Connexin43 expression during Xenopus development.

The spatio-temporal expression of connexin43 in Xenopus laevis embryos was studied by in situ hybridization. Cx43 expression is first detected at stage 25 in the developing eye. In stage 32, expression was found in the margin of the lens placode, the cement gland, notochord, and in stage 37 in the branchial arches. Early limb buds show strong expression of Cx43 distally while later on expression is confined to sites of precartilage condensation.

Regulated expression of the X. tropicalis connexin43 promoter.

The spatio-temporal expression pattern of the connexin43 gene during Xenopus development has been described (Van der Heyden et al. 2001). To further investigate the regulation and function of connexin43 (Cx43) in amphibians, we have isolated the gene from Xenopus tropicalis (Xt) and determined its structure. The X. tropicalis Cx43 gene displays the typical two exon-one intron connexin configuration, where the first exon is non-coding. The predicted amino acid sequence of the XtCx43 protein is highly homologous to that of X. laevis, chicken and mammals. Expression of XtCx43 cDNA in N2A cells results in gap-junction plaque formation. Promoter activity of a 3.5 kb upstream region of the X. tropicalis Cx43 gene, including exon 1, mimics endogenous timing of expression after injection of reporter constructs in X. laevis embryos.

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells.

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

Translation of maternal TATA-binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition.

Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is translation of maternally stored TBP RNA. We show that TBP is rate-limiting in egg extract under conditions that titrate nucleosome assembly. Precocious translation of TBP mRNA in Xenopus embryos facilitates transcription before the MBT, without requiring TBP to be prebound to the promoter before injection. This effect is transient in the absence of chromatin titration and is sustained when chromatin is titrated. These data show that translational regulation of TBP RNA contributes to limitations on the transcriptional capacity before the MBT. Second, we examined the ability of trans-acting factors to contribute to promoter activity before the MBT. Deletion of cis-acting elements does not affect histone H2B transcription in egg extract, a finding indicative of limited trans-activation. Moreover, in the context of the intact promoter, neither the transcriptional activator Oct-1, nor TBP, nor TFIID enable transcriptional activation in vitro. HeLa cell extract, however, reconstitutes activated transcription in mixed extracts. These data suggest a deficiency in egg extract cofactors required for activated transcription. We show that the capacity for activated H2B transcription is gradually acquired at the early gastrula transition. This transition occurs well after the blastula stage when the basal transcription machinery can first be complemented with TBP.

The Oct-1 POU domain directs developmentally regulated nuclear translocation in Xenopus embryos.

Early embryonic development in Xenopus is characterized by transcriptional repression which is relieved at the mid-blastula stage. Here we show that most of the maternally inherited POU domain transcription factor Oct-1 is retained in the cytoplasm during early development, and that it gradually translocates to the nucleus around the mid-blastula transition. Overexpressed epitope-tagged Oct-1 exhibits highly similar localization properties compared to endogenous protein. The amino acid sequence that directs this developmentally regulated nuclear translocation resides in the POU domain. Our findings may suggest that cytoplasmic retention of Oct-1 facilitates or contributes to the repression of Oct-1 target genes before the mid-blastula transition.

Analysis of HIV-1 Tat effects in Xenopus laevis embryos.

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo. We adopted the Xenopus system since expression of putative regulatory factors in the embryo has been widely used as a quick and effective first screen for protein function. Xenopus' early development is well characterized by stage-specific phenotypes, therefore, an in vivo HIV-1 Tat-mediated aberrant phenotype can easily be detected and analyzed. HIV-1 Tat protein expression through injection of synthetic mRNA into zygotes produced a marked delay in gastrulation leading to altered specification of the anterior-posterior axis and to partial or total loss of anterior structures. HIV-1 Tat effects resulted in a general suppression of gene expression, including that of Xbra and gsc, two early genes whose expression is required for proper gastrulation. The specificity of Tat effects was demonstrated by injecting a 'loss of function' mutant (Tat-C37S), lacking a single cysteine residue, which did not yield any effect. Both Tat and Tat-C37S were found to be localized mainly in the nucleus. The importance of subcellular targeting for the effects caused by HIV-1 Tat was demonstrated by injecting a second mutant (Tat-BDM), carrying an altered nuclear localization signal sequence. The Tat-BDM protein localized in the cytoplasm and accumulated at the cell membrane. Embryos injected with Tat-BDM mRNA did not develop beyond gastrulation. The importance of proper protein conformation and subcellular localization in determining Tat effects is discussed.

