A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.     

Identification and cloning of localized maternal RNAs from Xenopus eggs

A central question in developmental biology is to explain how cells in different regions of an embryo acquire different developmental fates. We have begun to address this question by investigating whether specific RNAs are localized within a frog egg. Differential screening of a cDNA library shows that most maternal RNAs are uniformly distributed along the animal-vegetal axis. However, we find that a rare class of maternal RNAs is localized. cDNA clones of four localized RNAs have been characterized. Three of these cDNAs are derived from maternal RNAs that are concentrated in the animal hemisphere of unfertilized eggs and remain localized through the early blastula stage. One cDNA is derived from a maternal RNA found almost exclusively in the vegetal hemisphere at both stages. These studies show that some informational molecules, specifically RNAs, are localized in eggs and are inherited by particular blastomeres.     

Evolutionary conservation of DNA sequences expressed in sea urchin eggs and early embryos

Summary DNA sequence divergence measurements indicate thatStrongylocentrotus franciscanus is more distinct fromS. purpuratus andS. drobachiensis than these two species are from each other, in agreement with paleontological and morphological evidence. The evolutionary divergence of several classes of expressed DNA sequences was compared with that of total single-copy DNA. BetweenS. franciscanus andS. purpuratus the divergence of cDNA made from gastrula cytoplasmic poly(A)⁺ RNA is about half that of total single-copy DNA. Similar results were obtained for cDNA made from unfertilized egg poly(A)⁺ RNA. In contrast, sequences expressed in gastrula nuclear RNA have diverged almost as much as total single-copy DNA.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originatefrom the axis of organelles and cytoskeletal elements establishedin the oocyte as it divides from the oogonium. Along this axis,cytoplasmic materials are localized during oogenesis: yolk platelets,for example, are translocated toward the vegetal pole, increasingtheir amount and size in that region. In the first cell cycleafter fertilization, the egg cortex rotates 30° relativeto the cytoplasmic core, modifying animal-vegetal organization.The direction of this rotation, biased by the point of spermentry, defines the site of development of anatomical structuresof the dorsal midline of the embryo. As its immediate effect,rotation activates the cytoplasm of a subregion of the vegetalhemisphere, causing cells cleaved from this subregion to bemore effective than other vegetal parts in inducing marginalzone cells to initiate gastrulation movements. The most stronglyinduced part of the marginal zone begins gastrulation first(the dorsal lip of the blastopore) and proceeds through a seriesof cell interactions leading to its determination as the anteriordorsal mesoderm of the embryo. If these cell movements are inhibitedin the gastrula stage, or if vegetal induction is inhibitedin the blastula stage, or if cortical rotation is inhibitedin the first cell cycle after fertilization, the embryo alwaysfails to develop dorsal structures of the anterior end of itsbody axis; the more inhibition, the more posterior is the levelof truncation, until a radial ventralized embryo develops, derivedfrom the animal-vegetal organization of the oocyte.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Early development and axis specification in the sea anemone Nematostella vectensis

We investigated the early development of the sea anemone Nematostella vectensis, an emerging model system of the Cnidaria. Early cleavage stages are characterized by substantial variability from embryo to embryo, yet invariably lead to the formation of a coeloblastula. The coeloblastula undergoes a series of unusual broad invaginations-evaginations which can be blocked by cell cycle inhibitors suggesting a causal link of the invagination cycles to the synchronized cell divisions. Blastula invagination cycles stop as cell divisions become asynchronous. Marking experiments show a clear correspondence of the animal-vegetal axis of the egg to the oral-aboral axis of the embryo. The animal pole gives rise to the concave side of the blastula and later to the blastopore of the gastrula, and hence the oral pole of the future polyp. Asymmetric distribution of granules in the unfertilized egg suggest an animal-vegetal asymmetry in the egg in addition to the localized position of the pronucleus. To determine whether this asymmetry reflects asymmetrically distributed determinants along the animal-vegetal axis, we carried out blastomere isolations and embryonic divisions at various stages. Our data strongly indicate that normal primary polyps develop only if cellular material from the animal hemisphere is included, whereas the vegetal hemisphere alone is incapable to differentiate an oral pole. Molecular marker analysis suggests that also the correct patterning of the aboral pole depends on signals from the oral half. This suggests that in Nematostella embryos the animal hemisphere contains organizing activity to form a normal polyp.     

