Dopamine signaling architecture in Caenorhabditis elegans

1. AIMS: In this review, we highlight the identification and analysis of molecules orchestrating dopamine (DA) signaling in the nematode Caenorhabditis elegans, focusing on recent characterizations of DA transporters and receptors. 2. METHODS: We illustrate the isolation and characterization of molecules important for C. elegans DA synthesis, packaging, reuptake and signaling and examine how mutations in these proteins are being exploited through in vitro and in vivo paradigms to yield novel insights of protein structure, DA signaling pathways and DA-supported behaviors. 3. RESULTS: DA signaling in the worm, as in man, arises by synaptic and nonsynaptic release from a small number of cells that exert modulatory control over a larger network underlying C. elegans behavior. 4. CONCLUSIONS: The C. elegans model system offers unique opportunities to elucidate ill-defined pathways that support DA release, inactivation, and signaling in addition to clarifying mechanisms of DA-mediated behavioral plasticity. Further use of the model offers prospects for the identification of novel genes and proteins whose study may yield benefits for DA-supported neural disorders in man.     

The Drosophila Toll signaling pathway

The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.     

Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.     

An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.

Mutations in daf-2 and age-1 cause a dramatic increase in longevity as well as developmental arrest at the dauer diapause stage in Caenorhabditis elegans. daf-2 and age-1 encode components of an insulin-like signaling pathway. Both daf-2 and age-1 act at a similar point in the genetic epistasis pathway for dauer arrest and longevity and regulate the activity of the daf-16 gene. Mutations in daf-16 cause a dauer-defective phenotype and are epistatic to the diapause arrest and life span extension phenotypes of daf-2 and age-1 mutants. Here we show that mutations in this pathway also affect fertility and embryonic development. Weak daf-2 alleles, and maternally rescued age-1 alleles that cause life span extension but do not arrest at the dauer stage, also reduce fertility and viability. We find that age-1(hx546) has reduced both maternal and zygotic age-1 activity. daf-16 mutations suppress all of the daf-2 and age-1 phenotypes, including dauer arrest, life span extension, reduced fertility, and viability defects. These data show that insulin signaling, mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates reproduction and embryonic development, as well as dauer diapause and life span, and that DAF-16 transduces these signals. The regulation of fertility, life span, and metabolism by an insulin-like signaling pathway is similar to the endocrine regulation of metabolism and fertility by mammalian insulin signaling.     

Generation of an external guide sequence library for a reverse genetic screen in Caenorhabditis elegans

Background  

A method for inhibiting the expression of particular genes using external guide sequences (EGSs) has been developed in bacteria, mammalian cells and maize cells.     

A mutational analysis of Caenorhabditis elegans in space

The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.     

A uniform genetic nomenclature for the nematode Caenorhabditis elegans.

Summary A uniform system of genetic nomenclature for the nematode Caenorhabditis elegans is described. Convenient ways are specified to designate genes, mutations and strains, and to attempt to avoid name duplications.     

Quantitative genetic analysis of life-history traits of Caenorhabditis elegans in stressful environments

In Figure 1 of [Harvey et al (Evolutionary Biology 2008, 8:15)] the plotted data were inverted. The correct Figure is shown below. The text and statistical analyses in [Harvey et al (Evolutionary Biology 2008, 8:15)] are correct.     

High-throughput reverse genetics: RNAi screens in Caenorhabditis elegans

Two recent chromosome-wide screens for phenotypes caused by RNA-mediated interference (RNAi) in Caenorhabditis elegans have increased our understanding of essential genes in nematodes. These papers represent a major advance in functional genomics.     

5'-AMP-activated protein kinase signaling in Caenorhabditis elegans

5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.     

Targets of TGF-beta signaling in Caenorhabditis elegans dauer formation

Caenorhabditis elegans dauer formation is controlled by multiple environmental factors. The chemosensory neuron ASI regulates dauer formation by secretion of DAF-7/TGF-beta, but the molecular targets of the DAF-7 ligand are incompletely defined and the cellular targets are unknown. We genetically characterized and cloned a putative transducer of DAF-7 signaling called daf-14 and found that it encodes a Smad protein. DAF-14 Smad has a highly unusual structure completely lacking the N-terminal domain found in all other Smad proteins known to date. daf-14 genetically interacts with daf-8, which encodes another Smad, and the interaction suggests partial functional redundancy between these two Smad proteins. We also studied the cellular targets of DAF-7 signaling by studying the sites of action of daf-14 and daf-4, the putative receptor for DAF-7. daf-14::gfp is expressed in multiple tissues that are remodeled during dauer formation. However, analysis of mosaics generated by free duplication loss and tissue-specific expression constructs indicate cell-nonautonomous function of daf-4, arguing against direct DAF-7 signaling to tissues throughout the animal. Instead, these experiments suggest the nervous system as a target of DAF-7 signaling and that the nervous system in turn regulates dauer formation by other tissues.     

