Embryonic Development Of Xenopus Laevis Research Articles

Proopiomelanocortin gene expression as a neural marker during the embryonic development of Xenopus laevis

Abstract Proopomelanocortin (POMC) is the precursor protein for a number of peptide hormones and neuropeptides, and the POMC gene is transcriptionally very active in the pars intermedia of the pituitary of the amphibian Xenopus laevis (Xenopus). We analysed the expression of this gene during Xenopus embryogenesis, in order to examine whether it can function as a (novel) neural marker. We investigated the spatio-temporal distribution of POMC mRNA, using a single-stranded probe that corresponds to the 3'untranslated region of Xenopus POMC gene B mRNA. Gene transcripts were first detected at stage 25 of development via RNase protection assays. In situ hybridization analysis performed at stage 46 showed clearly that these transcripts are localised in a region representing the future pars intermedia of the pituitary. Experiments using Xenopus explants indicate that the POMC gene can be used successfully as an indirect marker in studies on neural induction: in the absence of interactions with mesoderm, ectoderm fails to express the POMC gene, whereas POMC transcripts are readily detectable in conjugates of ectoderm and mesoderm. Artificial application of two different signals, which are likely to be relevant for neural differentiation (namely retinoic acid and the activation of protein kinase C via phorbol ester), was not effective in evoking POMC gene expression in cultured ectoderm explants. However, retinoic acid treatment of conjugates of Xenopus ectoderm and mesoderm successfully prevented POMC expression. We conclude that POMC gene expression can be used as an indirect marker for anterior neural differentiation in Xenopus.

Spatially restricted expression of fibroblast growth factor receptor-2 during Xenopus development.

The fibroblast growth factors (FGFs) play a role in Xenopus laevis embryonic development, particularly in the induction of ventral-type mesoderm. We have isolated a full-length cDNA from Xenopus that we have designated Xenopus fibroblast growth factor receptor-2 (XFGFR-2), with significant amino acid sequence similarity to the previously described bek gene (FGFR-2). We expressed the XFGFR-2 cDNA in COS1 cells and showed that it functions as an FGF receptor by binding radiolabeled FGF-2. RNA gel blot analysis demonstrates that unlike Xenopus fibroblast growth factor receptor-1 (XFGFR-1), XFGFR-2 mRNA expression begins during gastrulation and continues through early tadpole stages. Whole-mount in situ hybridization demonstrates that XFGFR-2 mRNA is localized to the anterior neural plate in early neurula stage embryos. Later in development, XFGFR-2 expression is found in the eye anlagen, midbrain-hindbrain boundary and the otic vesicle. In addition, XFGFR-2 transcripts are expressed in animal caps in a manner that is independent of mesoderm-inducing factors. These results indicate that XFGFR-2 may have a role in development that is distinct from that of XFGFR-1.

Biological Effects of Magnetic Fields

The biological effects of fields are classified into three categories: the effects of time-varying fields, the effects of static fields, and effects caused by compounding static fields with other energy forms such as light or radiation. In each category, a different approach is required in investigations of biomagnetic effects. Magnetic brain stimulation has been achieved with a resolution of 5 mm by eddy currents concentrated in the target, induced by a pair of opposing time-varying fields. No clear biological effects of static fields have been observed, except for a few special effects such as the orientation of fibrin. Static fields of up to 6.34 T do not affect the embryonic development of Xenopus laevis. An experiment has been performed which demonstrates the phenomenon of extinction of a candle flame by a field. A magnetic curtain model has been introduced to explain this phenomenon. Paramagnetic oxygen is indispensable to the formation of the curtain.

The thyroid hormone receptor gene (c-erbA alpha) is expressed in advance of thyroid gland maturation during the early embryonic development of Xenopus laevis.

