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.     

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.     

Molecular changes associated with tuberisation in Solanum tuberosum. Differential expression of α-tubulin isotypes

Changes in gene expression that occur in the stolon tips of potato ( Solanum tuberosum L.) cv. Record during tuberisation were investigated. Protein extracts from stolon tips at various stages in the tuberisation process were analysed by two-dimensional gel electrophoresis. A number of quantitative and qualitative changes in polypeptide composition accompanied the very early stages of tuberisation. In vitro translation of RNA extracted from stolon tips also revealed quantitative and qualitative changes associated with tuberisation. Immunoblotting of protein extracts with monoclonal antibodies raised against α- and β-tubulin showed quantitative changes in the relative level of β-tubulin, but not α-tubulin, as the stolon tips tuberised. Changes in the pattern of α-tubulin isotype expression were shown to occur at early stages in the tuberisation process.     

Gene Expression from Endogenou and Exogenously-introduced DNAs in Early Embryogenesis of Xenopus laevis

In Xenopus laevis embryogenesis, gene expression from endogenous and exogenously-introduced DNAs is reported to start only after 12 rounds of cleavage, at the stage called midblastula transition (MBT). In isotopic labeling experiments, however, we found that the synthesis of heterogeneous mRNA-like RNA occurs from the early cleavage stage, and that gene expression from zygotic genomes occurs sequentially in three characteristically different phases: the pre-MBT phase, in which heterogeneous mRNA-like RNA and small amounts of small-molecular-weight RNAs are synthesized; the MBT phase, in which there is a large increase in 4S RNA synthesis, and the post-MBT phase, in which rRNA is also synthesized. Moreover in our studies on the expression of exogenously introduced DNA, we found that circular forms of bacterial chloramphenicol acetyltransferase (CAT) genes connected to viral promoters were expressed from the early cleavage stage, whereas circular forms of genes connected to Xenopus cardiac α-actin promoter were expressed only after the embryos reached the neurula stage, when the endogenous α-actin gene started to be expressed. We, therefore, conclude that in Xenopus embryogenesis, DNA-dependent RNA polymerases II, III and I are activated in this order, and that the promoter, not changes associated with the MBT, probably determine the timing of gene expression.     

Inducible gene expression in transgenic Xenopus embryos

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.     

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.     

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

A Database of microRNA Expression Patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

Assembly of tight junctions during early vertebrate development

Tight junction formation during development is critical for embryonic patterning and organization. We consider mechanisms of junction biogenesis in cleaving mouse and Xenopus eggs. Junction assembly follows the establishment of cell polarity at 8-cell (mouse) or 2-cell (Xenopus) stages, characterized by sequential membrane delivery of constituents, coordinated by embryonic (mouse) or maternal (Xenopus) expression programmes. Cadherin adhesion is permissive for tight junction construction only in the mouse. Occludin post-translational modification and membrane delivery, mediated by delayed ZO-1 alpha(+)isoform expression in the mouse, provides a mechanism for completion of tight junction biogenesis and sealing, regulating the timing of blastocoel cavitation.