Functional genomic screen identifies novel mediators of collagen uptake

Tissue fibrosis occurs when matrix production outpaces matrix degradation. Degradation of collagen, the main component of fibrotic tissue, is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis. These pathways are poorly characterized. Here we identify novel mediators of the intracellular pathway of collagen turnover through a genome-wide RNA interference screen in Drosophila S2 cells. Screening of 7505 Drosophila genes conserved among metazoans identified 22 genes that were required for efficient internalization of type I collagen. These included proteins involved in vesicle transport, the actin cytoskeleton, and signal transduction. We show further that the flotillin genes have a conserved and central role in collagen uptake in Drosophila and human cells. Short hairpin RNA–mediated silencing of flotillins in human monocyte and fibroblasts impaired collagen uptake by promoting lysosomal degradation of the endocytic collagen receptors uPARAP/Endo180 and mannose receptor. These data provide an initial characterization of intracellular pathways of collagen turnover and identify the flotillin genes as critical regulators of this process. A better understanding of these pathways may lead to novel therapies that reduce fibrosis by increasing collagen turnover.     

A molecular view of onychophoran segmentation

This paper summarizes our current knowledge on the expression and assumed function of Drosophila and (other) arthropod segmentation gene orthologs in Onychophora, a closely related outgroup to Arthropoda. This includes orthologs of the so-called Drosophila segmentation gene cascade including the Hox genes, as well as other genetic factors and pathways involved in non-drosophilid arthropods.Open questions about and around the topic are addressed, such as the definition of segments in onychophorans, the unclear regulation of conserved expression patterns downstream of non-conserved factors, and the potential role of mesodermal patterning in onychophoran segmentation.     

Identification of a novel insect neuropeptide,CNMa and its receptor

To identify ligands for orphan GPCRs, we searched novel neuropeptide genes in the Drosophila melanogaster genome. Here, we describe CNMa, a novel cyclic neuropeptide that is a highly potent and selective agonist for the orphan GPCR, CG33696 (CNMaR). Phylogenetic analysis revealed that arthropod species have two paralogous CNMaRs, but many taxa retain only one. Drosophila CNMa potently activates CNMaR-2 from Apis mellifera, suggesting both receptors are functional. Although CNMa is conserved in most arthropods, Lepidoptera lack the CNMa gene. However, they retain the CNMaR gene. Bombyx CNMaR showed low sensitivity to Drosophila CNMa, hinting toward the existence of additional CNMaR ligand(s).     

Late Replication Domains Are Evolutionary Conserved in the Drosophila Genome

Drosophila chromosomes are organized into distinct domains differing in their predominant chromatin composition, replication timing and evolutionary conservation. We show on a genome-wide level that genes whose order has remained unaltered across 9 Drosophila species display late replication timing and frequently map to the regions of repressive chromatin. This observation is consistent with the existence of extensive domains of repressive chromatin that replicate extremely late and have conserved gene order in the Drosophila genome. We suggest that such repressive chromatin domains correspond to a handful of regions that complete replication at the very end of S phase. We further demonstrate that the order of genes in these regions is rarely altered in evolution. Substantial proportion of such regions significantly coincide with large synteny blocks. This indicates that there are evolutionary mechanisms maintaining the integrity of these late-replicating chromatin domains. The synteny blocks corresponding to the extremely late-replicating regions in the D. melanogaster genome consistently display two-fold lower gene density across different Drosophila species.     

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.     

Can colonization by an endophytic fungus transform a plant into a challenging host for insect herbivores?

Endophytic growth of arthropod pathogenic fungi can parasitize insect herbivores without causing damage to the crop. However, studies addressing this tritrophic interaction are absent. Here, the endophytic arthropod pathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordyciptaceae), the polyphagous two-spotted spider mite Tetranychus urticae Koch (Trombidiformes: Tetranychidae), and its preferred plant host Phaseolus vulgaris L. (Fabales: Fabaceae) were selected to study the multi-kingdom interactions among plants, arthropods, and entomopathogenic fungi. Real-Time PCR analysis of nine defense-related genes revealed that a broad range of plant defense mechanisms is activated in response to the endophytic growth of B. bassiana. Moreover, we studied the molecular mechanism adapted by the two-spotted spider mite that underlies resistance. The analysis of 41 detoxification genes revealed that relatively moderate, high, and few numbers of genes were changed in the adults, nymphs, and eggs stages of T. urticae, respectively, after inoculation on colonized tissues of P. vulgaris. The endophytic growth of B. bassiana can have a negative effect on the growth and performance of the pest, in a developmental stage-dependent manner, by priming plant defense pathways. In parallel, the herbivore induces a broad range of detoxification genes that could potentially be involved in adaptation to endophytically colonized plant tissues.     