Identification of connexin43 as a functional target for Wnt signalling.

Wnt mediated signal transduction is considered to regulate activity of target genes. In Xenopus embryos, ectopic Wnt1 and Wnt8 expression induces gap-junctional communication. During murine brain formation, Wnt1 and the gap-junctional protein connexin43 (Cx43) are co-expressed at the mid/hindbrain border, while interference with Wnt1 or Cx43 expression during embryogenesis leads to severe brain defects in the mid/hindbrain region. In PC12 cells, Wnt1 expression leads to an apparent increase in cell-cell adhesion. We investigated the effects of Wnt1 overexpression on gap-junctional communication in PC12 cells. Wnt1 expressing clones displayed an increased electrical and chemical coupling. This coincides with an increased expression of Cx43 mRNA and protein, while other connexins, Cx26, Cx32, Cx37, Cx40 and Cx45, were not up-regulated. Also, induction of Wnt1 expression in a mammary epithelial cell line leads to an increase in gap-junctional communication and Cx43 protein expression. In transient transactivation assays in P19 EC cells we found that Wnt1 and Li+, an ion that mimics Wnt signalling, increased transcription from the rat Cx43 promoter, potentially via TCF/LEF binding elements, in a pathway separate from cAMP-induced Cx43 transactivation. The results demonstrate that Cx43 acts as a functional target of Wnt1 signalling, and Cx43 expression can be regulated by Wnt1 at the transcriptional level. Our data suggest that Wnt1-induced cell fate determination is likely to involve regulation of gap-junctional communication.

Non-cell autonomous induction of apoptosis and loss of posterior structures by activation domain-specific interactions of Oct-1 in the Xenopus embryo.

Oct-1, a member of the POU family of transcription factors, is expressed at relatively high levels in ectodermal and mesodermal cell lineages during early Xenopus embryogenesis (Veenstra et al, 1995). Here we show that overexpression of Oct-1 induces programmed cell death concomitant with the loss of the posterior part of the body axis. Truncated Oct-1 variants, missing either the C-terminal or N-terminal trans-activation domain, exhibit a different capacity to cause such developmental defects. Oct-1-induced cell death is rescued in unilaterally injected embryos by non-injected cells, indicative of the non-cell autonomous character of the developmental effects of Oct-1. This was confirmed by marker gene analysis, which showed a significant decrease in brachyury expression, suggesting that Oct-1 interferes with an FGF-type signalling pathway.

Plasmid-mediated gene transfer in neurons using the biolistics technique.

Biolistics has been developed as a system for gene delivery into plant cells, but has recently been introduced for transfection into mammalian tissue, including few attempts in neural cells. Basically, in this system the plasmid DNA of interest is coated onto small particles, that are accelerated by a particular driving force. The combination of several so-called 'ballistic' parameters and tissue parameters determine the transfection efficiency. The main advantage of the system is that it is, unlike other available transfection methods, a mechanical way to cross the plasma membrane and therefore less dependent on target cell characteristics. In terms of transfection efficiency, biolistics seems favorable above conventional techniques, like calcium phosphate precipitation and lipofection. Compared to viral techniques biolistics may be less efficient, but is quicker and easier to handle and seems to produce fewer complications for in vivo gene delivery. Therefore, although the technique is only in a developmental stage, preliminary results seem promising, and optimalization of the method may prove useful in scientific research and/or clinical use.

B-50/growth-associated protein-43, a marker of neural development in Xenopus laevis.

To study the regulation and function of the growth-associated protein B-50/growth-associated protein-43 (mol. wt 43,000) in Xenopus laevis, B-50/growth-associated protein-43 complementary DNAs were isolated and characterized. The deduced amino acid sequence revealed potential functional domains of Xenopus B-50/growth-associated protein-43 that may be involved in G-protein interaction, membrane-binding, calmodulin-binding and protein kinase C phosphorylation. The expression of B-50/growth-associated protein-43 at the RNA and protein level during development was investigated using the Xenopus complementary DNA and the monoclonal B-50/growth-associated protein-43 antibody NM2. The antibody NM2 recognized the gene product on western blot and in whole-mount immunocytochemistry of Xenopus embryos. Moreover, visualization of the developmentally regulated appearance of B-50/growth-associated protein-43 immunoreactivity showed that this mode of detection may be used to monitor axonogenesis under various experimental conditions. In the adult Xenopus, XB-50/growth-associated protein-43 messenger RNA was shown to be expressed at high levels in brain, spinal cord and eye using northern blotting. The earliest expression detected on northern blot was at developmental stage 13 with poly(A) RNA. By whole-mount immunofluorescence, applying the confocal laser scanning microscope, the protein was first detected in embryos from stage 20, where it was expressed in the developing trigeminal ganglion. Also later in development the expression of the B-50/growth-associated protein-43 gene was restricted to the nervous system in Xenopus, as was previously found for the mouse. In conclusion, we find that XB-50/growth-associated protein-43 is a good marker to study the development of the nervous system in Xenopus laevis.

