Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis.
ABSTRACT: We have isolated a cDNA clone encoding the Xenopus homologue of the transcription factor AP-2 (XAP-2). The predicted amino acid sequence derived from the Xenopus cDNA shows very strong conservation with the amino acid sequence of human AP-2, suggesting that this protein is evolutionarily conserved, at least among vertebrates. This is further substantiated by the demonstration that an in vitro translation product of XAP-2 cDNA bound specifically to an AP-2 binding site from the human MT-IIA gene. Northern blot analysis of Xenopus embryo RNA revealed the existence of three major XAP-2 mRNA species that were only detectable after the midblastula transition (when embryonic transcription is activated), with peak accumulation of the transcripts occurring during gastrulation. Therefore, in contrast to other Xenopus transcription factors, XAP-2 is not maternally derived but arises exclusively from zygotic transcription. Unlike the situation in cultured human teratocarcinoma (NT2) cells, retinoic acid treatment did not induce XAP-2 mRNA in Xenopus embryos, even though the treatment had a pronounced morphogenetic effect on the embryos. Our results suggest that XAP-2 may play a distinctive role during Xenopus embryogenesis.
Project description:The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.
Project description:The transforming growth factor beta (TGF-beta)-related products of the Xenopus Vg-1 and Drosophila decapentaplegic (DPP) genes have been implicated in the control of growth and differentiation during embryogenesis. We have isolated a mouse cDNA, Vgr-1, that encodes a polypeptide structurally related to Xenopus Vg-1. Sequence comparisons indicate that the Vgr-1 protein belongs to a family of DPP-like gene products within the TGF-beta superfamily. The levels of Vgr-1 RNA were determined in embryos and tissues isolated at various stages of development. A 3.5-kilobase mRNA increases throughout development and into adulthood in many tissues and in F9 teratocarcinoma cells differentiating into endoderm in response to retinoic acid and cAMP. The amino acid homologies and patterns of expression suggest that, like the DPP gene product, Vgr-1 plays a role at various stages of development.
Project description:A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.
Project description:In Xenopus embryos, maternal cyclins drive the first 12 cell divisions after which several cyclins are terminally degraded, including cyclin B2. Cyclin B2 disappearance is due to transcription-mediated mRNA deadenylation at the midblastula transition, when transcription initiates and the cell cycle lengthens. To further define the mechanism, we characterized proteins capable of binding cyclin B2 3'UTR. We show that ElrA and AUF1 compete for binding to regions containing cytoplasmic polyadenylation elements (CPEs), with AUF1 binding increasing at the midblastula transition. Deletion of both CPEs abrogates polyadenylation but has no effect on deadenylation or binding of ElrA or AUF1. Overexpression of ElrA or AUF1 does not alter cyclin B2 mRNA stability. These results show that ElrA and AUF1 bind to cyclin B2 mRNA independent of CPEs and function by binding other elements.
Project description:Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos.
Project description:During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.
Project description:While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.
Project description:Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells.We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time.These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.
Project description:In Xenopus laevis zygotic transcription begins at the midblastula transition (MBT). Prior to this the genome is organized into chromatin that facilitates rapid cycles of DNA replication but not transcription. Here we demonstrate that DNA methylation contributes to the overall transcriptional silencing before MBT. Transient depletion of the maternal DNA methyltransferase (xDnmt1) by anti sense RNA during cleavage stages is associated with a decrease in the genomic 5-methyl-cytosine content and leads to the activation of zygotic transcription approximately two cell cycles earlier than normal. Hypomethylation allows the early expression of mesodermal marker genes such as Xbra, Cerberus, and Otx2, which are subsequently down-regulated during gastrulation of the xDnmt1-depleted embryos. The temporal switch in gene expression may account for the appearance of body plan defects that we observe. Loss of xDnmt1 can be rescued by the coinjection of mouse or human Dnmt1 protein. These results demonstrate that DNA methylation has a role in the regulation of immediately early genes in Xenopus at MBT.
Project description:Fertilization of Xenopus laevis eggs triggers a period of rapid cell division comprising 12 nearly synchronous mitoses. Protein synthesis is required for these divisions, and new proteins appear after fertilization. Others proteins however, which are synthesized in the unfertilized egg, are no longer made in the early embryo. To identify such proteins, a differential screen of an egg cDNA library gave nine clones corresponding to mRNAs that are deadenylylated soon after fertilization. The sequence of one of these clones (Eg1) revealed a high homology to p34cdc2, the kinase subunit of maturation-promoting factor. Only 12 amino acids in the deduced amino acid sequence were unique to Eg1 when its sequence was compared to all other known examples of cdc2. Despite this strong similarity, however, Eg1 was unable to complement a yeast cdc2- mutant in Schizosaccharomyces pombe or a cdc28 mutant of Saccharomyces cerevisiae. Four Eg1 transcripts, two major and two minor, were found in Xenopus oocytes and early embryos. These RNAs appeared very early (stage I) in oogenesis and their level remained constant until the midblastula transition, at which time they declined. Eg1 RNA is found in the poly(A)+ fraction of oocytes only between the time of meiotic maturation and fertilization--that is to say, in the unfertilized egg. At fertilization the RNA loses its poly(A) tail and at the same time leaves the polyribosomes.