Bessou C et al. (1997) International C. elegans Meeting "SEROTONIN-CONTROLLED BEHAVIORS IN C. elegans"

Segalat LS, Bessou C

[International C. elegans Meeting, 1997]

Serotonin is a neuromodulator which acts mainly through seven transmembrane receptors (7-TMRs) coupled to heterotrimeric G-proteins. Upon ligand binding, G-proteins modulate the level of cytoplasmic second messengers, which in turns alter the activity of ion channels. Despite its importance in human physiology, there is still little known about the ion channels whose activity is regulated by serotonin. We are taking advantage of the fact that serotonin modulates locomotion and foraging, two behaviors of C.elegans easy to follow, to screen for mutants with altered behaviors. In the past, such screens have led to the identification of Go as an component of the serotonin signaling cascade in the worm (1). However, Go-deficient mutants are still partly responsive to serotonin, suggesting that serotonin might signal through other pathways as well. We have now screened for EMS-mutagenized animals that behave like wild-type animals exposed to ketanserin, a 5HT2 antagonist. We isolated 10 mutants displaying the same ketanserin-like phenotype, and representing 4 to 5 genes. Interestingly, none of the other serotonin controlled behaviors of the worm (i.e. eating, egg-laying, mating) seem affected by the mutations. We are now testing the response of these mutants to various drugs including serotonin and we have started to map them. (1) Segalat et al. Science, 267, 1648.

L Segalat et al. (1999) International C. elegans Meeting "Towards a genome-wide collection of transposon insertions"

M Labouesse, S Sookhareea, L Segalat, T Alvarez, C Bessou, O Hauser, R Legouis, F Schurr

[International C. elegans Meeting, 1999]

With the genome sequence being complete, it is essential to determine the function of each predicted gene. To facilitate the cloning of genes, it is also important to create as many polymorphisms as possible. With these goals in mind, we have launched a large scale project whose long-term objective is to isolate insertions of transposable elements in most C. elegans genes. To do so, we have chosen to use techniques based on the random insertion of natural worm transposons, an approach which has been pioneered in the Plasterk lab. Starting from mutator backgrounds, we generate clones and determine the position of new insertions by a modification of the transposon insertion display protocol described in the WBG (Vol 14, ndeg4 page 20), in which DNA flanking transposons can be amplified by anchored PCR, and sequenced. Assuming ideal statistical conditions (non-biased insertion sites, independence of insertion events and no intergenic regions), the Poisson distribution would predict that 30,000 independent sequence reads would be enough to hit 80% of the estimated 19,000 C. elegans at least once. In practice, since the last of these three conditions cannot be met (and assuming the other two are), 30,000 insertions should lead to an insertion every few kilobases, which would give a polymorphism coverage of the genome much higher than the current one. It is expected that approximately a quarter of these insertions will be in coding sequences and UTRs (representing. potential mutations). This project is a complementary alternative to the gene-directed PCR-based search for deletions. We also believe that this project (currently estimated at $2-4 Million for 30,000 sequence reads) is competitive for cost and labor compared to gene targeting. Furthermore, transposon insertions should potentially provide a wider spectrum of alterations, which will be needed as genetic tools besides the knock-outs. We are currently running small-scale pilot experiments on a few hundred clones to optimize the protocols and validate the approach.

CJ Franks et al. (1999) International C. elegans Meeting "Evaluation of Excitation-Contraction Coupling in C. elegans Muscle."

L Holden-Dye, C Rogers, ET Claverol, JE Chad, RJ Walker, CJ Franks

[International C. elegans Meeting, 1999]

Electrophysiology is a vital technique for the phenotypic analysis of C. elegans muscle (Bessou et al . 1998). However, excitation-contraction coupling consists of a series of phenomena, only some of which manifest themselves as measureable change in the electrical properties of the cells. For example, systems that modulate realease of calcium from intracellular compartments, or calcium sensitivity of the contractile apparatus, can profoundly affect muscle function. We are currently developing an automated system for the correlation of electrophysiological and imaging data that provides a robust measure of the latency between action potentials and the muscle contractions with which they are associated. Video images of the muscle preparation are acquired using a standard black and white CCD camera and the data are recorded to hard disk via a proprietary frame grabber. Single electrode, membrane potential recordings are made from pharyngeal muscle in the manner described by Franks et al . (1997) and data are recorded using an Axon instruments Digidata multichannel acquisition system. One channel is used to record the electrophysiological data while the second records the output from a circuit designed and built 'in-house'. This circuit is connected between the CCD and the frame grabber. It essentially splits the video signal in two. The first output from the circuit, contains the full video signal and is recorded by the frame grabber. The second output contains only the vertical sync signal, stripped from the original video feed. This timing signal is recorded on channel two of the acquisition system producing a timestamp for each frame of the video. We have also designed an image processing algorithm that makes it possible to quantify the degree of muscle contraction in each video frame. Thus we have a standardised and automated system that can correlate electrophysiological and video data with a temporal resolution limited only by that of the CCD. We are using the system to analyse disruption of E-C coupling by pharmacological agents and to assess phenotypic variation between wild-type and mutant C. elegans . We will report on our progress at the meeting. Bessou, et al . (1998). Neurogenetics . In Press. Franks et al . (1997) J. Physiol. Lond. 504P: 15.

