Expression of <Emphasis Type="Italic">periostin</Emphasis> during <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryogenesis

Periostin (postn) is a secreted, extracellular matrix protein containing an EMI domain as well as four fasciclin I-like (Fas1) domains. Postn protein functions in cell adhesion, cell mobility, cell proliferation and gene regulation. Earlier studies have shown that postn is involved in different developmental processes such as somitogenesis, cardiogenesis and bone formation. Intriguingly, postn seems to be a very good candidate to establish novel therapies against cancer and chronic heart defects. Here we describe for the first time the spatio-temporal expression profile of postn during early development of Xenopus laevis. By semi-quantitative RT-PCR approaches, we demonstrate that postn is maternally expressed. Zygotic expression starts during early gastrulation and increases until stage 40. Whole mount in situ hybridization experiments revealed that postn is detectable in somites, the sensory layer of the epidermis, the roof plate, the notochord, the heart, migrating neural crest cells, cranial ganglia and forming cranial cartilage structures. Our results implicate a role of postn during Xenopus embryogenesis and represent a good starting point for future functional analyses.     

Molecular cloning and characterization of novel ficolins from<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.     

Sexually differentiated,androgen-regulated,larynx-specific myosin heavy-chain isoforms in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>; comparison to <Emphasis Type="Italic">Xenopus laevis</Emphasis>

We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles (Nasipak and Kelley, Dev Genes Evol, in press, 2008). The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Mutation in<Emphasis Type="Italic"> slowmo</Emphasis> causes defects in<Emphasis Type="Italic"> Drosophila</Emphasis> larval locomotor behaviour

We have identified a mutant slowmotion phenotype in first instar larval peristaltic behaviour of Drosophila. By the end of embryogenesis and during early first instar phases, slowmo mutant animals show a marked decrease in locomotory behaviour, resulting from both a reduction in number and rate of peristaltic contractions. Inhibition of neurotransmitter release, using targeted expression of tetanus toxin light chain (TeTxLC), in the slowmo neurons marked by an enhancer-trap results in a similar phenotype of largely absent or uncoordinated contractions. Cloning of the slowmo gene identifies a product related to a family of proteins of unknown function. We show that Slowmo is associated with mitochondria, indicative of it being a mitochondrial protein, and that during embryogenesis and early larval development is restricted to the nervous system in a subset of cells. The enhancer-trap marks a cellular component of the CNS that is seemingly required to regulate peristaltic movement.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

<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.     

Expression analysis of <Emphasis Type="Italic">epb41l4a</Emphasis> during <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryogenesis

Epbl41l4a (erythrocyte protein band 4.1-like 4a, also named Nbl4) is a member of the band 4.1/Nbl4 (novel band 4.1-like protein 4) group of the FERM (4.1, ezrin, radixin, moesin) protein superfamily. Proteins encoded by this gene family are involved in many cellular processes such as organization of epithelial cells and signal transduction. On a molecular level, band 4.1/Nbl4 proteins have been shown to link membrane-associated proteins and lipids to the actin cytoskeleton. Epbl41l4a has also recently been identified as a target gene of the Wnt/β-catenin pathway. Here, we describe for the first time the spatio-temporal expression of epbl41l4a using Xenopus laevis as a model system. We observed a strong and specific expression of epb41l4a in the developing somites, in particular during segmentation as well as in the nasal and cranial placodes, pronephros, and neural tube. Thus, epbl41l4a is expressed in tissues undergoing morphogenetic movements, suggesting a functional role of epbl41l4a during these processes.     

Survey of <Emphasis Type="Italic">O</Emphasis>-GlcNAc level variations in <Emphasis Type="Italic">Xenopus laevis</Emphasis> from oogenesis to early development

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I–V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.     

Characterization of a novel insertion sequence,IS<Emphasis Type="Italic">Bp1</Emphasis>, in<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis>

During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, IS Bp1, was found by sequence similarity searches. IS Bp1 contains two overlapping ORFs of 261 bp ( orfA) and 852 bp ( orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat. The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS 3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by IS D1 of Desulfovibrio vulgaris vulgaris. The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS 3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS 1222 of Enterobacter agglomerans. Sequence analysis of OrfA showed the presence of an alpha-helix-turn-alpha-helix motif, as well as the putative leucine zipper at its 3' end, for possible DNA binding to the terminal inverted repeats. Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS 3 family. Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed. PCR amplification of the IS Bp1 gene showed a specific band in 65% of the 26 B. pseudomallei strains tested. Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization. The number of copies of IS Bp1 in those strains that possessed the insertion sequence ranged from three to 12. Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B. pseudomallei.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

New sections in<Emphasis Type="Italic">Taraxacum</Emphasis>

Sectional taxonomy ofTaraxacum in steppe or subsaline habitats in Central Asia is revised based on material collected during expeditions, cultivated or studied in herbarium. Two new sections are described from that area:T. sect.Stenoloba similar toT. sect.Leucantha (syn.:T. sect.Sinensia), andT. sect.Suavia allied toT. sect.Dissecta. The type species of the sectionSuavia is described asTaraxacum formosissimum Kirschner etŠtěpánek. Widespread mountain dandelions of the Caucasus, intermediate between the sect.Piesis andT. stevenii, are described asT. sect.Confusa. Taraxacum species dominating dry habitats in S Ukraine and Crimea are described asT. sect.Borysthenica. Species belonging to the new sections were found to be polyploid and agamospermous.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Callus induction and regeneration in<Emphasis Type="Italic"> Spirodela</Emphasis> and<Emphasis Type="Italic"> Lemna</Emphasis>

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.Abbreviations ABA: (±)-Abscisic acid - BA: N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba: 3,6-Dichloro-2-methoxybenzoic acid - 2iP: N⁶-(2-Isopentenyl)adenine - NAA: -Naphthaleneacetic acid - PCA: p-Chlorophenoxy acetic acid - Picloram: 4-Amino-3,5,6-trichloropicolinic acid - TDZ: Thidiazuron Communicated by A. AltmanJ. Li and M. Jain contributed equally to the research reported in this article.     

Ultraweak emissions of developing <Emphasis Type="Italic">Xenopus laevis</Emphasis> eggs and embryos

We measured ultraweak emissions of the Xenopus laevis eggs and embryos during normal development and under the influence of stress factors in a spectral range of 250 to 800 nm using a photomultiplier. The registered emissions were analyzed by several basic characteristics: mean intensity, histograms, kurtosis, linear trends, and Fourier spectra. We followed relationships between these parameters and developmental stage, as well as the number of individuals in optic contact with each other. The ultraweak emissions did not differ from the background at all developmental stages according to the mean intensity. But Fourier analysis revealed the reliable presence of a number of spectral lines of ultraweak emission, predominantly in the range of 10^?2–50 Hz, in the embryos at developmental stages 2 to 11. The intensity of ultraweak emissions reliably decreased within the first 10 min after egg activation and fertilization, as well as in the case of optic interaction between groups of embryos. Sharp cooling, increase in osmotic medium pressure, and transfer in a Ca²⁺ and Mg²⁺-free medium induced a short term (～1–5 min) increase in the mean intensity of ultraweak emission. We studied specific features of ultraweak emissions from different parts of the embryo. The intensity of emission from the animal part of early blastula exceeded those from the vegetal area and entire embryo. Separated fragments of the lateral ectoderm at the neurula stage had higher mean intensities of ultraweak emission than intact embryos at the same developmental stages.