Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C. elegans

MicroRNAs (miRNAs) are regulatory molecules that share both biosynthetic derivation (cleavage from short hairpin precursor RNAs) and functional roles (downregulation of specific mRNAs through targeted degradation and/or translational inhibition). A distinct family of small RNAs, termed siRNAs, have some common characteristics but exhibit distinct modes of biosynthesis and function. In this study, we report procedures for purification of a predominant species of miRNA-containing ribonucleoprotein complexes from Caenorhabditis elegans and demonstrate that this population is distinct from the predominant pool of siRNA-containing ribonucleoprotein complexes. An observed miRNP-associated RNA population consisting predominantly (>95%) of miRNAs supported the unique identity of miRNPs as biological effectors within the cell, provided clean material for analysis of changes in miRNA spectra during development, and provided strong evidence of miRNA character for a number of novel small RNAs. Likewise, the RNA spectrum derived from partial siRNP purification was useful in defining functional characteristics of this more diverse population of small RNAs.     

Distinct protein domains regulate ciliary targeting and function of C. elegans PKD-2

TRPP2 (transient receptor potential polycystin-2) channels function in a range of cells where they are localized to specific subcellular regions including the endoplasmic reticulum (ER) and primary cilium. In humans, TRPP2/PC-2 mutations severely compromise kidney function and cause autosomal dominant polycystic kidney disease (ADPKD). The Caenorhabditis elegans TRPP2 homolog, PKD-2, is restricted to the somatodendritic (cell body and dendrite) and ciliary compartments of male specific sensory neurons. Within these neurons PKD-2 function is required for sensation. To understand the mechanisms regulating TRPP2 subcellular distribution and activity, we performed in vivo structure-function-localization studies using C. elegans as a model system. Our data demonstrate that somatodendritic and ciliary targeting requires the transmembrane (TM) region of PKD-2 and that the PKD-2 cytosolic termini regulate subcellular distribution and function. Within neuronal cell bodies, PKD-2 colocalizes with the OSM-9 TRP vanilloid (TRPV) channel, suggesting that these TRPP and TRPV channels may function in a common process. When human TRPP2/PC-2 is heterologously expressed in transgenic C. elegans animals, PC-2 does not visibly localize to cilia but does partially rescue pkd-2 null mutant defects, suggesting that human PC-2 and PKD-2 are functional homologs.     

Distinct innate immune responses to infection and wounding in the C. elegans epidermis

BACKGROUND: In many animals, the epidermis is in permanent contact with the environment and represents a first line of defense against pathogens and injury. Infection of the nematode Caenorhabditis elegans by the natural fungal pathogen Drechmeria coniospora induces the expression in the epidermis of antimicrobial peptide (AMP) genes such as nlp-29. Here, we tested the hypothesis that injury might also alter AMP gene expression and sought to characterize the mechanisms that regulate the innate immune response. RESULTS: Injury induces a wound-healing response in C. elegans that includes induction of nlp-29 in the epidermis. We find that a conserved p38-MAP kinase cascade is required in the epidermis for the response to both infection and wounding. Through a forward genetic screen, we isolated mutants that failed to induce nlp-29 expression after D. coniospora infection. We identify a kinase, NIPI-3, related to human Tribbles homolog 1, that is likely to act upstream of the MAPKK SEK-1. We find NIPI-3 is required only for nlp-29 induction after infection and not after wounding. CONCLUSIONS: Our results show that the C. elegans epidermis actively responds to wounding and infection via distinct pathways that converge on a conserved signaling cassette that controls the expression of the AMP gene nlp-29. A comparison between these results and MAP kinase signaling in yeast gives insights into the possible origin and evolution of innate immunity.     

Distinct roles for two C. elegans anillins in the gonad and early embryo

Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.     

Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development

The C. elegans PUF and FBF proteins regulate various aspects of germline development by selectively binding to the 3' untranslated region of their target mRNAs and repressing translation. Here, we show that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulvaI development. Loss-of-function mutations in puf-8 cause ectopic vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling. PUF-8 acts cell-autonomously in the vulval cells to limit their temporal competence to respond to the extrinsic patterning signals. fbf-1 and fbf-2, however, redundantly inhibit primary vulval cell fate specification in two distinct pathways acting in the soma and in the germline. The FBFs thereby ensure that the inductive signal selects only one vulval precursor cell for the primary cell fate. Thus, translational repressors regulate various aspects of vulval cell fate specification, and they may play a conserved role in modulating signal transduction during animal development.     

