Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer.

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2(+)-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2(+)-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2(+)-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.     

Developmental regulation of catecholamine levels during sea urchin embryo morphogenesis

Results of a number of pharmacological studies suggest that catecholamines play a regulatory role in cleavage, morphogenesis and cell differentiation during early animal embryonic development. Few studies, however, have actually assayed for levels of catecholamines in these early embryos by methods that are both sensitive and specific. In this investigation the catecholamines dopamine, norepinephrine and epinephrine and their precursor, dopa and metabolites were determined in eight different embryonic stages of the sea urchin, Lytechinus pictus from hatched blastula to late pluteus larva, using high performance liquid chromatography with electrochemical detection. Levels of each of the catecholamines exhibited unique developmental profiles and are consistent with a role for epinephrine in blastula and early gastrula embryos and for norepinephrine in gastrulation. Changes in levels of catecholamine precursor and metabolites suggest a changing pattern of synthetic and metabolic enzyme activity, which can, for the most part, explain the fluctuations in catecholamine levels during development from blastula to the pluteus larva stage.     

An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.     

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes)

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.     

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.     

Achromosomal Division of Early Starfish Embryos Cultured in the Presence of Actinomycin D

Embryos of the starfish Asterina pectinifera were examined for their ability to undergo the early events of embryonic development in the presence of actinomycin D, a most widely used inhibitor of RNA synthesis. Fertilized eggs continued to divide eight or nine times in the presence of 25 μg ml⁻¹actinomycin D, although delay of development was observed. Chromatin disintegrated in the blastomeres of actinomycin D-treated embryos specifically at the 32-cell stage and the nucleus was undetectable at later stages. Before the 32-cell stage, RNA synthesis was not affected by the presence of actinomycin D whereas DNA synthesis was severely inhibited. The stage when achromosomal divisions cease and embryos begin to die corresponds to the period just before onset of blastulation, suggesting that the presence of the nucleus and chromosomes is a prerequisite for blastula formation and development beyond the 512-cell stage in this species.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.     

Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.     

Chromatin proteins from normal,vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.     

Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo.

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.     

Mitochondrial profile densities and areas in different developmental stages of the sea urchin Sphaerechinus granularis

Mitochondrial profile densities in electronmicrographs were counted in the swimming blastula, mesenchyme blastula, gastrula and prism stages of the sea urchin embryos Sphaerechinus granularis. No numerical changes were statistically apparent. When profile areas were investigated, the mean values of the swimming blastula, the gastrula and the prism stage showed no statistical differences. However, increased areas were measured in the mesenchyme blastula stage. This increase might be related to an increase of the embryonic volumina in the mesenchyme blastula stage. In contrast to earlier reported data, the results indicate that the mitochondrial density in S. granularis embryos does not alter during development in these stages.