The c-erbA proto-oncogene encodes the thyroid hormone receptor, a ligand-dependent transcription factor which plays an important role in vertebrate growth and development. To define the role of the thyroid hormone receptor in developmental processes, we have begun studying c-erbA gene expression during the ontogeny of Xenopus laevis, an organism in which thyroid hormone has well-documented effects on morphogenesis. Using polymerase chain reactions (PCR) as a sensitive assay of specific gene expression, we found that polyadenylated erbA alpha RNA is present in Xenopus cells at early developmental stages, including the fertilized egg, blastula, gastrula, and neurula. By performing erbA alpha-specific PCR on reverse-transcribed RNAs from high-density sucrose gradient fractions prepared from early-stage embryos, we have demonstrated that these erbA transcripts are recruited to polysomes. Therefore, erbA is expressed in Xenopus development prior to the appearance of the thyroid gland anlage in tailbud-stage embryos. This implies that erbA alpha/thyroid hormone receptors may play ligand-independent roles during the early development of X. laevis. Quantitative PCR revealed a greater than 25-fold range in the steady-state levels of polyadenylated erbA alpha RNA across early stages of development, as expressed relative to equimolar amounts of total embryonic RNA. Substantial increases in the levels of erbA alpha RNA were noted at stages well after the onset of zygotic transcription at the mid-blastula transition, with accumulation of erbA alpha transcripts reaching a relative maximum in advance of metamorphosis. We also show that erbA alpha RNAs are expressed unequally across Xenopus neural tube embryos. This differential expression continues through later stages of development, including metamorphosis. This finding suggests that erbA alpha/thyroid hormone receptors may play roles in tissue-specific processes across all of Xenopus development.

Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis

We have used a quantitative reverse transcription-polymerase chain reaction assay to detect MyoD mRNA during early embryonic development of Xenopus laevis. We find that during a short period of time following the midblastula transition MyoD becomes transcriptionally activated at a low level ubiquitously throughout the embryo. Restriction of MyoD expression to muscle precursor cells appears as a subsequent event, in which the process of mesoderm induction stabilizes transcription only in the marginal zone of the embryo, the presumptive mesoderm.

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84 % homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.

Transcription of endogenous and injected cytoskeletal actin genes during early embryonic development in Xenopus laevis

Abstract The transcriptional regulation of a cytoskeletal actin gene during Xenopus laevis embryonic development has been investigated. New transcripts of this gene begin to accumulate at approximately the mid-gastrula stage, between 12 and 16 hours after fertilization, replenishing maternal supplies of this transcript. To study the molecular processes which act to determine the timing of transcriptional activation of this gene, a gene-injection assay was devised, utilizing a cloned copy of the gene which has been marked by a small DNA insertion. Accurately-initiated transcripts of the injected gene accumulate in concert with those of the endogenous gene, showing that injected genes can undergo developmental regulation. As little as 485 nucleotides of upstream sequence is sufficient for proper temporal control of activation of an injected gene. The results presented here demonstrate the feasibility of a microinjection assay for the identification of regulatory gene sequences and transacting regulatory factors in amphibian embryos. Such an assay will be useful in achieving an understanding of general transcriptional control mechanisms acting in early development, and should also provide a means to study certain aspects of long-standing developmental problems, such as cytoplasmic localization and embryonic induction.

Membrane skeleton protein 4.1 in developing Xenopus: Expression in postmitotic cells of the retina

Membrane skeleton protein 4.1 plays a key role in modulating the interactions of spectrin, actin, and integral membrane proteins in erythroid and nonerythroid cells. We have investigated its structure and expression during embryonic development of Xenopus laevis. An analysis of the complete 2758-nucleotide sequence and predicted translation of 801 amino acids (85.5 kDa) of X. laevis oocyte protein 4.1 reveals that, within overlapping regions, oocyte protein 4.1 is 74% identical to a composite amino acid sequence of human erythroid and lymphoid protein 4.1 and has an identity similar to that of amino acid motifs variably expressed in either human erythroid or lymphoid protein 4.1 S1 nuclease protection analysis demonstrates the presence of a single species of protein 4.1 transcript in embryos. Antibodies produced against X. laevis protein 4.1 fusion protein recognize two bands of 180 and 115 kDa on Western blots of X. laevis embryos and retina and, using immunocytochemical techniques, label the developing retina most intensely. In vitro transcription of a cDNA constrnet fully encoding X. laevis protein 4.1 yields a synthetic mRNA which, when translated in vitro, produces a polypeptide that comigrates on SDS-polyacrylamide gels with the 115-kDa form of embryos and retina. Protein 4.1 is found exclusively in photoreceptors following the terminal mitoses of retinal neurons. When retinal synaptogenesis is complete, protein 4.1 is also expressed in the inner retina. In adult amphibian retinas, protein 4.1 is detected in photoreceptors, bipolar cells, and ganglion cell axons. As these cell types have previously been shown to express spectrin, actin, and ankyrin, it is likely that the membrane skeleton of erythrocytes and retinal cells share functional similarities.

