strongly suggest that increased aryl sulfatase activity is under nuclear control. Thus, total activity is intermediate to that of the two parents in both reciprocal crosses. Gel electrophoresis of the sulfatase in the hybrid embryos reveals a band of intermediate mobility to that of the two parents. Anomalously, however, there are no electrophoretic bands corresponding to the parental enzymes in the hybrid embryos. Although nuclear control is implied, inhibition of 85% of the protein synthesis by puromycin does not immediately arrest the increase in sulfatase activity. This suggests some sort of activation process which is independent of protein synthesis. Several models involving modification of protein after its synthesis are proposed to explain the lack of immediate effect of puromycin and the absence of the parental bands in the hybrid embryos. Although less probable, it is also proposed that the increase in sulfatase activity may result from the modification of a pre-existing protein by a factor or factors whose synthesis is under genetic control during development.

Protein synthesis and development in the absence of cytoplasmic RNA synthesis in nonnucleate egg fragments and embryos of sea urchins: Effect of ethidium bromide

Nonnucleate fragments of eggs of the sea urchin Strongylocentrotus purpuratus were made by sucrose density gradient centrifugation. The synthesis of RNA and of protein was studied in the nonnucleate fragments after artificial activation with butyric acid. Ethidium bromide inhibited the incorporation of uridine-5- 3H into RNA by activated nonnucleate fragments and by cleaving embryos more effectively than did dactinomycin. The incorporation of uridine-5- 3H into high molecular weight RNA was inhibited by 10 μg/ml of ethidium bromide by about 97% in nonnucleate fragments, 85% in fertilized eggs and 53% in blastulae. The electrophoretic pattern of proteins labeled in activated nonnucleate fragments, in sodium dodecyl sulfate polyacrylamide gels, was not qualitatively altered by 6 hours of preincubation with 10 μg/ml of ethidium bromide. The range of molecular weights for the labeled proteins was 9 to 40 thousand for those found in the 15,000 g supernatant fraction of the homogenate and 6 to 60 thousand for those in the corresponding pellet. These results establish that most, or possibly all, of the types of proteins labeled after artificial activation of nonnucleate fragments are synthesized upon RNA templates which were present in the unfertilized egg. Continuous exposure to various concentrations of ethidium bromide affected development. Two micrograms per milliliter produced a prism stage larva with an abbreviated gut and 5 μg/ml arrested development at the blastula stage. At 10 μg/ml of development did not proceed beyond the 4- to 8-cell stage. When mesenchyme blastulae or gastrulae were treated with 10 μg/ml of ethidium bromide, development ceased, but the embryos continued to swim for at least 30 hours.

Rates of synthesis and degradation of protein in the sea urchin embryo

A rapid expansion of the amino acid pool by an influx of exogenous labeled amino acid is the basis for the direct measurement of the amount of protein synthesized in the sea urchin embryo. The amount of newly synthesized protein is calculated from data on incorporation rates of labeled amino acids and protein amino acid analyses. The average rate of protein synthesis for the early gastrula, based on protein composition and incorporation rates of valine, alanine, proline, histidine, threonine, and phenylalanine, is 8.4 × 10 −4 μg protein/embryo/hour, or 0.96% of the total protein content of the embryo/hour. Turnover rates of proteins synthesized in the sea urchin embryo were determined by pulse labeling the proteins with 14C-valine and following the decrease of the label during subsequent development. Proteins labeled shortly after fertilization and in the mesenchyme blastula undergo an average degradation of 0.8%/h.

Serological studies of two hybrid sea urchins

Embryos of the intergeneric hybrid of Lytechinus variegatus eggs and Tripneustes esculentus sperm and the interordinal hybrid of Strongylocentrotus purpuratus eggs and Dendraster excentricus sperm were described, their developmental rate and morphology were compared, and the influence of the paternal nucleus was observed. The antigens present in the unfertilized eggs, mesenchyme blastulae, gastrulae, and larvae of the two hybrids and the four parental species were investigated with the Ouchterlony double diffusion technique, immunoelectrophoresis, and passive cutaneous anaphylaxis in conjunction with specific absorption of the antisera. No paternal antigens or hybrid-specific antigens were detected in extracts of the embryos of either hybrid. Thus, hybrid pluteus larvae of two different crosses which show paternal characteristics are formed without any indication that the zygote nucleus directs the synthesis of any major class of antigenic protein. No molecules of new antigenic specificity were detected in developmental stages of the four parental species investigated. The implications of these findings were discussed.

Transfer RNA changes associated with early development and differentiation of the sea urchin, Stronglyocentrotus purpuratus

Transfer RNA alterations were found to occur during early development and differentiation of the sea urchin, Strongylocentrotus purpuratus. A quantitative difference was observed between egg and blastula leucyl-tRNA, a new species of lysyl-tRNA appeared in the mesenchyme blastula, and eultion profile “shifts” were observed for seryl- and lysyl-tRNAs from eggs and blastulae. A possible regulatory role for these tRNA changes during differentiation was discussed. Art. no. 30

Effect of actinomycin D on antigen synthesis during sea urchin development.

AbstractEggs and developmental stages of the sea urchin Paracentrotus lividus were incubated with radioactive (14C) amino acids. Incorporation of label into antigen was studied by means of rabbit antisera against sea urchin material. In an attempt to block genetic control of protein synthesis, eggs and embryos were treated with actinomycin D during different periods of development.During normal development the antigen‐labeling pattern, studied by immunoelectrophoresis and autoradiography, remained essentially unchanged until hatching. In the mesenchyme blastula, however, a new labeled antigen was detected, and one which earlier was unlabeled became heavily labeled. As seen in autoradiographs, during the same period, the intensity of labeling of other antigens also changed. When the embryos were exposed to actinomycin D (10–20 μg/ml) from hatching or some hours before, the development stopped at the mesenchyme blastula stage and these embryos never developed the labeling pattern characteristic of normal mesenchyme blastula.The results suggest that in contrast to the early development, gastrulation requires new production of templates. These templates seem to be essential for the changing pattern of antigen‐labeling. It is suggested that alterations in amino acid incorporation into antigens reflect changes in synthesis of proteins necessary for gastrulation.

Gene expression in sea urchin development.

Actinomycin D has been administered for various lengths of time and at various concentrations to developing sea urchin embryos. The results obtained support the following suggestions. (1) The shape of respiratory curve up to the mesenchyme blastula stage is not under direct gene control. (2) The RNA needed for gastrulation seems to be made during the period between early and late blastula. (3) The RNA needed for skeleton differentiation is continously made from some time before early gastrula throughout skeleton growth. (4) The process of development of skeleton is extremely sensitive to actinomycin.

Ribosomal ribonucleic acid of the sea urchin egg and its fate during embryogenesis

The sedimentation properties of the ribosomal RNA of eggs and that synthesized by late-stage embryos are identical in three species of sea urchin. However, uniquely in the eggs of Strongylocentrotus purpuratus, the 18 s ribosomal RNA can be converted to approximately 13 s (presumably two fragments) by heating briefly at 60 °C. This fragility is peculiar to the RNA of the egg and is not a property of ribosomal RNA synthesized by late-stage embryos. The specific concentration of this 13 s RNA decreased during embryonic development, but not until the 20-hour mesenchyme blastula stage had been reached. The initiation at this stage of substantial ribosomal RNA synthesis, previously demonstrated, suggested the hypothesis that the decline in 13 s RNA content was due to a replacement of egg ribosomes. Hence, we propose that during development, from the 20-hour mesenchyme blastula stage, one-half the original egg ribosomes are replaced in 30 hours.