Extralenticular expression of Xenopus laevis alpha-, beta-, and gamma-crystallin genes.

PURPOSE: Extralenticular expression of alpha- and beta-crystallin genes has been demonstrated in mammals and expression of gamma-crystallin genes has been shown in Xenopus laevis. To determine a possible correlation between lens determination and crystallin gene expression, the site of expression of (a member of) the alpha-, beta-, and gamma-crystallin gene families was observed before and during lens formation in X. laevis. METHODS: The partial complementary DNAs (cDNAs) of alpha A- and beta A4-crystallin and a gamma-crystallin were cloned from an X. laevis lens cDNA library. The corresponding antisense RNAs were used to analyze the expression of these genes during X. laevis development by wholemount in situ hybridization. RESULTS: Expression of the beta A4- and gamma-crystallin (but not alpha-crystallin) genes could first be detected in the animal cap of the X. laevis gastrula. The beta A4- and gamma-crystallin messengers were also found in the first stage of lens development, when the ectodermal tissue overlying the optic vesicle thickens to form the lens placode. alpha A-crystallin messenger RNAs were only detectable when the lens epithelial cells were formed. CONCLUSIONS: In contrast to observations in most vertebrates, expression of the beta A4- and gamma-crystallin genes was observed to precede that of the alpha A-crystallin gene during lens development of X. laevis, reflecting the determination that in amphibians, the (presumptive) fiber cells are formed before the epithelial cells, whereas in vertebrates, the order is reversed. Expression of beta A4- and gamma-crystallin genes in the ectodermal tissue of the X. laevis gastrula shows that these genes are expressed when this tissue gains competence for lens formation.

Regulation of the zebrafish goosecoid promoter by mesoderm inducing factors and Xwnt1.

Goosecoid is a homeobox gene that is expressed as an immediate early response to mesoderm induction by activin. We have investigated the induction of the zebrafish goosecoid promoter by the mesoderm inducing factors activin and basic fibroblast growth factor (bFGF) in dissociated zebrafish blastula cells, as well as by different wnts in intact embryos. Activin induces promoter activity, while bFGF shows a cooperative effect with activin. We have identified two enhancer elements that are functional in the induction of the goosecoid promoter. A distal element confers activin responsiveness to a heterologous promoter in the absence of de novo protein synthesis, whereas a proximal element responds only to a combination of activin and bFGF. Deletion experiments show that both elements are important for full induction by activin. Nuclear proteins that bind to these elements are expressed in blastula embryos, and competition experiments show that an octamer site in the activin responsive distal element is specifically bound, suggesting a role for an octamer binding factor in the regulation of goosecoid expression by activin. Experiments in intact embryos reveal that the proximal element contains sequences that respond to Xwnt1, but not to Xwnt5c. Furthermore, we show that the distal element is active in a confined dorsal domain in embryos and responds to overexpression of activin in vivo, as well as to dorsalization by lithium. The distal element is to our knowledge the first enhancer element identified that mediates the induction of a mesodermal gene by activin.

Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B.

COUP-TFs (Chicken Ovalbumin Upstream Promoter Transcription Factors) have been proposed to be negative regulators of retinoid receptor-mediated transcriptional activation. In a previous paper we reported the cloning of a Xenopus (x) COUP-TF (Matharu, P.J. and Sweeney, G.E. (1992) Biochim. Biophys. Acta 1129, 331-334). Here we describe the cloning of a second xCOUP-TF. Amino acid sequence comparison between these two Xenopus COUP-TFs revealed a high level of similarity. Extensive amino acid sequence conservation was found among all Drosophila, Xenopus, zebrafish and mammalian COUP-TF genes examined. Phylogenetic tree analyses indicate that the vertebrate COUP-TFs fall into three classes. The two Xenopus COUP-TF genes show similar temporal expression patterns: both are expressed from the end of gastrulation. In situ hybridization studies reveal complex expression patterns in the developing central nervous system (CNS), besides expression in the eye and in some mesodermal tissues. Retinoic acid (RA) treatment enhances xCOUP-TF-A expression in neurula stage embryos, whereas the expression of xCOUP-TF-B is inhibited during the same developmental period. The strictly conserved amino acid sequences and the strong similarities between the expression patterns of the two different xCOUP-TFs on the one hand, and other vertebrate COUP-TF homologues on the other, make it likely that COUP-TFs have a conserved role in patterning the nervous system.

