Changes in mitochondrial respiration during the development of Xenopus laevis

Mitochondria isolated from Xenopus laevis embryos at various developmental stages show a good oxidative capacity and an acceptable respiratory control provided that certain requirements are fulfilled. The rates of respiration with pyruvate and Krebs' cycle intermediates, especially with citrate and isocitrate, are very low during cleavage stages and increase after gastrulation. Glutamate in the presence of malate is the only substrate to be readily oxidized during early development and its rate of oxidation decreases after gastrulation. These results, together with the altered sensitivity of embryonic mitochondria towards azide, support the view that the oxidative metabolism undergoes important changes around gastrulation and is associated with mitochondrial differentiation.     

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 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.     

Expression of the N-myc proto-oncogene during the early development of Xenopus laevis

The N-myc proto-oncogene is expressed in a wide range of tissues during mammalian embryogenesis. This observation, along with the oncogenic capacity of this gene, has led to the suggestion that N-myc plays an important role in early development. However, due to the complexity of the expression pattern and the difficulty of manipulating mammalian embryos, little progress has been made towards understanding the developmental function of this gene. To enable a more detailed analysis of the role of this gene in early development, a study of the Xenopus homologue of N-myc was undertaken. Xenopus N-myc cDNA clones were isolated from a neurula library using a murine N-myc probe. Analysis of the timing of expression of N-myc mRNA and of the distribution of N-myc protein during Xenopus development indicate that this gene may be playing an important role in the formation of a number of embryonic structures, including the nervous system. N-myc is initially expressed as a maternal RNA, but this mRNA is degraded by the gastrula stage of development. Zygotic expression does not commence until late neurula. Examination of the distribution of the N-myc protein by whole-mount immunohistochemistry indicates that the early embryonic expression occurs in the central nervous system, the neural crest, the somites and the epidermis. Later expression is mostly within the head and somites. Specific structures within the head that express the protein include the eye, otic vesicle, fore and hindbrain and a number of cranial nerves. The results demonstrate that while N-myc is expressed in the developing nervous system of Xenopus, the timing of expression indicates that it is unlikely to be involved in regulation of the very first stages of neurogenesis.     

Absence of dorsal-ventral differences in energy metabolism in early embryos of Xenopus laevis.

The establishment of dorsoventral polarity in amphibian development begins prior to the first cleavage with the formation of the gray crescent. It has been suggested that quantitative differences exist in the metabolic activity of dorsal and ventral regions at these stages and that this metabolic activity gradient is involved in the maintenance of dorsoventral polarity. In this study regional metabolism has been examined by the microinjection of various radioactive compounds into dorsal or ventral cells of the four-celled Xenopus laevis embryo. The activities of the Embden-Meyerhof pathway, the pentose shunt, and the Krebs cycle and phosphate metabolism have been measured. No difference was detected in the rate or pattern of metabolism between dorsal and ventral regions, suggesting that metabolic activity gradients do not exist in these pathways during early Xenopus embryogenesis.     

Antioxidant metabolism of Xenopus laevis embryos during the first days of development

Reactive oxygen species (ROS) are formed and degraded in all aerobic organisms, but their role during embryonic development has not yet been well established. In this paper, we report the activities of various enzymes involved in antioxidant metabolism during the first 7 days of embryonic development of Xenopus laevis embryos. During the first two days of development, embryo antioxidant metabolism is based on catalase and superoxide dismutase activities. Later, the glutathione system is activated, and the activity of all the enzymes involved increases. The results presented in this study, together with previously reported data, support the hypothesis that antioxidant defences may include enzymes that are genetically regulated, while the other systems that appear to be environmentally modulated become relevant later in development, probably to protect embryos from environmental and toxic factors.     

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 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.     

Emerin expression in early development of Xenopus laevis

Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24 kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of approximately 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism.     

Transforming growth factor-beta5 expression during early development of Xenopus laevis

Members of the transforming growth factor-beta (TGF-beta) superfamily play various roles during development in both vertebrates and invertebrates. Two isoforms, TGF-beta2 and -beta5, have been isolated from Xenopus laevis. We describe here the localization of TGF-beta5 mRNA in early embryos of X. laevis, assessed by whole-mount in situ hybridization. The first detectable expression of TGF-beta5 was seen in the stage 14 embryo at the posterior tip of notochord, which continued to later stages, accompanied by the expression in bilateral regions of posterior wall in the tail region next to the notochord. At later stages, transient expression was seen in the cement gland (around stage 21) and in the somites (stages 24-27). In addition, expression was present in the branchial arches (stage 29-36) and olfactory placodes (stage 36).     

Intercellular communication is cell cycle modulated during early Xenopus laevis development

We investigated intercellular communication during the seventh and tenth cell cycles of Xenopus laevis development using microinjection of Lucifer yellow and FITC-dextran as well as freeze-fracture electron microscopy. We found that gap junction-mediated dye coupling visualized using Lucifer yellow was strongly cell cycle modulated in the tenth cell cycle. Cytoplasmic bridge-mediated dye coupling visualized via FITC-dextran was also, of course, cell cycle modulated. The basis of cell cycle-modulated gap junctional coupling was investigated by measuring the abundance of morphologically detectable gap junctions through the tenth cell cycle. These proved to be six times more abundant at the beginning than at the end of this cell cycle.