Cuticle collagen genes. Expression in Caenorhabditis elegans

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. The exoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractable to genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter the shape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinct collagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagen genes are expressed in distinct temporal periods, which might facilitate the formation of specific interactions between distinct collagens.     

Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle

The mechanical properties of cells and tissues play a well-known role in physiology and disease. The model organism Caenorhabditis elegans exhibits mechanical properties that are still poorly understood, but are thought to be dominated by its collagen-rich outer cuticle. To our knowledge, we use a novel microfluidic technique to reveal that the worm responds linearly to low applied hydrostatic stress, exhibiting a volumetric compression with a bulk modulus, κ = 140 ± 20 kPa; applying negative pressures leads to volumetric expansion of the worm, with a similar bulk modulus. Surprisingly, however, we find that a variety of collagen mutants and pharmacological perturbations targeting the cuticle do not impact the bulk modulus. Moreover, the worm exhibits dramatic stiffening at higher stresses—behavior that is also independent of the cuticle. The stress-strain curves for all conditions can be scaled onto a master equation, suggesting that C. elegans exhibits a universal elastic response dominated by the mechanics of pressurized internal organs.     

Optimal Mating Strategies for Preferentially Outcrossing Simultaneous Hermaphrodites in the Presence of Predators

The optimal timing for initiating reproduction (i.e., the age at first reproduction) is a critical life history trait describing aspects of an individual’s resource-allocation strategy. Recent theoretical and empirical work has demonstrated that this trait is also tied to mating system expression when individuals have the opportunity to reproduce via both self-fertilization and cross-fertilization. A strategy of “delayed selfing” has emerged as a “best of both worlds” arrangement where, in the absence of a mate, an individual will delay reproduction (selfing) to “wait” for a mate. Herein, we extend previously developed predictive optimization models for the timing of reproduction to a situation where organisms can allocate their resources to size-dependent and size-independent defensive strategies to counter the threat of predation. By incorporating inducible defenses into a predictive framework for analyzing life history expression and evolution, we can more accurately evaluate the role that allocation strategy plays in altering the optimal waiting time. We compare our model to previous models and empirical results highlighting that incorporation of inducible defenses into the model broadens the parameter space in which a waiting time is expected and often leads to a predicted waiting time that is longer than in the situation without inducible defenses. In particular, a waiting time is predicted to exist regardless of the strength of inbreeding depression in the population.     

Rapid Experimental Evolution of Pesticide Resistance in C. elegans Entails No Costs and Affects the Mating System

Pesticide resistance is a major concern in natural populations and a model trait to study adaptation. Despite the importance of this trait, the dynamics of its evolution and of its ecological consequences remain largely unstudied. To fill this gap, we performed experimental evolution with replicated populations of Caenorhabditis elegans exposed to the pesticide Levamisole during 20 generations. Exposure to Levamisole resulted in decreased survival, fecundity and male frequency, which declined from 30% to zero. This was not due to differential susceptibility of males. Rather, the drug affected mobility, resulting in fewer encounters, probably leading to reduced outcrossing rates. Adaptation, i.e., increased survival and fecundity, occurred within 10 and 20 generations, respectively. Male frequency also increased by generation 20. Adaptation costs were undetected in the ancestral environment and in presence of Ivermectin, another widely-used pesticide with an opposite physiological effect. Our results demonstrate that pesticide resistance can evolve at an extremely rapid pace. Furthermore, we unravel the effects of behaviour on life-history traits and test the environmental dependence of adaptation costs. This study establishes experimental evolution as a powerful tool to tackle pesticide resistance, and paves the way to further investigations manipulating environmental and/or genetic factors underlying adaptation to pesticides.     

The C. elegans Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

Background

Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.

Methodology/Principal Findings

Here we exploit the simplicity of the nematode Caenorhabditis elegans to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes acr-18, acr-16 and unc-29) in favor of the backward command cells.

Conclusion/Significance

Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.     

Cuticle of Caenorhabditis elegans: its isolation and partial characterization

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross-linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.     

The Occurrence of a Secondary Cuticle in Libertia elegans (Iridaceae)

A study of the formation of cuticle in Libertia elegans hasshown that the cuticle develops centripetally and is producedcontinuously at its juncture with the underlying pectic layer.Peroxidase activity may be demonstrated in this region duringdevelopment. Growth occurs in two discrete phases; initiallywhilst the underlying epidermal cells are expanding and laterwhen such expansion is complete. The phases are morphologicallydistinct and it is proposed that the terms primary and secondarybe applied to them. Cuticular channels with a pit-like appearancein T.S. are described here for the first time as is the fissuringand active replacement of inelastic secondary cuticular materialto accommodate costal expansion and the growth of papillae.     

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.     

Characterization of the C. elegans erlin homologue

Background

Ovarian cancer is one of the most lethal malignancies in women, as it is frequently detected at an advanced stage, and cancers often become refractory to chemotherapy. Evidence suggests that dysregulation of pro-apoptotic genes plays a key role in the onset of chemoresistance. The secreted Frizzled-Related Protein (sFRP) family is pro-apoptotic and also a negative modulator of the Wnt signalling cascade. Studies have demonstrated that the re-expression of sFRPs, in particular sFRP4, is associated with a better prognosis, and that experimentally induced expression results in cell death.

Results

In vitro experimental models determined that sFRP4 was differentially expressed in chemosensitive (A2780) and chemoresistant (A2780 ADR and A2780 Cis) ovarian cell lines, with chemosensitive cells expressing significantly higher levels of sFRP4. Transfection of the chemoresistant cell lines with sFRP4 significantly increased their sensitivity to chemotherapy. Conversely, silencing of sFRP4 expression in the chemosensitive cell line resulted in a corresponding increase in chemoresistance. Comparison of sFRP4 expression in tumour biopsies revealed a positive trend between sFRP4 expression and tumour grade, with mucinous cyst adenocarcinomas exhibiting significantly decreased sFRP4 levels compared to mucinous borderline tumours.

Conclusions

This study indicates a role for sFRP4 as a predictive marker of chemosensitivity in ovarian cancer and suggests that this pathway may be worth exploiting for novel therapies.     

Genome-wide profiling of the C. elegans dsRNAome

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3′ UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.     

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.     

Neuronal polarity in C. elegans

Neuronal polarity sets the foundation for information processing and signal transmission within neural networks. However, fundamental question of how a neuron develops and maintains structurally and functionally distinct processes, axons and dendrites, is still an unclear. The simplicity and availability of practical genetic tools makes C. elegans as an ideal model to study neuronal polarity in vivo. In recent years, new studies have identified critical polarity molecules that function at different stages of neuronal polarization in C. elegans. This review focuses on how neurons guided by extrinsic cues, break symmetry, and subsequently recruit intracellular molecules to establish and maintain axon-dendrite polarity in vivo.     

Chemosensory signaling in C. elegans

The nematode Caenorhabditis elegans can sense and respond to hundreds of different chemicals with a simple nervous system, making it an excellent model for studies of chemosensation. The chemosensory neurons that mediate responses to different chemicals have been identified through laser ablation studies, providing a cellular context for chemosensory signaling. Genetic and molecular analyses indicate that chemosensation in nematodes involves G protein signaling pathways, as it does in vertebrates, but the receptors and G proteins involved belong to nematode-specific gene families. It is likely that about 500 different chemosensory receptors are used to detect the large spectrum of chemicals to which C. elegans responds, and one of these receptors has been matched with its odorant ligand. C. elegans olfactory responses are also subject to regulation based on experience, allowing the nematode to respond to a complex and changing chemical environment.