Early Embryos Of Xenopus Laevis Research Articles

Brix from xenopus laevis and brx1p from yeast define a new family of proteins involved in the biogenesis of large ribosomal subunits.

A clone was isolated from a cDNA library from early embryos of Xenopus laevis that codes for a highly charged protein containing 339 amino acids. Two putative nuclear localization signals could be identified in its sequence, but no other known motifs or domains. Closely related ORFs are present in the genomes of man, C. elegans, yeast and Arabidopsis. A fusion protein with GFP expressed in HeLa cells or Xenopus oocytes was found to be localized in the nucleolus and coiled (Cajal) bodies. Moreover, immunoprecipitation experiments demonstrated that the new Xenopus protein interacts with 5S, 5.8S and 28S RNAs of large ribosomal subunits. The name Brix (biogenesis of ribosomes in Xenopus) is proposed for this protein and the corresponding gene. In Saccharomyces cerevisiae, the essential gene YOL077c, now named BRX1, codes for the Brix homolog, which is also localized in the nucleolus. Depletion of Brx1 p in a conditional yeast mutant leads to defects in rRNA processing, and a block in the assembly of large ribosomal subunits.

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos.

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant's layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant's cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell's trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar "embryoid" shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events.

Mitochondrial Gene Expression Is Regulated at the Level of Transcription during Early Embryogenesis of Xenopus laevis

Mitochondrial transcription in the early Xenopus laevis embryo resumes several hours before active mtDNA replication, effectively decoupling mtDNA transcription and replication. This developmental feature makes Xenopus embryogenesis an appealing model system to investigate the regulation of mitochondrial transcription. Studies reported here refine our understanding of the timing, magnitude, and mechanism of this transcriptional induction event. Northern analyses of six mitochondrial mRNAs (normalized to mtDNA) reveal that transcript levels remain basal between fertilization and gastrulation and then undergo a coordinate induction, culminating in a 20-28-fold increase over egg levels by 48 h of development. Measurement of mitochondrial run-on transcription rates demonstrates a good correlation between transcription rates and transcript levels, showing that transcription itself is the primary determinant of transcript abundance. Experimental increases in mitochondrial ATP and energy charge also correlate with patterns of transcript levels and transcription rates, suggesting that developmental changes in the biochemical composition of the mitochondrial matrix could be regulating transcriptional activity. Consistent with this idea, transcriptional run-on rates in mitochondria of early embryos can be stimulated by the addition of tricarboxylic acid cycle intermediates to the run-on reaction. However, mitochondria of later stages do not show this response to the addition of metabolite. In combination, these data suggest that mitochondrial transcription is under metabolic regulation during early Xenopus embryogenesis.

Interaction of EphB2-tyrosine kinase receptor and its ligand conveys dorsalization signal in Xenopus laevis development.

The Eph class is the largest family of receptor tyrosine kinases and has been shown to play various roles in neural development including axon pathfinding and neural crest migration. EphB2 associates with transmembrane ligands Ephrin-B1 and -B2, which leads to tyrosine phosphorylation of both the ligands and receptor and is presumed to regulate cell-to-cell interactions by bidirectional signaling. We have investigated the biological effects of the EphB2-induced signal in the early stage of Xenopus laevis development. Xenopus EphB2 transcripts were detected maternally and were expressed at equal levels between the ventral and dorsal halves of the gastrulae, with expression increasing after the late gastrula stage. EphB2 mRNA expression in dorsal marginal zone explants from gastrulae increases during later development while that in ventral explants does not. We show here that microinjection of RNA encoding EphB2 into a ventral blastomere of embryos induced a partial secondary dorsal axis which consisted of neural tissues, notochord and somites. Analysis with molecular markers verified that the microinjected EphB2 dorsalized the mesoderm of ventral marginal zone explants. These dorsalizing effects of EphB2 in both the whole embryo and ventral explants were inhibited by the coinjection of RNA encoding the soluble form of Ephrin-B1. Furthermore, co-injection of EphB2 and Ephrin-B1 RNAs synergistically enhanced the dorsalization effect. These data show that the interaction between EphB2 and its ligands including Ephrin-B1 causes signaling events which lead to dorsal development, and strongly suggests the existence of proteins which mediate the dorsalization induced by EphB2 in early stage embryos of Xenopus laevis.

