Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

A novel protein involved in heart development in <Emphasis Type="Italic">Ambystoma mexicanum</Emphasis> is localized in endoplasmic reticulum

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.     

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast

In fission yeast the Weel kinase and the functionally redundant Mikl kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Weel and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate weel or mikl homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both weel and mikl. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Weel kinase function. The expression of this clone in a weel/mikl-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Weel kinase activity.     

Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16–18000) proteins of gap junctions

Summary Gap junctions have been isolated from four murine tissues, from rat and Xenopus laevis liver, and from Nephrops norvegicus (Norway lobster) hepatopancreas. The preparations of gap junctions from each vertebrate tissue contain a single major protein, M_r 16000, and those from Nephrops hepatopancreas a protein, M_r 18000. Immunocytochemical studies using affinity-purified antibodies raised against gap junctions from Nephrops show the junctional origin of the 18k protein. Immunological studies using Western blotting and biochemical studies using tryptic peptide mapping show no significant differences between the 16k junctional proteins of mouse and hence provide no evidence of tissue variation. These studies also suggest that the mouse, rat, and Xenopus 16 k proteins and the Nephrops 18 k protein share some common structural features.     

cDNA cloning and functional characterization of Xenopus laevis DNase γ

We report here the cDNA cloning and functional analysis of Xenopus DNase γ (xDNase γ). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase γ protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase γ mRNA is expressed in spleen, liver, testis, and ovary. xDNase γ expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase γ results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase γ in apoptosis during amphibian metamorphosis. The nucleotide sequence of Xenopus DNase γ has been submitted to DDBJ/EMBL/GenBank database under the accession number AF059612     

Cloning,functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k)

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.     

Xenopus laevis L-14 lectin is expressed in a typical pattern in the adult, but is absent from embryonic tissues

Soluble, dimeric, lactose-binding lectins with subunit Mr ofñ 14–16 x 10³, here called L-14s, are expressedin multiple tissues in all vertebrates that have been examined.L-14s have particular affinity for polylactosamine chains onlaminin, co-localize with laminin in some basement membranes,and influence adhesion to laminin and proliferation for somecultured cells. In previous studies of mammals and chickens,L-14s have been found at high levels in a variety of adult tissues,such as muscle and peripheral nerve, but at much higher levelsin many embryonic tissues, suggesting a special role in development.To further explore possible roles of L-14 in embryogenesis,we have studied the expression of L-14 in embryonic and adultXenopus laevis tissues. Except for the abundant L-14 in poisonglands in adult Xenopus skin, we find that Xenopus L-14 is expressedin the same general distribution as its mammalian homologues.However, we could detect no expression of L-14 in Xenopus embryosusing either a sensitive immunoassay for the protein or a sensitiveRNase protection assay for its mRNA. Furthermore, use of affinitychromatography to identify other lactose-binding lectins inembryonic tissue revealed only scarce proteins with higher subunitmolecular weights. These results suggest that in X.laevis L-14functions in adult tissues and is not involved in embryogenesis. ß-galactoside-binding lectin L-14 lectin Xenopus laevis.     

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)–cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.     

TISSUE AND DEVELOPMENTAL SPECIFICITY OF A POLYSIALO-GANGLIOSIDE SPECIES IN THE AMPHIBIAN XENOPUS

Xenopus embryos contain a considerable amount of a polysialo-ganglioside not yet fully characterized; in this paper, we will refer to it as ganglioside X1. Preliminary experiments indicate asialo-GM1 as the core structure of the ganglioside X1 and palmitic and oleic acid as the fatty acids of the ceramide moiety. Further analyses by comparative 2D-TLC with adult fish and chick embryo brains indicate the pentasialilated ganglioside GP1c as the possible structure of X1. In the adult Xenopus, X1 characterizes the ganglioside pattern of the central nervous system while is absent in all the other tested tissues. At least two other more polar (presumably richer in sialic acid) bands are often visible under X1, both in embryos and in brain and spinal cord tissues of adult Xenopus. The persistence of polysialo-gangliosides in the brain and spinal cord of adult amphibians could serve to guarantee a proper functioning of the central nervous system at low body temperature.     

EBF proteins participate in transcriptional regulation of Xenopus muscle development

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells.     

Developmentally regulated expression of a cell surface protein,neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons. © 1996 John Wiley & Sons, Inc.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.