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm.

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.

Mitochondrial gene expression during Xenopus laevis development: a molecular study.

Mitochondrial gene expression has been analysed during embryonic development of Xenopus laevis; the relative amounts of 12S and 16S ribosomal RNAs and of most mitochondrial messenger RNAs were determined by slot-blot and Northern-blot hybridization experiments with specific mitochondrial DNA probes. The rRNA content per embryo remained constant during early development, confirming earlier results of Chase and Dawid (1972, Dev. Biol., 27, 504-518.); on the contrary, all mRNAs decreased abruptly after fertilization within a few hours (by a factor of 5-10), remained at a very low level up to the late neurula stages and increased again during organogenesis. Since the mitochondrial DNA content does not vary during this period molecular analyses as well as biological observations suggest that the mitochondrial genome is completely inactivated at the beginning of embryonic development. The amounts of rRNAs and mRNAs evolve therefore as a function of time only according to their half-lives. Mitochondrial RNA accumulation resumes subsequently with a high rate when the general transcription in the embryo is starting again, and this occurs before the resumption of DNA replication in the organelle. It appears that the Xenopus embryonic development represents a quite clear example of regulation of the mitochondrial expression at the level of transcription.

S-4-4 - Role of cell interaction, in embryonic development of Xenopus laevis studied in a cell culture system with tissue-specific monoclonal antibodies.

S-4-4 - Role of cell interaction, in embryonic development of Xenopus laevis studied in a cell culture system with tissue-specific monoclonal antibodies.

Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis.

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in the expression of alpha-fodrin during embryonic development of Xenopus laevis.

Fodrin (nonerythroid spectrin) and its associated proteins have been previously implicated in the establishment of specialized membrane-cytoskeletal domains in differentiating cells. Using antiserum which is monospecific for the alpha-subunit of fodrin, we demonstrate that alpha-fodrin is present in oocytes and adult tissues of Xenopus laevis. Analyses of the de novo synthesis of alpha-fodrin during embryonic development reveal that alpha-fodrin is synthesized in oocytes, but not during early development. To investigate the level of control of alpha-fodrin expression, we isolated two cDNA clones for oocyte alpha-fodrin. The oocyte cDNA clones were identified as encoding portions of alpha-fodrin based on DNA sequence analysis and on the comparison of the predicted amino acid sequence of the cDNAs with the known sequence of human erythrocyte alpha-spectrin. The Xenopus alpha-fodrin cDNAs hybridize to a transcript of approximately 9 kb on RNA blots, and probably to a single gene type on genomic DNA blots. Both RNA blot analyses and S1 nuclease protection assays with the Xenopus alpha-fodrin cDNAs demonstrate that the observed decline in the de novo synthesis of alpha-fodrin polypeptides is controlled by a dramatic decrease in the abundance of alpha-fodrin transcripts after fertilization. In contrast, levels of actin transcripts do not decrease during this period. Inasmuch as steady-state levels of alpha-fodrin transcripts rise by the neurula stage of development, these results suggest that the synthesis of alpha-fodrin polypeptides during embryonic development of Xenopus is regulated, rather than constitutive, and that the primary level of control is the steady-state abundance of mRNA.

Altered levels of a 5 S gene-specific transcription factor (TFIIIA) during oogenesis and embryonic development of Xenopus laevis.