Structural and functional analysis of the BMP-4 promoter in early embryos of Xenopus laevis

The Xenopus laevis BMP-4 gene shows an evolutionary conserved structure containing two coding exons and a leader exon. The transcripts which are detected after zygotic activation of the gene in ventral mesoderm of late blastula stage embryos do either contain the leader exon or begin within the first intron. Luciferase reporter/promoter studies revealed multiple elements being required for the activation and for the spatial control of transcription. These elements are located within the upstream region and within the second intron and they interact with a most proximal located basal promoter being indispensable for transcriptional activation. The auto-activatory capacity of BMP-4 is mediated by several enhancer elements being responsive not only to BMP-4 but also to BMP-2 signaling. BMP-2 might thus function as a natural activator of the BMP-4 gene in the early embryo. Since reporter activity obtained with distinct BMP-2/4 responsive promoter deletion mutants is simultaneously inhibited by the dominant negative BMP receptor as well as by chordin, we suggest that down-regulation of the BMP-4 gene by chordin results from an interference with the auto-regulatory loop at the level of protein–protein interactions.

Cloning and developmental regulation of tissue inhibitor of metalloproteinases-3 (TIMP3) in Xenopus laevis early embryos.

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn184, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.

5'-3' Interactions in regulation of translation in Xenopus early embryos

We have investigated the effects of 3' noncoding elements in enhancing translation of messengers having translation-inhibiting 5' untranslated regions (UTRs). The translation of transcripts bearing the 5' UTRs of either human c-myc or a synthetic hairpin structure upstream of a chloramphenicol acetyltransferase (CAT) reporter sequence is greatly attenuated in early embryos of Xenopus laevis. Translation of transcripts bearing the human c-myc-5' UTR was markedly stimulated by the presence of 3' poly(A). Transcripts bearing the 5' hairpin element were insensitive to the presence of poly(A), but they were extremely sensitive to the composition of the 3' UTR. A GC-rich distal sequence repressed translation, whereas a proximal GGAAU sequence promoted translation of these transcripts. Our results support the concept that long-range interactions between the 5' and 3' ends of transcripts are important in regulating translation in Xenopus embryos. Key words: translational regulation, oncogenes, c-myc, Xenopus laevis, development, embryo.

Regulation of replicon size in Xenopus egg extracts.

Once a specific number of cells have been produced in the early Xenopus laevis embryo, replicon size during the S phase of the cell cycle increases. Here, it is reported that similar increase in replicon size occurred when the concentration of nuclei in replication-competent Xenopus egg extracts exceeded a critical threshold. In this system, the origin recognition complex (ORC) did not become stoichiometrically limiting for initiation, and similar amounts of this complex bound to chromatin regardless of replicon size. These data suggest that in early development, an unidentified factor controls how many preformed ORC-DNA complexes initiate DNA replication.

5'-3' Interactions in regulation of translation in <i>Xenopus</i> early embryos

We have investigated the effects of 3' noncoding elements in enhancing translation of messengers having translation-inhibiting 5' untranslated regions (UTRs). The translation of transcripts bearing the 5' UTRs of either human c-myc or a synthetic hairpin structure upstream of a chloramphenicol acetyltransferase (CAT) reporter sequence is greatly attenuated in early embryos of Xenopus laevis. Translation of transcripts bearing the human c-myc-5' UTR was markedly stimulated by the presence of 3' poly(A). Transcripts bearing the 5' hairpin element were insensitive to the presence of poly(A), but they were extremely sensitive to the composition of the 3' UTR. A GC-rich distal sequence repressed translation, whereas a proximal GGAAU sequence promoted translation of these transcripts. Our results support the concept that long-range interactions between the 5' and 3' ends of transcripts are important in regulating translation in Xenopus embryos.

601 Molecular compartmentation expressed in developing cerebellar purkinje cells

Primordial germ cells (PGCs) have been removed from their normal migratory route in early embryos of Xenopus laevis, and their behaviour studied in vitro. They adhere to, and move over the upper surface of, layers of outgrowing cells from expiants of adult Xenopus mesentery. They move by the extrusion of single filopodia, elongation, forward streaming of the yolky cytoplasm and retraction of their trailing ends. When the underlying cells are polarized in one direction only, PGCs always elongate and move along the same direction. Furthermore, when PGCs elongate and move over less obviously polarized cells, they always do so in the direction of ‘stress fibres’ (actin bundles) in the underlying cells. A substrate-guidance hypothesis for PGC migration is only tenable if there is some orientation in their natural substrate in vivo. Using the scanning electron microscope, we demonstrate that the coelpmic lining cells, beneath which PGCs migrate up the dorsal mesentery of the gut, are orientated in the direction of travel. Furthermore, this orientation changes at the time of gonadal ridge formation. This raises the intriguing possibility that PGCs are guided for at least part of their migration in Xenopus laevis embryos by a substrate-guidance mechanism.

