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.

Characterization of a functional promoter for the Xenopus wnt-1 gene on vivo.

The Xenopus homolog of the proto-oncogene wnt-1 (int-1) is transiently expressed during neurula and tailbud stages of early development. To determine the mechanisms involved in the transcriptional regulation of the Xwnt-1 gene, we isolated Xwnt-1 genomic sequences. The promoter activity of the 5' flanking region of the gene was analysed by microinjection of chimeric luciferase reporter constructs into embryos. It is shown that the proximal 220 bp of the Xwnt-1 promoter is able to confer activation of the reporter gene at the early neurula stage. DNaseI footprinting analysis of the proximal promoter region with nuclear protein extracts from neurula stage embryos revealed five protein binding regions. Deletion and mutation analysis shows that one of these five protein binding regions is essential for promoter activity. Gel shift experiments indicate that a sequence element resembling the GT-I and GT-II motifs of the SV40 enhancer is important for the Xwnt-1 promoter activity in vivo.

Histones of Xenopus laevis erythrocytes. Purification and characterization of the lysine-rich fractions.

Lysine-rich histones have been isolated from the terminally differentiated erythrocytes of Xenopus laevis. Three major proteins have been separated by ion-exchange chromatography. These proteins have been characterized by electrophoresis, amino acid analysis and immunochemical techniques. It is concluded that two 'typical' lysine-rich subfractions are present in Xenopus erythrocytes and, in addition, a serine-rich histone, that shares no common antigenic determinants with the other lysine-rich histones.

Cytoplasmic origin of the so-called nuclear neutral histone protease.

1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.

Organization of Xenopus histone gene variants within clusters and their transcriptional expression.

Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene containing clones identified, 36 were selected as possibly containing H1 histone genes by hybridization to a probe derived from a sea urchin H1 histone gene. These 36 clones were further analyzed by hybrid-selected translation for the definitive presence of H1 histone genes. The genes for three different H1 histone variants were found: H1A , H1B and H1C . Mapping of the histone genes within each clone showed that at least three different gene arrangements can occur within a cluster and that the type of H1 histone variant present in a cluster may be related to the cluster type. S1-mapping experiments indicated that histone genes found in different cluster-types can be expressed in oocytes. Also, the H1 gene found in one cluster-type was expressed in at least three different cell-types: oocytes, gastrula-stage embryos, and erythroblasts.

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.

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.

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.

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.

The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme.

The murine S8 gene, originally identified by Kongsuwan et al. [EMBO J. 7(1988)2131-2138] encodes a homeodomain which resembles those of the paired family. We studied the expression pattern during mid-gestation embryogenesis of S8 by in situ hybridization. Expression was detected locally in craniofacial mesenchyme, in the limb, the heart and the somites and sclerotomes all along the axis, and was absent from the central and peripheral nervous system, splanchnopleure, and endodermal derivatives. This pattern differs considerably from that of most previously described homeobox containing genes. By genetic analysis, the gene was located on chromosome 2, about 20 cM from the HOX-4 cluster.

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.

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.

Histone genes from Xenopus laevis: molecular cloning and initial characterization.

Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris. The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E. coli plasmid pCR1. A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA. A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII. The fragment was not cleaved by KpnI, AvaI, SalI and HindIII. Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA. The gene order was found to be H3, H4, H2A and H2B. The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence. Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.

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.

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.

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.