Complete mitochodrial genome of the crab spider Ebrechtella tricuspidata (Araneae: Thomisidae): A novel tRNA rearrangement and phylogenetic implications for Araneae

Mitochondrial genomes are widely used for phylogenetic and phylogeographic analyses among arthropods, but there is a lack of sufficient mitochondrial genome sequence data for spiders. Herein, we sequenced and characterized the complete mitochondrial genome of a crab spider Ebrechtella tricuspidata (Araneae: Thomisidae). The circular mitochondrial genome is 14,352 bp long, including a standard set of 37 genes and an A + T-rich region. Nucleotide composition is highly biased toward A + T nucleotides (77.3%). A novel gene order rearrangement is detected by a tRNA (trnL1) translocation. Tandem repeats are not identified in the A + T-rich region. Most of the tRNAs are greatly reduced in size and cannot be folded into typical cloverleaf-shaped secondary structures. The phylogenetic analysis confirms that the mitochondrial genome sequences are useful in resolving higher-level relationship of Araneae. Overall, our data present in this study will elevate our knowledge on the architecture and evolution of spider mitochondrial genome.     

Gene silencing in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: dsRNA and siRNA parental silencing of the <Emphasis Type="Italic">Distal-less</Emphasis> gene

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.     

Notch-mediated segmentation of the appendages is a molecular phylotypic trait of the arthropods

Arthropod limbs are arguably the most diverse organs in the animal kingdom. Morphological diversity of the limbs is largely based on their segmentation, because this divides the limbs into modules that can evolve separately for new morphologies and functions. Limb segmentation also distinguishes the arthropods from related phyla (e.g. onychophorans) and thus forms an important evolutionary innovation in arthropods. Understanding the genetic basis of limb segmentation in arthropods can thus shed light onto the mechanisms of macroevolution and the origin of a character (articulated limbs) that defines a new phylum (arthropods). In the fly Drosophila limb segmentation and limb growth are controlled by the Notch signaling pathway. Here we show that the Notch pathway also controls limb segmentation and growth in the spider Cupiennius salei, a representative of the most basally branching arthropod group Chelicerata, and thus this function must trace from the last common ancestor of all arthropods. The similarities of Notch and Serrate function between Drosophila and Cupiennius are extensive and also extend to target genes like odd-skipped, nubbin, AP-2 and hairy related genes. Our data confirm that the jointed appendages, which are a morphological phylotypic trait of the arthropods and the basis for naming the phylum, have a common developmental genetic basis. Notch-mediated limb segmentation is thus a molecular phylotypic trait of the arthropods.     

Functional dissection of an innate immune response by a genome-wide RNAi screen

The innate immune system is ancient and highly conserved. It is the first line of defense and the only recognizable immune system in the vast majority of metazoans. Signaling events that convert pathogen detection into a defense response are central to innate immunity. Drosophila has emerged as an invaluable model organism for studying this regulation. Activation of the NF-κB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria. To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens. We identified numerous inhibitors and activators of immune reporters in a cell culture model. Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish. We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway. Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop. We propose that interruption of this feedback loop contributes to signal transduction.     

Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: Genetic and biochemical characterization of two crustacean cyclooxygenases

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world.     

Singular value decomposition-based regression identifies activation of endogenous signaling pathways in vivo

Background

The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression.

Results

We have catalogued ordered duplicated genes in Drosophila melanogaster, and found that one in five of all genes is organized as tandem arrays. Furthermore, among arrays that have been spatially conserved over longer periods than would be expected on the basis of random shuffling, a disproportionate number contain genes encoding developmental regulators. Using in situ gene expression data for more than half of the Drosophila genome, we find that genes in these conserved clusters are co-expressed to a much higher extent than other duplicated genes.

Conclusions

These results reveal the existence of functional constraints in insects that retain copies of genes encoding developmental and regulatory proteins as neighbors, allowing their co-expression. This co-expression may be the result of shared cis-regulatory elements or a shared need for a specific chromatin structure. Our results highlight the association between genome architecture and the gene regulatory networks involved in the construction of the body plan.     

Molecular evolution of mobile elements of the <Emphasis Type="Italic">gypsy</Emphasis> group: A homolog of the <Emphasis Type="Italic">gag</Emphasis> gene in <Emphasis Type="Italic">Drosophila</Emphasis>

Retrotransposons of the gypsy group of Drosophila melanogaster that are structurally similar to retroviruses of vertebrates occupy an important place among retroelements of eukaryotes. The infectious abilities of some retrotransposons of this group (gypsy, ZAM, and Idefix) have been demonstrated experimentally, and therefore they are true retroviruses. It is supposed that retrotransposons can evolve acquiring new components, the sources of which remain to be elucidated. In this work, the CG4680gene (Gag related protein, Grp) homologous to gag of retrotransposons of the gypsy group has been identified in the genome of D. melanogaster and characterized. The Grp gene product has a highly conserved structure in different species of the Drosophilidae family and is under of purifying selection, which suggests its important genomic function in Drosophila. In view of the earlier data, it can be concluded that homologous genes of all components of gypsy retrotransposons are present in the Drosophila genome. These genes can be both precursors and products of domestication of retrovirus genes.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling