Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Expression analysis of the peroxiredoxin gene family during early development in Xenopus laevis

Development in the frog, Xenopus laevis, requires the utilization of yolk glyco-lipo-proteins in a temporally- and spatially-dependent manner. The metabolism of the yolk produces hydrogen peroxide (H₂O₂), a potent reactive oxygen species (ROS). Peroxiredoxins (prdxs) are a family of six anti-oxidant enzymes that, amongst other roles, reduce H₂O₂. Prdxs reduce H₂O₂ through a thiol-redox reaction at conserved cysteine residues which results in the creation of disulfide bonds. Recently the thiol-redox reaction of Prdxs has also been implicated in several cell signaling systems. Here we report the cloning and expression patterns during development of six peroxiredoxin homologs from the frog X. laevis. Sequence analysis confirmed their identity as well as their evolutionary relationship with peroxiredoxins from several other species. Using RT-PCR and in situ hybridization analysis we have shown that there is early and robust expression of all six homologs during development. All six X. laevis peroxiredoxins are expressed in neural regions including the brain, eyes, as well as the somites. Different expression patterns for each peroxiredoxin are also observed in the pronephric region, including the proximal and distal tubules. Expression of several peroxiredoxins was also observed in the blood precursors and the olfactory placode. These results suggest important roles for all six peroxiredoxins during early development. These roles may be restricted to their functions as anti-oxidant enzymes, but may also be related to their emerging roles in redox signaling.     

Characteristics of immunoglobulin synthesized early in the development of Xenopus laevis.

A radioimmune assay has been used to detect the onset of immunoglobulin synthesis during development. In Xenopus laevis, immunoglobulin is first produced at stage 35 of embryonic development or about the time of emergence of the embryo from its jelly coat. At all embryonic stages measured, beginning at stage 35, both 19 and 7 S immunoglobulin are found. These immunoglobulins contain heavy and light chains identical in size to adult Xenopus IgM. These observations suggest the presence of both cell-associated and circulating IgM in Xenopus embryos.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Identification and expression analysis of GPAT family genes during early development of Xenopus laevis

Production of lysophosphatidic acid (LPA) is the first step in the de novo pathway for glycerolipid biosynthesis, which is mainly catalyzed by the glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15). DHAPAT (EC2.3.1.42) also contributes in a minor way, using dihydroxyacetone phosphate as substrate. Final products and intermediates of the glycerolipid synthesis pathway are the main structural components of cellular membranes, and provide signalling molecules that regulate diverse biological processes, including cell proliferation, differentiation and growth. Here we identified the four orthologs of the mammalian GPATs (1-4) and DHAPAT in Xenopus, including a novel, short variant of GPAT2, and analyzed their expression pattern during embryonic development. Xenopus GPAT1/2 localized to mitochondria, while GPAT3/4 associated with the endoplasmic reticulum. All are similarly expressed in the early embryonic nervous system. A more tissue specific pattern emerges during organogenesis, including liver expression for GPAT1/4, and testis expression for GPAT2. All acyltransferases were expressed in kidney, though GPAT3 was excluded from the pronephric ducts. Our results suggest important roles of GPATs and DHAPAT during early organogenesis.     

Expression of muscle LIM protein during early development in Xenopus laevis

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.     

Changes in energy metabolism during the early development of Xenopus laevis

Xenopus embryos at various development stages were incubated in the presence of labelled substrates and the 14CO2 production determined. From the rates of oxidation of glucose labelled in positions 1 and 6 and from that of radioactive acetate, pyruvate and glutamate, it was concluded that the Embden-Meyerhof pathway and the Krebs cycle are functional during early embryogenesis, but that their relative participation in the metabolic processes is limited and increases from gastrulation onwards. Early development is characterized by the predominance of the pentose cycle and the glutamate-aspartate cycle. Furthermore, it was shown that glutamate may be the main energy source up to gastrulation.     

Genes and mechanisms involved in early embryonic development in Xenopus laevis

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.     

Modulation of Na,K-ATPase expression during early development of Xenopus laevis.

In amphibian and mammalian systems, regulation of Na+ transport via the Na,K-ATPase plays an important role in distinct developmental processes such as blastocoele formation and neurulation. In this study, we have followed the Na,K-ATPase activity, the biosynthesis, and the cellular accumulation of catalytic alpha-subunits after fertilization of Xenopus laevis eggs up to neurula formation. Our data show that Na,K-ATPase activity increases significantly between stages 4 and 6 and again between stages 13 and 24. The four-fold rise in Na,K-ATPase activity during blastocoele formation is not mediated by an increased cellular pool of alpha-subunits. On the other hand, a five-fold increase of the biosynthesis rate around midblastula precedes a progressive accumulation up to neurula stage mainly of alpha 1-subunits and to a lesser extent of a second alpha-immunoreactive species. In contrast, newly synthesized glycoproteinic beta 1-subunits of Na,K-ATPase cannot be detected up to late neurula. These data indicate that (1) upregulation of Na,K-ATPase activity during blastocoele and neurula formation are mediated by different regulation mechanisms and (2) alpha- and possibly beta-isoforms are expressed in a developmentally regulated fashion during early Xenopus development.     

The distribution of nucleoplasmin in early development and organogenesis of Xenopus laevis

Summary The fate of the germinal vesicle-derived protein, nucleoplasmin, was followed in embryos and tadpoles of Xenopus using monoclonal antibodies and indirect immunofluorescent staining. Nucleoplasmin was found in all nuclei up to feeding tadpole stages. Thereafter its level decreased in all nuclei. It was not detected in nuclei of advanced tadpoles or of adults. Contrasting with another protein, N₁, that was previously monitored in the nuclei of dividing gonia of both sexes, nucleoplasmin was only detected in the nuclei of ovarian oocytes starting at diplotene. Traces of nucleoplasmin have also been found in a rapidly-dividing fibroblastic cell-line by immunohistology and protein blotting.     

Appearance of DNA polymerase activities during early development of Xenopus laevis

Extracts of large oocytes of Xenopus laevis contain high levels of one major DNA polymerase activity. After maturation into eggs, the overall level of DNA polymerase activity in extracts increases fourfold and a second major activity appears on Sephadex G-200 or DEAE cellulose columns. Although intense DNA synthesis occurs as the number of cells increase from one to over 100,000, no further increases in the level of either DNA polymerase activity are observed in cleavage, gastrula or early neurula stage embryos. In extracts of late neurulae or hatched embryos, however, a third major DNA polymerase activity appears coincident with an increase in the ability of the extracts to utilise native DNA templates in vitro.     

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.     

Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.