Sushant Khandekar et al. (2001) International C. elegans Meeting "A role of sarcoglycans in C. elegans"

John Plenefisch, Sushant Khandekar

[International C. elegans Meeting, 2001]

In vertebrates, muscle-wasting disorders arise from mutation in dystrophin and dystrophin associated proteins such as the sarcoglycans. Sarcoglycans are trans-membrane proteins that are part of the dystroglycan complex that links dystrophin to the plasma membrane in muscle cells (1). In humans, limb girdle muscular dystrophy arises from mutation of sarcoglycan proteins, which leads to sarcolemma dysfunction and ultimately necrosis of the skeletal muscle cells (1). In C elegans many of the components of the dystrophin/dystroglycan complex have been conserved, including dystrophin, dystroglycan and three sarcoglycans (alpha, beta, gamma) (2). Surprisingly, the phenotype of mutated dystrophin in C. elegans is mild; mutants display a hyperactive phenotype but no muscle degeneration (3), whether mutating other genes within the C elegans dystrophin/dystroglycan complex has a more severe effect remains to be determined. We are investigating the functional role of the sarcoglycans using RNAi in variety of different muscle mutant background. Moreover, antibody staining and/or GFP reporter gene analysis will examine the distribution and expression of all three sarcoglycans. In addition to examining the functions of the C elegans homologs of classic vertebrate muscular dystrophy genes, we are interested in molecular characterization of genes in C elegans initially identified through a dystrophic paralytic phenotype. These genes include the mua-1 (for m uscle a ttachment-1) gene. MUA-1 is a zinc finger protein of the EKLF family and is required for normal muscle attachment to hypodermis. Mutations in the mua-1 results in postembryonic paralysis and progressive detachment of body wall muscles from the hypodermis. We are determming the molecular lesions of mua-1 mutants alleles as well as using RNAi to help define the null phenotype of this gene. Culligan et.al. International Journal of Molecular Medicine (1998) 2:639-648. Hutter et.al. Science (2000) 287:989-994 Bessou et.al. Neurogenetics (1998) 2:61-72

Kathrin Gieseler et al. (2003) International Worm Meeting "DYSTROPHIN AND ASSOCIATED PROTEINS IN C. elegans"

Marie-Christine Mariol, Kathrin Gieseler, Karine Grisoni, Maite Carre-Pierrat, Laurent Segalat, Edwige Martin

[International Worm Meeting, 2003]

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy, a neuromuscular disease leading to muscle necrosis. The function of the dystrophin protein is not known. In mammals, dystrophin is located under the muscle plasma membrane, and is associated with a protein complex spanning the membrane. The C. elegans genome contains a dystrophin homologue named dys-1. Our goal is to understand dystrophin function in C. elegans. Loss-of-function mutations in the dys-1 gene do not alter the muscle structure, but make animals hyperactive and slightly hypercontracted. In a forward genetics approach, we isolated mutations with dys-1-like phenotypes affecting four additional genes: dyb-1 (Gieseler et al., 2001), dyc-1 (Gieseler et al. 2000), dys-4 and slo-1. dyb-1 is the C. elegans homologue of dystrobrevins, a family of proteins belonging to the dystrophin complex and known to bind dystrophin directly through coiled-coil domains present on each protein The DYC-1 protein is not homologous to any known mammalian proteins, but comprises two regions of similarity to CAPON, a nNOS-binding protein. Like DYS-1 and DYB-1, DYC-1 contains a putative coiled-coil domain. A search for protein motifs with the Prosite program reveals a high number of putative PKA- and PKC-dependent phosphorylation sites in DYC-1. Since C. elegans possesses a dystrophin-like and a dystrobrevin gene, we investigated whether other homologues of dystrophin associated proteins known in mammals could also be found in the C. elegans genome. Our results strongly suggest that a protein complex comprising functional analogies with that of mammals exists in C. elegans (Grisoni et al. 2002). The DYC-1, SLO-1 and DYS-4 proteins may be part of this complex in C. elegans. In order to verify this hypothesis and to identify other members of the dystrophin associated protein complex , we are using the yeast two hybrid system. Bessou et al. (1998) Neurogenetics 2: 61-72. Gieseler et al. (2000) Current biology 10:1092-7. Gieseler et al. (2001) J Mol Biol.307 :107-17. Grisoni et al. (2002) Gene 294 : 77-84.

Jennifer L Hueston et al. (2005) International Worm Meeting "Identification of a new genetic modifier of the dystrophin phenotype in Caenorhabditis elegans"

Matthew J Buechner, Erik A Lundquist, Kathy A Suprenant, Jennifer L Hueston

[International Worm Meeting, 2005]

While mutation of the dystrophin gene is critical for the development of muscular dystrophy, accumulating evidence indicates that additional genes contribute to the modulation of the dystrophin mutant phenotype. C. elegans adults with knockout mutations in the single dystrophin gene (dys-1(cx18)) display behavioral defects that include hyperactivity, an exaggerated bending of the head, and a tendency to be slightly hypercontracted. There is no sign of muscle degeneration and the animals are viable (Bessou et al., 1998). We find that loss of a novel microtubule binding protein, ELP-1, acts as a genetic modifier of the dystrophin phenotype. To elucidate the function of ELP-1, we carried out a pilot RNA interference screen to search for candidate ELP-1 interactors in muscle cells. While loss of ELP-1 alone does not cause a severe phenotype, loss of ELP-1 greatly exacerbates the dystrophin mutant motility and egg laying defects that eventually die. The body wall muscles, vulval muscles, and proximal myoepithelia surrounding the gonads all exhibit varying degrees of hypercontractility and/or attachment defects and in severe cases large vacuoles surround the gonads. We conclude that ELP-1 may contribute to the mechanical stability or cell signaling events that originate at cell adhesion sites.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.