Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

Molecular detection and phylogenetic analysis of the alkane 1-monooxygenase gene from Gordonia spp.

The alkB gene encodes for alkane 1-monooxygenase, which is a key enzyme responsible for the initial oxidation of inactivated alkanes. This functional gene can be used as a marker to assess the catabolic potential of bacteria in bioremediation. In the present study, a pair of primers was designed based on the conserved regions of the AlkB amino acid sequences of Actinobacteria, for amplifying the alkB gene from the genus Gordonia (20 Gordonia strains representing 13 species). The amplified alkB genes were then sequenced and analyzed. In the phylogenetic tree based on the translated AlkB amino acid sequences, all the Gordonia segregated clearly from other closely related genera. The sequence identity of the alkB gene in Gordonia ranged from 58.8% to 99.1%, which showed higher sequence variation at the inter-species level compared with other molecular markers, such as the 16S rRNA gene (93.1–99.8%), gyrB gene (77.5–97.3%) or catA gene (72.4–99.5%). The genetic diversity of four selected loci also showed that the alkB gene might have evolved faster than rrn operons, as well as the gyrB or catA genes, in Gordonia. All the available actinobacterial alkB gene sequences derived from the whole genome shotgun sequencing projects are phylogenetically characterized here for the first time, and they exclude the possibility of horizontal gene transfer of the alkB gene in these bacterial groups.     

Altered Notch signaling resulting from expression of a WAMTP1-MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin's tumors

Chromosome translocations in neoplasia commonly result in fusion genes that may encode either novel fusion proteins or normal, but ectopically expressed proteins. Here we report the cloning of a novel fusion gene in a common type of salivary and bronchial gland tumor, mucoepidermoid carcinomas (MEC), as well as in benign Warthin's tumors (WATs). The fusion, which results from a t(11;19)(q21-22;p13) translocation, creates a chimeric gene in which exon 1 of a novel gene of unknown function, designated WAMTP1, is linked to exons 2-5 of the recently identified Mastermind-like Notch coactivator MAML2. In the fusion protein, the N-terminal basic domain of MAML2, which is required for binding to intracellular Notch (Notch ICD), is replaced by an unrelated N-terminal sequence from WAMTP1. Mutation analysis of the N-terminus of WAMTP1-MAML2 identified two regions of importance for nuclear localization (amino acids 11-20) and for colocalization with MAML2 and Notch1 ICD in nuclear granules (amino acids 21-42). Analyses of the Notch target genes HES5 and MASH1 in MEC tumors with and without the WAMTP1-MAML2 fusion revealed upregulation of HES5 and downregulation of MASH1 in fusion positive MECs compared to normal salivary gland tissue and MECs lacking the fusion. These findings suggest that altered Notch signaling plays an important role in the genesis of benign and malignant neoplasms of salivary and bronchial gland origin.     

Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes

Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.     

C. elegans RBX-2-CUL-5- and RBX-1-CUL-2-based complexes are redundant for oogenesis and activation of the MAP kinase MPK-1

Cul5-based complex is a member of ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) ubiquitin ligase family. The cellular function of the Cul5-based complex is poorly understood. In this study, we found that oocyte septum formation and egg production did not occur in either cul-5- or rbx-2-depleted cul-2 homozygotes, although control cul-2 homozygotes laid approximately 50 eggs. These phenotypes are reminiscent of those caused by the MAP kinase mpk-1 depletion. In fact, activation of MPK-1 was significantly inhibited in cul-5-depleted cul-2 mutant and cul-2-depleted cul-5 mutant. Yeast two-hybrid analysis and RNAi-knockdown experiments suggest that oocyte maturation from pachytene exit and MPK-1 activation are redundantly controlled by the RBX-2-CUL-5- and RBX-1-CUL-2-based complexes.     

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.     

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.     

鲤TYK2基因的克隆鉴定及组织表达分析

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