Centrosomes can initiate a polarity axis from any position within one-cell C. elegans embryos

The stereotyped asymmetry of one-cell C. elegans embryos has proven to be an important model for identifying molecular determinants of cell polarity. How polarity is initiated is less well understood. Polarity establishment depends on centrosomes, which use two molecularly distinct pathways to break symmetry. In both, the centrosome's position adjacent to the cell cortex is thought to determine where polarization starts. Defects in centrosome-cortex juxtaposition correlate with defects in polarity establishment in several mutants, suggesting that these processes may be linked, but there is no direct test of this. Here we assess how centrosome position relative to the cortex affects polarity establishment. We find that centrosomes can initiate polarity from any position within the embryo volume, but centrosome-cortex proximity decreases the time required to initiate polarity. Polarization itself brings about close centrosome-cortex proximity. Prior to polarization, cytoplasmic microtubules constrain centrosome movement near the cortex, expanding the controversial role of microtubules during polarity establishment. The ability of centrosomes to induce a single polarity axis from any position within the egg emphasizes the flexible, self-organizing properties of polarization in C. elegans embryos and contrasts the common view of C. elegans development as invariant.     

Distinct IFT mechanisms contribute to the generation of ciliary structural diversity in C. elegans

Individual cell types can elaborate morphologically diverse cilia. Cilia are assembled via intraflagellar transport (IFT) of ciliary precursors; however, the mechanisms that generate ciliary diversity are unknown. Here, we examine IFT in the structurally distinct cilia of the ASH/ASI and the AWB chemosensory neurons in Caenorhabditis elegans, enabling us to compare IFT in specific cilia types. We show that unlike in the ASH/ASI cilia, the OSM-3 kinesin moves independently of the kinesin-II motor in the AWB cilia. Although OSM-3 is essential to extend the distal segments of the ASH/ASI cilia, it is not required to build the AWB distal segments. Mutations in the fkh-2 forkhead domain gene result in AWB-specific defects in ciliary morphology, and FKH-2 regulates kinesin-II subunit gene expression specifically in AWB. Our results suggest that cell-specific regulation of IFT contributes to the generation of ciliary diversity, and provide insights into the networks coupling the acquisition of ciliary specializations with other aspects of cell fate.     

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-beta repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.     

Innexins in C. elegans

Innexins are functionally analogous to the vertebrate connexins, and the innexin family of gap junction proteins has been identified in many invertebrates, including Drosophila and C. elegans. The genome sequencing project has identified 25 innexins in C. elegans. We are particularly interested in the roles that gap junctions may play in embryonic development and in wiring of the nervous system. To identify the particular C. elegans innexins that are involved in these processes, we are examining their expression patterns using specific antibodies and translational GFP fusions. In addition we are investigating mutant, RNAi and overexpression phenotypes for many of these genes. To date, we have generated specific antibodies to the non-conserved carboxyl termini of 5 innexins. We have constructed GFP translational fusions for 17 innexins and observed expression patterns for 13 of these genes. In total we have characterized expression patterns representing 14 innexins. Mutations have been identified in 5 of these genes, and at least 3 others have RNAi mutant phenotypes. Generalities emerging from our studies include: 1) most tissues and many individual cells express more than one innexin, 2) some innexins are expressed widely, while others are expressed in only a few cells, and 3) there is a potential for functional pairing of innexins.     

YACs and the C. elegans genome

During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms. It is widely accepted that this undertaking must include the elucidation of the genetic blueprint – the genome sequence – of a number of model organisms. As a prelude to the determination of these sequences, clonebased physical maps of the genomes of a number of multicellular animals and plants are being constructed. Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps. The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone ‘islands’ to be linked together in an efficient manner. The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility. Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large.     

MyoD and myogenesis in C. elegans

One of the goals in developmental biology is the identification of key regulatory genes that govern the transition of embryonic cells from a pluripotent potential to a specific, committed cell fate. During vertebrate skeletal myogenesis, this transition is regulated by the MyoD family of genes. C. elegans has muscle analogous to vertebrate skeletal muscle and has a gene(hlh-1) related to the MyoD family. The molecular and genetic characterization of hlh-1 shows that it is very similar to the vertebrate MyoD family in many respects, including its expression pattern and DNA binding activity. The hlh-1 product is required for proper myogenesis, but it is not required for myogenic commitment during embryogenesis in the nematode. The role of this MyoD-related gene in nematode myogenesis is discussed and compared to those of the vertebrate MyoD family.     

Protein kinase C isotypes in C. elegans

The protein kinase C (PKC) family, consisting of multiple isotypes, plays a major role in cellular signaling. In the nematode Caenorhabditis elegans, four pkc genes, tpa-1, pkc-1, pkc-2 and pkc-3, have been identified and investigated. Molecular analysis of tpa-1, pkc-1, and pkc-2 has shown that each gene encodes multiple PKC isoforms with different expression patterns. One of the tpa-1 isoforms, which is expressed in vulval cells, is found to play a role in nicotine-induced adaptation. The expression of pkc-1 seems to be specific to neurons, while that of pkc-2 is detected in several types of cells including neurons and muscle cells. An aPKC member encoded by pkc-3 has been shown to play an essential role in establishing the polarity of the zygote. Recent studies have revealed that the mechanism of polarity establishment mediated by aPKC is evolutionarily conserved in diverse organisms from nematodes to mammals. C. elegans provides an excellent model system for molecular dissection of the cellular signaling pathways involving PKC.