Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).     

Developmental regulation,induction, and embryonic tissue specificity of sea urchin metallothionein gene expression

Metallothionein (MT) is shown to be present in sea urchin embryos on the basis of its characteristic properties as a small protein (6–7 Da) of extraordinarily high cysteine content, whose biosynthesis is readily induced by heavy metals. Induction by Zn²⁺ results in the accumulation of the cysteine-rich MT protein, a 0.8 kb MT mRNA and a 2.9 kb nuclear RNA. The amount of MT mRNA is regulated intrinsically through the course of embryogenesis to the pluteus stage: A maternal MT mRNA is poly(A)-deficient and is polyadenylated after fertilization. New MT mRNA begins to accumulate between the seventh and eighth cell cleavage, reaches a maximum at the mesenchyme blastula stage, decreases during gastrulation, and rises again in the early pluteus stage. “Animalizing” embryos with Zn²⁺ during early embryogenesis causes a sustained accumulation of MT mRNA to levels greater than 25 times the normal amount. MT mRNA is present in high amount in the ectoderm of the pluteus, but is barely detectable in the mesoderm-endoderm tissue fraction. Treatment of either the pluteus or its isolated tissue fractions with Zn²⁺ results in the induction of MT mRNA accumulation in the mesoderm-endoderm but not in the already MT mRNA-enriched ectoderm. Furthermore, differences in Zn²⁺ induction of the MT gene in the blastula and gastrula are consistent with a developmental pattern in which MT gene expression is maintained constitutively at a high level in the ectoderm and at a low level in the mesoderm-endoderm tissues, which are, however, preferentially inducible by Zn²⁺.     

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.     

3H]serotonin binding to blastula, gastrula, prism, and pluteus sea urchin embryo cells

1. The presence of serotonin binding sites in blastula, gastrula, prism, and pluteus embryos of the sea urchin, Arbacia punctulata, was investigated by the binding of radiolabelled serotonin to dissociated embryo cells. 2. [3H]serotonin binding sites were identified in prism, early pluteus, and advanced pluteus larvae, but not in blastula or gastrula embryos. 3. The ontogeny of [3H]serotonin binding activity closely parallels that of serotonin content as previously reported in Paracentrotus lividus embryos (Toneby, 1977a). 4. Results of this study support a regulatory role of serotonin in developmental processes in postgastrula sea urchin embryos.     

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.     

Association of 7 SL RNA and an SRP-like particle with polysomes and endoplasmic reticulum in the developing sea urchin embryo

We have identified the sea urchin cognate of the mammalian signal recognition particle (SRP). This particle contains the diagnostic 7 SL small RNA, sediments at a similar velocity to that reported for the mammalian particle, and is found associated with the ER and polysomes. We have examined its subcellular localization during embryogenesis in order to determine whether it could serve in a translational regulatory capacity for a subset of the stored maternal mRNAs. In these studies the 7 SL RNA was used as a marker for the particle, since we determined that the 7 SL RNA exists exclusively within the SRP-like particle at all developmental stages. The relative distribution of the SRP among cytoplasmic structures changes dramatically during development. This represents an actual change in subcellular localization because the 7 SL RNA level remains nearly constant per embryo until the pluteus stage, when it increases slightly. In eggs, the SRP exists almost entirely free in the cytoplasm as an 11 S particle. Very soon after fertilization and throughout development there is an increase in the association of the particle with rapidly sedimenting structures, until by the pluteus stage greater than 90% of the SRP exists in a bound state. The nature of the associations is complex, and the bound structures include, at least in part, ribosomes, polysomes, and microsomes. The SRP is associated with microsomal membranes in gastrula (36 hr) but not in blastula (12 hr) or earlier embryos. Using the criteria of sensitivity to Triton X-100, we determined that 16% of the SRP in a 10,000g cytoplasmic fraction was bound to membranes in a microsomal (endoplasmic reticulum)-containing fraction in the gastrula. In contrast, less than 1% was membrane associated in the blastula. The SRP was also found in a ribosome-polysome fraction in 12-, 36-, and 48-hr embryos, but not in eggs. Finally, a small but significant portion of the SRP was found associated with monosomes in cleavage stage embryos. The possible role the SRP could play in the elongation arrest of stored maternal messages for secreted proteins is discussed.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.     

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.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Lipid peroxidation in embryos and larvae of the sea urchinStrongylocentrotus intermedius

Lipid peroxidation (LP) and glutathione content were studied at different developmental stages of the sea urchinStrongylocentrotus intermedius: egg cell, fertilization, 4 blastomers, blastula, hatching, gastrula, prism, pluteus. A high rate of LP in the total membrane fraction of sea urchin embryos and larvae at the stages from the egg cell to hatching was observed at enzymatic and nonenzymatic activation of LP. The LP rate was significantly reduced at the gastrula stage and at subsequent stages, there was practically no further development of the process. The glutathione concentration remained unchanged at different stages. The alterations in LP seem to reflect participation of free radicals in regulation of individual development.     

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.     

TOTAL CELL NUMBER AND NUMBER OF THE PRIMARY MESENCHYME CELLS IN WHOLE, 1/2 AND 1/4 LARVAE OF CLYPEASTER JAPONICUS*

Total cell number and number of the primary mesenchyme cells of 1/2 and 1/4 larvae were counted at several developmental stages after hatching in comparison with those of a whole larva, using Clypeaster japonicus as material. To obtain partial larvae, blastomeres were isolated at the 2- or 4-cell stage in Ca-free sea water and cultured in natural sea water at around 23°C. Isolated blastomeres cleaved as in situ, namely, as a part of an embryo. Although each partial embryo tended to spread into a plate, it acquired spherical shape prior to hatching of control whole embryo and developed normally in terms of both developmental rate and morphogenesis. Total cell number of a whole larva was about 620 just after hatching and increased almost linearly until i t reached 1850 at the pluteus stage. A half and quarter larvae contained roughly 1/2 and 1/4, respectively, of the number of cells of whole larva through all stages counted. Numbers of the primary mesenchyme cells in the partial larvae, however, tended to be slightly larger than a half or a fourth of that in whole larva. In whole larva, 35, 50, 56 and 58 was counted at the mesenchyme blastula, early gastrula, late gastrula and pluteus stage, respectively.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.