A systematic analysis of protein palmitoylation in Caenorhabditis elegans

Background

Palmitoylation is a reversible post-translational protein modification which involves the addition of palmitate to cysteine residues. Palmitoylation is catalysed by the DHHC family of palmitoyl-acyl transferases (PATs) and reversibility is conferred by palmitoyl-protein thioesterases (PPTs). Mutations in genes encoding both classes of enzymes are associated with human diseases, notably neurological disorders, underlining their importance. Despite the pivotal role of yeast studies in discovering PATs, palmitoylation has not been studied in the key animal model Caenorhabditis elegans.

Results

Analysis of the C. elegans genome identified fifteen PATs, using the DHHC cysteine-rich domain, and two PPTs, by homology. The twelve uncategorised PATs were officially named using a dhhc-x system. Genomic data on these palmitoylation enzymes and those in yeast, Drosophila and humans was collated and analysed to predict properties and relationships in C. elegans. All available C. elegans strains containing a mutation in a palmitoylation enzyme were analysed and a complete library of RNA interference (RNAi) feeding plasmids against PAT or PPT genes was generated. To test for possible redundancy, double RNAi was performed against selected closely related PATs and both PPTs. Animals were screened for phenotypes including size, longevity and sensory and motor neuronal functions. Although some significant differences were observed with individual mutants or RNAi treatment, in general there was little impact on these phenotypes, suggesting that genetic buffering exists within the palmitoylation network in worms.

Conclusions

This study reports the first characterisation of palmitoylation in C. elegans using both in silico and in vivo approaches, and opens up this key model organism for further detailed study of palmitoylation in future.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-841) contains supplementary material, which is available to authorized users.     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(?) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(?) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(?) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(?), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(?) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frameshift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD.     

lon-1 regulates Caenorhabditis elegans body size downstream of the dbl-1 TGF beta signaling pathway

Although the primitive vasculature is identical in XX and XY genital ridges until 11.5 days postcoitum (dpc), by 12.5 dpc the XY gonad develops a distinct vasculature. This male-specific vasculature, which includes the development of a large coelomic vessel, develops coincident with expression of Sry and formation of testis cords. We show that similar levels of proliferation and vasculogenesis expand the primary vasculature in XX and XY gonads. However, soon after Sry expression begins, the XY gonad recruits a large number of endothelial cells from the adjacent mesonephros, a mechanism totally absent in XX gonads. These migrating cells do not contribute to venous or lymphatic development. Instead, these cells contribute to the arterial system, as indicated by expression of ephrinB2 and by elements of the Notch signaling pathway. This newly formed arterial system establishes a new pattern of blood flow in the XY gonad, which we speculate may have an important role in export of testosterone to masculinize the XY embryo.     

Targets of TGF beta-related signaling in Caenorhabditis elegans.

With the characterization of the Smads 5 years ago, it became possible to trace the TGFβ signal transduction pathway from the plasma membrane to the nucleus. Since that time, many Smad interaction partners, cofactors and target genes have been identified using a variety of experimental approaches and model systems. Understanding how these partners generate tissue specificity and crosstalk between pathways is an ongoing pursuit for the field of TGFβ signal transduction. The nematode Caenorhabditis elegans provides a simple, genetically tractable model organism in which to address this goal. This review will examine progress towards the identification of cellular and molecular targets of TGFβ-related signaling in C. elegans.     

High genetic diversity in the chemoreceptor superfamily of Caenorhabditis elegans

We investigated genetic polymorphism in the Caenorhabditis elegans srh and str chemoreceptor gene families, each of which consists of approximately 300 genes encoding seven-pass G-protein-coupled receptors. Almost one-third of the genes in each family are annotated as pseudogenes because of apparent functional defects in N2, the sequenced wild-type strain of C. elegans. More than half of these "pseudogenes" have only one apparent defect, usually a stop codon or deletion. We sequenced the defective region for 31 such genes in 22 wild isolates of C. elegans. For 10 of the 31 genes, we found an apparently functional allele in one or more wild isolates, suggesting that these are not pseudogenes but instead functional genes with a defective allele in N2. We suggest the term "flatliner" to describe genes whose functional vs. pseudogene status is unclear. Investigations of flatliner gene positions, d(N)/d(S) ratios, and phylogenetic trees indicate that they are not readily distinguished from functional genes in N2. We also report striking heterogeneity in the frequency of other polymorphisms among these genes. Finally, the large majority of polymorphism was found in just two strains from geographically isolated islands, Hawaii and Madeira. This suggests that our sampling of wild diversity in C. elegans is narrow and that identification of additional strains from similarly isolated regions will greatly expand the diversity available for study.