Analysis of Wnt/Engrailed signaling in Xenopus embryos using biolistics.

We adapted the Biolistics Particle Delivery System for the introduction of DNA into Xenopus embryos, allowing us to modulate the expression of different genes at specific time points during development. In the present study we applied the Biolistics method to the study of the wnt-engrailed signaling cascade in the developing Xenopus embryo. We show that ectopic expression of Xwnt-1 and Xwnt-5C is sufficient to activate specifically XEn-1 and not XEn-2.

Dynamic and differential Oct-1 expression during early Xenopus embryogenesis: persistence of Oct-1 protein following down-regulation of the RNA.

As a first step towards the elucidation of the role of the transcription factor Oct-1 in development, we prepared a monoclonal antibody to study the spatio-temporal distribution of Oct-1 protein in vivo. Here we report differential expression of the Oct-1 gene in the Xenopus embryo both at the RNA and the protein level. Transcripts and protein are detected in ectodermal and mesodermal cell lineages, in which the expression exhibits a pattern of progressive spatial restriction in the course of development. The Oct-1 expression as reported here is not correlated with cell density or cell proliferation in the embryo. Our results suggest a role of Oct-1 in the specification and differentiation of neuronal and neural crest cells. In many other cells, the developmental decision to down regulate Oct-1 is delayed, probably due to a high stability of the protein.

Comparative analysis of Engrailed-1 and Wnt-1 expression in the developing central nervous system of Xenopus laevis.

Expression of the Engrailed-1 (XEn-1) gene was studied in Xenopus embryogenesis by Northern blot analysis and whole-mount in situ hybridization. One transcript of 2.2 kb was detected from stage 17 (midneurula) onwards, until stage 47 (swimming tadpole). The expression pattern of the XEn-1 gene as revealed by in situ hybridization can be divided in three regions. The first domain of transient expression appears at the midneurula stage (st. 17) in the anterior part of the neural fold, forming a complete ring of positive cells at the mid/hindbrain border after neural tube closure. A second region of transient expression is detected as groups of ventro-lateral cells in the spinal cord and the hindbrain from late-neurula till tadpole stages. A third area of transient expression of XEn-1 is formed by the anterior part of the developing pronephros. Comparison of XEn-1 expression at the mid/hindbrain border with that of the Xenopus wnt-1 and engrailed-2 genes reveals that XEn-1 and Xwnt-1, in contrast to XEn-2, are both detected in a narrow stripe of positive cells in this region. Analysis in exogastrulated embryos reveals that expression of XEn-1 and Xwnt-1, but not XEn-2, is induced by planar signaling in the presumptive midbrain. Of the three genes only XEn-1 is expressed in the floorplate at the mid/hindbrain border, while Xwnt-1 is expressed in adjacent cells in the neural ectoderm. The results suggest that in vertebrates at the interface between cells in the floorplate and in the paraxial neuroectoderm, at the limited region of the mid/hindbrain border, En-1 interacts with wnt-1 in a signaling pathway analogous to the engrailed/wingless signaling in the parasegments of the Drosophila embryo.

Expression of the glucocorticoid receptor gene is regulated during early embryogenesis of Xenopus laevis.

To study the possible role of the glucocorticoid receptor (GR) in early embryogenesis, we isolated a Xenopus glucocorticoid receptor cDNA from an embryonic stage 17 cDNA library. Overexpression of this Xenopus GR in COS cells confers the ability to transactivate a GRE-tk CAT promoter construct in a ligand dependent manner. Expression of the Xenopus GR gene at the RNA level was analyzed by Northern blot hybridization. Transcripts of 4 and 6 kb are present in oocytes. The 4 kb mRNA is abundant and is degraded together with the 6 kb mRNA during cleavage stages of early development. Between stages 17 and 24, GR messengers are extremely rare. From stage 32 onwards, both GR transcripts start to be expressed again at intermediate levels. These results provide the first evidence that expression of the GR gene is regulated during early embryonic development.