Transient effects of microgravity on early embryos of Xenopus laevis

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity (μg) during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and μg. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in μg and on earth. Analysis of the video tapes revealed that the cortical contraction in μg starts earlier than at 1 g on earth. After recovery of the eggs fertilized in μg and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the μg embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

Analysis of tissue organization by microinjection of follistatin into early Xenopus laevis embryos

Xenopus laevis embryos injected with follistatin developed abnormally with enlarged anterior structures and defective tails. Histological sections reveal that these follistatin treated embryos contain large amount of notochordal tissue that fails to elongate. Embryonic responses to follistatin are dose and stage specific. These results suggested that follistatin contributes to body axis formation, with its most visible effect on notochord formation.

Developmental hazard assessment with FETAX: Aerobic metabolites in bacterial transformation of naphthalene

The underlying principle of bioremediation is the capability of microorganisms to biodegrade pollutants. When a contaminated site is biotreated, it is usually assumed that the disappearance of the pollutant means a reduction in the toxic effects of the contaminants. However, pollutants can undergo partial biodegradation or biotransformation. Microbial-mediated transformations play a critical role in the toxic effects of pollutants, as any alteration in structure can result in a change in physicochemical properties which influence toxicity. Therefore, a relevant question is; what is the toxicity of accumulative metabolites relative to the parent chemical? One class of chemicals that consistently appears at Superfund hazard waste sites is aromatic hydrocarbons. Studies of the aerobic bacterial metabolism of representative compounds, including benzene, naphthalene, and phenanthrene, have revealed similar oxidative pathways. Bacterial degradation of these aromatic hydrocarbons was initiated by the addition of two molecules of oxygen via a dioxygenase enzyme, with the resulting intermediate being converted to a catechol-like compound. From a biotransformation standpoint, one of the more thoroughly studied aromatic hydrocarbons has been naphthalene. Cerniglia (1984) has identified five major intermediates, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylic acid, gentisic acid and catechol in the aerobic bacterial degradation of naphthalene. In vitro test systems such as themore » Frog Embryo Teratogenesis Assay - Xenopus (FETAX) provide a time- and resource-effective means for assessing developmental toxicity on a preliminary basis. FETAX is a 96-hr static-renewal system that uses early embryos of the frog Xenopus laevis. The purpose of this investigation was to determine the developmental hazard, using FETAX, of exposure to the model aromatic hydrocarbon, naphthalene, and it`s known major aerobic metabolites from bacterial transformation. 18 refs., 2 tabs.« less

Expression Patterns of Hoxb Genes in the Xenopus Embryo Suggest Roles in Anteroposterior Specification of the Hindbrain and in Dorsoventral Patterning of the Mesoderm

Hox genes are thought to participate in patterning the anteroposterior (a-p) axis during vertebrate embryogenesis. In this investigation, the spatial expression of six Hoxb genes was analyzed in early embryos of Xenopus laevis by in situ hybridization. Hoxb gene expression was first detected in late gastrulae/early neurulae, by which stage, the characteristic spatially colinear Hoxb gene expression sequence was already apparent. Dissection experiments indicated that the establishment of these localized expression patterns coincides with the acquisition of anteroposterior positional information along the main body axis. The Hoxb genes continued to be expressed in similar domains along the anteroposterior axis at all developmental stages examined, although there were some changes in expression at the cellular level. Interestingly, the 3′ genes, Hoxb-1, Hoxb-3, and Hoxb-4 were expressed in very restricted domains in the future hindbrain, while Hoxb-5, Hoxb-7, and Hoxb-9 transcripts were present along the entire presumptive spinal cord. It was thus notable that the 5′ Hoxb genes exhibited different types of expression domain than the 3′ Hoxb genes. These observations suggest that there may be different mechanisms regulating the expression of the 3′ and 5′ Hoxb genes. Expression of all of the Hoxb genes analyzed, except Hoxb-4, was predominantly detectable in the central nervous system or in neural crest-derived structures. Hoxb-4 mRNA was detected in the central nervous system, but interestingly, the major expression site for this gene was the somites. The other Hoxb genes tested failed to show significant expression in the somitic mesoderm, although transcripts from genes 5′ from Hoxb-4 were detected in other mesodermal tissues. In the vertebrate trunk, anteroposterior patterning of the CNS is thought to be regulated by the somites. The results obtained here for Xenopus embryos did not explicitly support the idea of a Hoxb code for the somites, although we cannot rule this out. Instead, interestingly, the data were consistent with a role for Hoxb genes in dorsoventral patterning of the mesoderm.

