Tunicamycin-inducible polypeptide synthesis during Xenopus laevis embryogenesis

Tunicamycin treatment of Xenopus laevis embryos enhanced the synthesis of a specific set of polypeptides with molecular masses of 98, 78, 59 and 58 kDa. The 78-kDa polypeptide was tentatively identified as glucose-regulated protein (GRP) 78 on the basis of molecular mass, pl (5.2), and tunicamycin inducibility, which took place upon treating embryos after the midblastula transition (MBT). The synthesis of a polypeptide with this electrophoretic mobility was detected but was not tunicamycin-inducible at stages prior to the MBT. GRP78 mRNA was detectable before the MBT but was not inducible by tunicamycin until the tailbud stage. A comparison of tunicamycin-induced polypeptide synthesis in Xenopus embryos, A6 cell line, and white blood cells by 2D-PAGE and fluorography revealed three spots in the GRP78 region of the gel. One was observed in both embryos and adult cells; another was adult-specific; and the third one was possibly an embryo-specific form. These results suggest that GRP78 synthesis might undergo a switch from an embryonic to an adult pattern during Xenopus development.     

The U18 snRNA is not essential for pre-rRNA processing in Xenopus laevis.

The U18 small nuclear RNA (snRNA) is one of several newly discovered intron-encoded nucleolar RNAs whose function is unknown. We have studied the accumulation and function of the U18 snRNA in oocytes of the vertebrate, Xenopus laevis. The U18 snRNA contains 13 nt complementary to a highly conserved sequence in 28S ribosomal RNA (rRNA). Three oligonucleotides, selected to contain all or some of the complementary sequence, deplete the U18 snRNA upon injection into Xenopus oocytes. Injection of two of the oligonucleotides has no effect on pre-rRNA processing or ribosome transport. Injection of the third oligonucleotide does interrupt pre-18S rRNA processing, but this is due to coincidental simultaneous depletion of the U22 snRNA. The U18 snRNA is the first nucleolar snRNA that is not essential for ribosome biogenesis in vertebrates.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Cloning and developmental expression of WSTF during Xenopus laevis embryogenesis

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams-Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.     

Behavior of individual maternal pA+ RNAs during embryogenesis of Xenopus laevis

Of the 10 Xenopus oocyte cDNA clones previously examined in this laboratory (L. Golden, U. Schafer, and M. Rosbash, 1980, Cell22, 835–844), 5 are complementary to RNAs which which decrease in abundance during early development. We have further examined the behavior during embryogenesis of these 5 sets of clone-complementary RNAs. The results indicate that for 3 of these 5 sets of RNAs there is an increase in the per embryo levels of RNA. Thus, 8 of the 10 clones originally examined are complementary to RNAs which increase in amount during early embryogenesis. One of the remaining two clones is complementary to (at least) 4 RNAs which vary somewhat in their levels during embryogenesis. The last clone (XOC 2–7) is complementary to an RNA species which is largely destroyed at late blastula or early gastrula. This RNA is therefore the only maternal sequence, of the ten clones examined, which unambiguously decreases in amount during embryogenesis. The data also show that XOC 2–7 RNA is largely adenylated at oocyte maturation and then deadenylated during subsequent embryogenesis while another clone, XOC 1–2, is largely dead-enylated at oocyte maturation. The results also suggest that a large fraction of oocyte RNAs are present in early embryos (and in liver) and are largely the same size as in oocytes.     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus laevis.

A method is described which permits the preparation of descrete classes of oocytes of different sizes from all stages of oogenesis in Xenopus laevis. This technique is used in the determination of the content of microtubule protein in oocytes during the course of oogenesis. These experiments show that microtubule protein is present in oocytes of all sizes assayed and that the amount is simply related to the volume of the oocyte. In the largest oocytes microtubule protein constitutes 1% of the soluble protein and this amount does not change on maturation and fertilization. These results show that the changes occurring in the oocyte on maturation which allow the cytoplasm to support microtubule polymerization occur as a result of a modification of the pre-existing microtubule protein, not from protein synthesis de novo. These experiments also indicate that the synthesis of microtubule protein either form 'masked' mRNA or from newly synthesized mRNA plays an insignificant role in microtubule protein synthesis at maturation, ovulation and immediately post-fertilization.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22

We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22. A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes. In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes. Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki