Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus

The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.     

Cold-inducible RNA binding protein is required for the expression of adhesion molecules and embryonic cell movement in Xenopus laevis

We have previously shown that the Xenopus homologue of cold-inducible RNA binding protein, XCIRP-1, is required for the morphogenetic migration of the pronephros during embryonic development. However, the underlying molecular mechanisms remain elusive. Here, we report that XCIRP is essential for embryonic cell movement, as suppression of XCIRP by microinjection of anti-sense mRNA and morpholino antisense oligonucleotides (MOs) significantly reduced protein expression, inhibited the cell migration rate, and inhibited eFGF and activin-induced animal cap elongation. By immunoprecipitation and RT-PCR, we further showed that the mRNA of a panel of adhesion molecules, including alphaE- and beta-catenin, C- and E-cadherin, and paraxial proto-cadherin, are the targets of XCIRP. Consistently, in animal cap explant studies, suppression of XCIRP by MOs inhibited the expression of these adhesion molecules, while over-expression of sense XCIRP-1 mRNA fully rescued this inhibition. Taken together, these results suggest for the first time that XCIRP is required to maintain the expression of adhesion molecules and cell movement during embryonic development.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.     

Cloning and heterologous expression of new xANO2 from Xenopus laevis

We have successfully isolated a novel anoctamin (xANO2), Ca²⁺-activated chloride channel (ANO1, TMEM16A), from Xenopus laevis. The cDNA sequence was determined to belong to the anoctamin family by comparison with the xTMEM16A sequence in a previous report. Full length cDNA synthesis was performed by repeating 5′- and 3′-rapid amplification of cDNA end (RACE). We successfully completed the entire cDNA sequence and transiently named this sequence xANO2. The xANO2 cDNA is 3884 base pair (bp) long and codes 980 amino acid (aa) proteins. According to an aa homology search using the Basic Local Alignment Search Tool (BLAST), xANO2 showed an overall identity of 92% to xTMEM16A (xANO1) independently sub-cloned in our laboratory. A primary sequence of xANO2 revealed typical characteristics of transmembrane proteins. In tissue distribution analysis, the gene products of anoctamins were ubiquitously detected by real-time PCR (RT-PCR). The expression profiles of each anoctamin were different among brain, oocytes, and digestive organs with relatively weak expression. To clarify the anoctamin activity, physiological studies were performed using the whole cell patch-clamp technique with HEK293T cells, enhanced green fluorescent protein (EGFP), and expression vectors carrying anoctamins. Characteristics typical of voltage-dependent chloride currents were detected in cells expressing both xANO2 and xTMEM16A but not with EGFP alone. Sensitive reactions to the anion channel blocker niflumic acid (NFA) were also revealed. Considering these results, xANO2 was regarded as a new TMEM16A belonging to the Xenopus anoctamin family.     

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.     

Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.     

The expression of creatine kinase isozymes in Xenopus tropicalis,Xenopus laevis laevis,and their viable hybrid

Starch gel electrophoresis of creatine kinase (CK) isozymes of Xenopus tropicalis shows that at least two different genes code for CK in this diploid (2n=20) species. These genes seem to be orthologous to the CK-A and CK-C genes of extant crossopterygian fish. Additional isozymes may be interpreted either as products of duplicate genes or, more probably, as epigenetically modified forms of the homodimers A^tA^t and C^tC^t, respectively. The originally tetraploid species X. laevis laevis (2n=36), which may have arisen by hybridization of diploid ancestors some 30–40 million years ago, has retained expression of all duplicate CK-A and CK-C genes. Differential expression during ontogenesis (CK-A genes) and in different adult tissues (CK-C genes) indicates that divergence occurred not only with respect to the primary sequence of these duplicate genes, but also with respect to the regulation of their expression. In the interspecific hybrid X. 1. laevis × X. tropicalis, all parental CK genes appear to be expressed simultaneously in the heart. However, several subunit combinations cannot be detected on the zymograms.This work was supported by Swiss National Foundation for Scientific Research Grant 3.775.0.80.     

Identification and preliminary function study of Xenopus laevis DRR1 gene

Xenopus laevis has recently been determined as a novel study platform of gene function. In this study, we cloned Xenopus DRR1 (xDRR1), which is homologous to human down-regulated in renal carcinoma (DRR1) gene. Bioinformatics analysis for DRR1 indicated that xDRR1 shared 74% identity with human DRR1 and 66% with mouse DRR1, and the phlogenetic tree of DRR1 protein was summarized. The xDRR1 gene locates in nuclei determined by transfecting A549 cells with the recombinant plasmid pEGFP-N1/xDRR1. RT-PCR analysis revealed that xDRR1 gene was expressed in all stages of early embryo development and all kinds of detected tissues, and whole-mount in situ hybridization showed xDRR1 was mainly present along ectoderm and mesoderm. Furthermore, xDRR1 expression could suppress A549 cell growth by transfecting with plasmid pcDNA3.1(+)/xDRR1. xDRR1 probably plays important roles involving in cell growth regulation and Xenopus embryo development.     

Differentiation and characterization of embryonic stem cells into three germ layers

Embryonic stem cells differentiated on M15 cells have previously been shown to give rise to cells of the mesendodermal and definitive endodermal lineages. Here we demonstrate that neuroectodermal and mesodermal lineages can be derived from ES cells cultured on M15 cells and subsequently subjected to specific culture conditions, as confirmed by the expression of molecular markers. Prospective isolation and microarray analyses showed that neuroectodermal cells expressed anterior-to-posterior, as well as dorso-ventral regional markers, suggesting that this procedure could be used for the induction of cells belonging to a wide variety of neural lineages. Lateral mesoderm and paraxial mesoderm cells were also produced and their gene expression profiles were confirmed by microarray analyses. These results indicate that the M15 cell system provides a valuable tool for generating ES cell-derived lineage-specific cell types belonging to the three germ layers, namely neuroectoderm, mesoderm, and definitive endoderm.