Xenopus laevis oocytes contain a 38,000-Da protein which serves both as a 5 S gene-specific transcription initiation factor (TFIIIA) and to stabilize 5 S RNA in ribonucleoprotein complexes. Using an antibody to this protein we have measured the levels of TFIIIA during oogenesis and embryonic development in X. laevis. The maximal steady state level (approximately 10(12) molecules/oocyte) is reached early in oogenesis but drops 10- to 20-fold in later stages and another 10- to 20-fold during ovulation. The reduced amount present in the unfertilized egg remains at a nearly constant level throughout early development, but with cell division the cellular concentration drops from 3 X 10(9) to about 10(4) molecules/cell. An immunoreactive protein of the same size is also found in liver tissues and in cultured kidney cells, which also contain about 10(4) molecules/cell. By both structural (CNBr peptide analysis) and functional (transcription of 5 S genes) analyses the embryonic and kidney cell 38,000-Da factors appear indistinguishable from oocyte TFIIIA. In addition a second antigenically related protein of about 40,000 Da is found in late stage embryos, liver tissues, and adult kidney cells (where it is severalfold more abundant than the 38,000-Da TFIIIA). The chromatographic fractionation and functional analysis of the kidney cell extracts has shown that fractions containing the 38,000-Da protein support 5 S RNA synthesis while fractions containing the 40,000-Da protein do not. The significance of these findings for 5 S gene regulation is discussed with respect to the dual function of TFIIIA, the presence of rate-limiting amounts of TFIIIA, and the possibility of stage-specific factors.

Silver positivity of the NORs during embryonic development of Xenopus laevis

Transcriptional activity of ribosomal RNA (rRNA) genes is detectable around blastulagastrula transition during the embryonic development of amphibians and other nonmammalian systems. The silver staining reaction, known to selectively stain transcriptionally active nucleolus organizer regions (NORs) both in interphase and metaphase chromosomes allowed us to follow the activation of the NORs during the embryonic development of Xenopus laevis.

Changes in Activity of Mitochondrial Enzymes during the Development of Xenopus laevis: (Mitochondria/emzymes/embryonic development/Xenopus).

Changes in activity of mitochondrial enzymes were studied during the embryonic development of Xenopus laevis. The following enzymes were determined: malate dehydrogenase (MDH), isocitrate dehydrogenase (NAD+ -dependent) (IDH), aspartate aminotransferase (GOT), cytochrome oxidase (COX), succinate dehydrogenase (SDH), rotenone-insensitive NADH cytochrome c reductase (NADH-red) and monoamine oxidase (MAO). IDH is constant throughout the period studied. COX and SDH, two enzymes of the inner membrane, are constant in pregastrula stages, and subsequently decrease significantly. MDH and NADH-red are highly active in the pregastrula stages and decline thereafter, while MAO is undetectable during early development and increases significantly only in the larvae. GOT increases during the cleavage stages, being most active in the gastrula stages, and decreases subsequently. The results are discussed in the sense of mitochondrial differentiation during the early development of the amphibian embryo.

Changes in tRNA nucleotidyltransferase activity during embryonic development of Xenopus laevis

The level of tRNA nucleotidyltransferase activity was measured in eggs and embryos of Xenopus laevis. The enzyme activity increases after fertilization reaching a maximum level at gastrulation after which there is a dramatic decrease in activity. This decrease in tRNA nucleotidyltransferase activity occurs after tRNA synthesis has begun in the developing embryo. The decrease in the measured enzyme activity does not appear to be due to the presence of stage-specific inhibitors of tRNA nucleotidyltransferase. The results suggest that maturation of precursor tRNA may not be the major role of the enzyme in this system.

LSD: its effect upon 5-hydroxytryptamine in embryonic development of Xenopus laevis.

Nach LSD-Einfluss auf Embryonen vonXenopus laevis zu verschiedenen Entwicklungsstadien wurden langandauernd veranderte 5-HT-Stufen im Gehirn und im ganzen Embryo gefunden. Die LSD-Empfindlichkeitsperioden waren fur das Gehirn und den ganzen Embryo voneinander verschieden.

A study of ribosomes during embryonic development of Xenopus laevis

The ribosomes of Xenopus laevis were studied during embryonic development. The RNA of the ribosomal fraction constituted only 20% of the total RNA. Approx. 70% of the total RNA of eggs is found in the first sediment of centrifugation. If the supernatant containing ribosomes is centrifuged on a sucrose gradient, at a concentration of 10 −3 M Mg 2+ only one peak is detectable. During the embryonic development, the sedimentation constant of ribosomes is approx. 70 S. The base composition of ribosomes reveals the usual high G-C content.