Tooth development in vitro in two teleost fish,the cichlid <Emphasis Type="Italic">Hemichromis bimaculatus</Emphasis> and the cyprinid <Emphasis Type="Italic">Danio rerio</Emphasis>

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.The in vitro infrastructure at the Ghent laboratory was financed through a grant of the Bijzonder Onderzoeksfonds of Ghent University (BOF: 01102995) and a Krediet aan navorsers (no. 31513695) of the Fonds voor Wetenschappelijk onderzoek (FWO-Vlaanderen). This study also benefitted from an exchange program between the Centre National de Recherche Scientifique (CNRS) and the Ministerie van de Vlaamse Gemeenschap. Research performed by C. Van der heyden was partly financed through a specialization grant of the Flemish Institute for the Advancement of Scientific-Technological Research in Industry (IWT).     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Temperature Effects on the Temporal Properties of Calling Songs in the Crickets <Emphasis Type="Italic">Gryllus fultoni</Emphasis> and <Emphasis Type="Italic">G. vernalis</Emphasis>: Implications for Reproductive Isolation in Sympatric Populations

Two closely related wood-cricket species, Gryllus fultoni (Orthoptera: Gryllidae) and G. vernalis, produce similar calling songs, consisting of 3-pulse chirps. Analysis of field and laboratory recordings of calling songs showed that, after correction to a common temperature, there was a divergence in chirp and pulse rates between far allopatric populations of G. fultoni and populations sympatric with G. vernalis. To determine whether the divergence in calling songs potentially provides reproductive isolation between G. fultoni and G. vernalis throughout the temperature range over which these insects sing, we recorded calling songs of lab-reared G. fultoni and G. vernalis populations between 18 and 28°C. Mean chirp rate significantly differed between sympatric and far allopatric G. fultoni populations as well as between sympatric G. fultoni and G. vernalis populations. Although there was a significant difference in mean pulse rate between sympatric G. fultoni and G. vernalis populations, pulse rate did not differ between sympatric and far allopatric G. fultoni populations in the laboratory study. Considering the magnitudes of differences in calling song characters discriminated by females of G. fultoni and the mean differences and the variability in calling song characters between the two species, the joint difference in chirp and pulse rates may be adequate for species discrimination over most of the range at which these crickets breed.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of the segmentation gene <Emphasis Type="Italic">hairy</Emphasis> in <Emphasis Type="Italic">Tribolium</Emphasis>

Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Two <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in <Emphasis Type="Italic">Chenopodium rubrum</Emphasis> differ in expression patterns

FLOWERING LOCUS T (FT) like genes are crucial regulators (both positive and negative) of flowering in angiosperms. We identified two FT homologs in Chenopodium rubrum, a short-day species used as a model plant for the studies of photoperiodic flower induction. We found that CrFTL1 gene was highly inducible by a 12-h dark period, which in turn induced flowering. On the other hand, photoperiodic treatments that did not induce flowering (short dark periods, or a permissive darkness interrupted by a night break) caused only a slight increase in CrFTL1 mRNA level. We demonstrated diurnal oscillation of CrFTL1 expression with peaks in the middle of a light period. The oscillation persisted under constant darkness. Unlike FT homologs in rice and Pharbitis, the CrFTL1 expression under constant darkness was very low. The CrFTL2 gene showed constitutive expression. We suggest that the CrFTL1 gene may play a role as a floral regulator, but the function of CrFTL2 remains unknown.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.     

Flavonoids in the leaves of <Emphasis Type="Italic">Oxalis corniculata </Emphasis>and sequestration of the flavonoids in the wing scales of the pale grass blue butterfly, <Emphasis Type="Italic">Pseudozizeeria </Emphasis><Emphasis Type="Italic">maha</Emphasis>

Three C-glycosylflavones in the leaves of Oxalis corniculata, the host plant of the lycaenid butterfly pale grass blue (Pseudozizeeria maha), were identified as 6-C-glucosylluteolin (isoorientin), 6-C-glucosylapigenin (isovitexin) and isovitexin 7-methyl ether (swertisin). Comparative spectral and HPLC analyses between the leaf extract of the host plants and the wings of P. maha showed selective uptake of the host-plant flavonoid isovitexin to the wings of the butterfly.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

<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