Fertilization alters the spatial distribution and the density of voltage-dependent sodium current in the egg of the ascidian Boltenia villosa

The spatial distribution of voltage-dependent ionic currents was characterized in Boltenia villosa eggs before and after fertilization using two-microelectrode voltage clamp of paired animal-vegetal halves of eggs (merogones) made surgically. Major voltage-dependent conductances in the Boltenia egg are a transient inward Na current, a transient inward Ca current, and an inwardly rectifying K current. These currents were randomly distributed along the animal-vegetal axis in the unfertilized egg. When paired merogones (surgically prepared egg fragments) were made at the vegetal cap stage, 15-30 min after fertilization, Ca and K currents remained randomly distributed along the animal-vegetal axis. In contrast, the relative Na current density was found to be twofold lower in the vegetal vs the animal merogones made at the vegetal cap stage. By making pairs of merogones from unfertilized eggs and subsequently fertilizing one merogone of a pair, we showed that this change in current density ratio was due to a loss of absolute Na current density in the vegetal hemisphere shortly after fertilization. These results also show that this loss was intrinsic to the vegetal hemisphere, rather than being determined solely by the point of sperm entry. A second decrease in Na current was observed during the hour before first cleavage, 60-120 min after fertilization (M.L. Block and W.J. Moody, 1987, J. Physiol. 393, 619-634), both in fertilized eggs and in animal merogones fertilized after isolation. This second loss of Na current was not observed in vegetal merogones fertilized after isolation or in either animal or vegetal merogones made from fertilized eggs at the vegetal cap stage. Possible mechanisms for te rapid (complete by 40 min after fertilization) and the late (occurring from ca. 60 to 120 minutes after fertilization) Na current losses are discussed.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos

To obtain information on the amounts and major classes of RNA stored in the mouse egg and accumulated during cleavage, we determined the contents of total RNA, total poly(A), and ribosomes from the 1-cell stage to blastocyst. Using purified RNA for assay, we obtained an RNA content of 0.35 ng in the unfertilized egg, 0.24 ng in 2-cell, 0.69 ng in 8- to 16-cell, and 1.47 ng in early bastocyst (32 cells). As derived from EM morphometry, the number of ribosomes accounts for 60–70% of the total RNA content at all these stages; the marked increase in ribosomal number during cleavage is attributable entirely to new synthesis. Hybridization with [³H]poly(U) in solution yielded a poly(A) content of 0.7 pg for the unfertilized egg and 0.83 pg for the 1-cell embryo. The poly(A) content dropped sharply, to 0.26 pg per embryo, by the late 2-cell stage and increased to 0.44 pg in 8- to 16-cell embryos and 1.42 pg in early blastocysts. Hybridization in situ gave a similar pattern and also revealed a heavy labeling of embryo nuclei from the 2-cell onward but very little, if any, labeling of the pronuclei of 1-cell embryos, suggesting an absence, or low level, of poly(A)+ RNA synthesis at the 1-cell but an active synthesis at the 2-cell and later stages. These findings and other available evidence(e.g., R. Bachvarova and V. De Leon, 1980, Develop. Biol.74, 1–8) suggest that the mouse embryo inherits a large supply of maternal mRNA but that the bulk of this RNA is eliminated in the 2-cell embryo. In situ hybridization was used to study the relative concentration of poly(A) in ovarian oocytes. In growing oocytes, the cytoplasmic concentration of poly(A) remains about the same, suggesting that the accumulation of poly(A)+ RNA is proportional to oocyte growth. The poly(A) content declines about twofold between the time of completion of oocyte growth and fertilization. The germinal vesicle continues to be labeled up to the time of ovulation, raising the possibility that poly(A)+ RNA synthesis (and presumably turnover) occurs in fully grown oocytes.     

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.