Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster

Diploid sexual reproduction involves segregation of allelic pairs, ensuring equal representation of genotypes in the gamete pool. Some genes, however, are able to “cheat” the system by promoting their own transmission. The Segregation distorter (Sd) locus in Drosophila melanogaster males is one of the best-studied examples of this type of phenomenon. In this system the presence of Sd on one copy of chromosome 2 results in dysfunction of the non–Sd-bearing (Sd⁺) sperm and almost exclusive transmission of Sd to the next generation. The mechanism by which Sd wreaks such selective havoc has remained elusive. However, its effect requires a target locus on chromosome 2 known as Responder (Rsp). The Rsp locus comprises repeated copies of a satellite DNA sequence and Rsp copy number correlates with sensitivity to Sd. Under distorting conditions during spermatogenesis, nuclei with chromosomes containing greater than several hundred Rsp repeats fail to condense chromatin and are eliminated. Recently, Rsp sequences were found as small RNAs in association with Argonaute family proteins Aubergine (Aub) and Argonaute3 (AGO3). These proteins are involved in a germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway, which specifically suppresses transposon activation in the germline. Here, we evaluate the role of piRNAs in segregation distortion by testing the effects of mutations to piRNA pathway components on distortion. Further, we specifically targeted mutations to the aub locus of a Segregation Distorter (SD) chromosome, using ends-out homologous recombination. The data herein demonstrate that mutations to piRNA pathway components act as enhancers of SD.     

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.     

Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A)

Staphylococcus capitis is a coagulase-negative staphylococcus (CoNS) commonly found in the human microflora. Recently, a clonal population of Staphylococcus capitis (denominated NRCS-A) was found to be a major cause of late-onset sepsis (LOS) in several neonatal intensive care units in France. Here, we report the complete genome sequence and annotation of the prototype Staphylococcus capitis NCRS-A strain CR01. The 2,504,472 bp long genome (1 chromosome and no plasmids) exhibits a G+C content of 32.81%, and contains 2,468 protein-coding and 59 tRNA genes and 4 rRNA genes.     

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.     

Linking Gene Expression in the Intestine to Production of Gametes Through the Phosphate Transporter PITR-1 in Caenorhabditis elegans

Inorganic phosphate is an essential mineral for both prokaryotic and eukaryotic cell metabolism and structure. Its uptake into the cell is mediated by membrane-bound transporters and coupled to Na⁺ transport. Mammalian sodium-dependent Pi cotransporters have been grouped into three families NaPi-I, NaPi-II, and NaPi-III. Despite being discovered more than two decades ago, very little is known about requirements for NaPi-III transporters in vivo, in the context of intact animal models. Here we find that impaired function of the Caenorhabditis elegans NaPi-III transporter, pitr-1, results in decreased brood size and dramatically increased expression of vitellogenin by the worm intestine. Unexpectedly, we found that the effects of pitr-1 mutation on vitellogenin expression in the intestine could only be rescued by expression of pitr-1 in the germline, and not by expression of pitr-1 in the intestine itself. Our results indicate the existence of a signal from the germline that regulates gene expression in the intestine, perhaps linking nutrient export from the intestine to production of gametes by the germline.     

Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family’s function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.     

Defining Specificity Determinants of cGMP Mediated Gustatory Sensory Transduction in Caenorhabditis elegans

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.     

A Novel Nondevelopmental Role of the SAX-7/L1CAM Cell Adhesion Molecule in Synaptic Regulation in Caenorhabditis elegans

The L1CAM family of cell adhesion molecules is a conserved set of single-pass transmembrane proteins that play diverse roles required for proper nervous system development and function. Mutations in L1CAMs can cause the neurological L1 syndrome and are associated with autism and neuropsychiatric disorders. L1CAM expression in the mature nervous system suggests additional functions besides the well-characterized developmental roles. In this study, we demonstrate that the gene encoding the Caenorhabditis elegans L1CAM, sax-7, genetically interacts with gtl-2, as well as with unc-13 and rab-3, genes that function in neurotransmission. These sax-7 genetic interactions result in synthetic phenotypes that are consistent with abnormal synaptic function. Using an inducible sax-7 expression system and pharmacological reagents that interfere with cholinergic transmission, we uncovered a previously uncharacterized nondevelopmental role for sax-7 that impinges on synaptic function.     

Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Genomic analysis of Skermanella stibiiresistens type strain SB22T

Members of genus Skermanella were described as Gram-negative, motile, aerobic, rod-shaped, obligate-heterotrophic bacteria and unable to fix nitrogen. In this study, the genome sequence of Skermanella stibiiresistens SB22^T is reported. Phylogenetic analysis using core proteins confirmed the phylogenetic assignment based on 16S rRNA gene sequences. Strain SB22^T has all the proteins for complete glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The RuBisCO encoding genes cbbL1S1 and nitrogenase delta subunit gene anfG are absent, consistent with its inability to fix carbon and nitrogen, respectively. In addition, the genome possesses a series of flagellar assembly and chemotaxis genes to ensure its motility.     

A Novel Gene Controlling the Timing of Courtship Initiation in Drosophila melanogaster

Over the past 35 years, developmental geneticists have made impressive progress toward an understanding of how genes specify morphology and function, particularly as they relate to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless protein function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal courtship and mating behavior. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. We describe the results of feminization of the Tre1-expressing neurons, as well as the effects on courtship behavior of mutation of Tre1. In addition, we show that Tre1 is expressed in a sexually dimorphic pattern in the central and peripheral nervous systems and investigate the role of the Tre1 cells in mate identification.     

Genome sequence of Phaeobacter caeruleus type strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564^T was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.