Induction of mesoderm in Xenopus laevis embryos by translation initiation factor 4E.

The microinjection of messenger RNA encoding the eukaryotic translation initiation factor 4E (eIF-4E) into early embryos of Xenopus laevis leads to the induction of mesoderm in ectodermal explants. This induction occurs without a stimulation of overall protein synthesis and is blocked by the co-expression of a dominant negative mutant of the proto-oncogene ras or a truncated activin type II receptor. Although other translation factors have been studied in vertebrate and invertebrate embryos, none have been shown to play a direct role in development. The results here suggest a mechanism for relaying and amplifying signals for mesoderm induction.

Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis: (ureotelism/urea concentration in frog blood/culture of frog embryos/mesoderm induction).

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus.

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations.

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Höger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.

Evaluation of the developmental toxicity of trichloroethylene and detoxification metabolites using Xenopus.

Potential mechanisms of trichloroethylene-induced developmental toxicity were evaluated using FETAX (Frog Embryo Teratogenesis Assay--Xenopus). Early Xenopus laevis embryos were exposed to trichloroethylene for 96 h in two separate definitive concentration-response assays with and without an exogenous metabolic activation system (MAS) and inhibited MAS. The MAS was treated with either carbon monoxide or cyclohexene oxide to modulate mixed-function oxidase (MFO) or epoxide hydrolase activity, respectively. Trichloroethylene metabolites: dichloroacetic acid, trichloroacetic acid, trichloroethanol, and oxalic acid were also evaluated in two separate definitive, static renewal tests. Addition of the MAS decreased the 96 h LC50 and EC50 (malformation) of trichloroethylene 1.8-fold and 3.8-fold, respectively. Addition of the carbon monoxide inhibited MAS decreased the developmental toxicity of activated trichloroethylene to levels approximating that of the parent compound. Cyclohexene oxide-inhibited MAS substantially increased the developmental toxicity of trichloroethylene. In addition, each of the metabolites tested were significantly less developmental toxic than the parent compound, trichloroethylene. Results indicate that a highly embryotoxic epoxide intermediate, trichloroethylene oxide, formed as the results of MFO mediated metabolism may play a significant role in the developmental toxicity of trichloroethylene in vitro.

Expression and distribution of SPARC in early Xenopus laevis embryos

SPARC (Secreted Protein, Acidic, Rich in Cysteine) is a highly conserved, calcium-binding, extracellular matrix protein. To investigate its role in early embryogenesis, we examined its tissue distribution in early Xenopus embryos. SPARC mRNA transcripts were detectable by Northern blot analysis beginning at the early neurula stage. SPARC transcripts then rapidly accumulated, reaching their highest embryonic level at the tailbud stage. The levels of SPARC mRNA were similar in dorsalized (LiCl-treated) and ventralized (UV-treated) embryos, indicating embryonic expression of SPARC mRNA was not obviously altered by changes in pattern formation. Immunofluorescence staining showed SPARC was an abundant protein in tissues of mesodermal and ectodermal origin in tailbud embryos, with the exception of the notochord. Of particular interest was the distribution of SPARC within the intersomitic clefts during somitogenesis. The widespread distribution of SPARC in early embryogenesis suggests it may play a role in the formation of a variety of tissues.

Identification of plakoglobin in oocytes and early embryos of Xenopus laevis: maternal expression of a gene encoding a junctional plaque protein

Abstract We have isolated a cDNA encoding the junctional plaque protein plakoglobin of Xenopus laevis and determined its amino acid sequence. Comparisons with sequences of related proteins of the same and other species revealed that in Xenopus plakoglobin and β-catenin are two different proteins, encoded by separate genes, that both genes are expressed in embryogenesis, and that the amphibian plakoglobin is more closely related to the human plakoglobin than to β-catenin of the same species. Using this cDNA as a probe, we also show that plakoglobin mRNA is produced and stored in Xenopus oocytes and eggs. We discuss the possibility that the maternal pool of this junctional protein contributes to the junctional structures connecting the oocyte with the follicle epithelium and to the rapid formation of desmosomes and other plaque-bearing junctions in pregastrulation embryogenesis.