Yandell MD et al. (1995) International C. elegans Meeting "EXPRESSION OF THE C. ELEGANS TGFb HOMOLOG CEG-1"

Yandell MD, Wood WB

[International C. elegans Meeting, 1995]

We have used degenerate PCR to clone a C. elegans homolog of the dpp/BMP2,4 subclass from the TGFb superfamily; we have located it on the left arm of LGV and provisionally called it ceg-1 (for C. elegans growth factor) (WBG 13 (4):65). Sequencing of genomic and cDNA clones has shown that the ceg-1 gene contains seven introns (previously incorrectly reported as 8) and encodes a 1.7 kb mRNA which is transspliced to SL1. In transgenic animals carrying a construct that includes 4.4kb of ceg-1 upstream sequence fused to lacZ, staining first appears in a quartet of cells at about the 100 cell stage of embryogenesis and by the comma stage is present in two rows of cells along the ventral midline. Pretzels and early L1s show staining in what appear to be neuronal and hypodermal nuclei in the head, the juvenile cells of the ventral nerve cord (VNC) and a single cell in the tail. By late L1 there are additional staining VNC cells, suspected to be the Pn cells which migrate ventrally into the VNC. In males, staining is also present in what may be the preanal equivalence group, the post-cloacal sensilla and the dorsal-rectal ganglion. Over-expression of ceg-1 in transgenic animals carrying a ceg-1-cDNA construct driven by a heat-shock promoter show a variety of phenotypes which are consistent with the ceg-1-lacZ expression pattern. Early heat shocks cause embryos to arrest at the comma stage, while later heat shocks produce severely constipated L1 larvae with marked hypodermal defects and notched heads. In progress are further identification of expressing cells, examination of ceg-1 overexpression effects on the male tail, and analysis of ceg-1 expression in Hox gene mutant backgrounds.

Yandell MD et al. (1991) International C. elegans Meeting "PSORALEN CAUSES SMALL UNC-22 DELETIONS AT A HIGH FREQUENCY"

Edgar LG, Wood WB, Yandell MD

[International C. elegans Meeting, 1991]

We have analysed 23 trimethylpsoralen (TMP)-induced alleles of unc- 22 by Southern blot for RFLPs, using the probes DM17, DM18, DM20, DM22, SL13, and SL14 (many thanks to Carol Trent and Guy Benian!), which cover the entire coding region and about 11 kb of 5' upstream sequence. Eighteen of these mutations were obtained in a single mutagenesis, screening for both homozygotes and heterozygotes (mutation rate of lx10-4). In this mutagenesis, 15 strains were homozygous-viable, and 8 were originally homozygous-lethal. Two of these, however, were homozygous-viable after backcrossing; a third was successfully balanced over sDf22, and 5 were lost before analysis. Of the mutants analysed (the 18 remaining strains and an additional 4 homozygous- viable mutations isolated in previous TMP screens), 9 of the homozygous-viable mutants and the balanced homozygous-lethal mutant show visible polymorphisms within the unc-22 locus. All but one, which remains-ambiguous, were deletions ranging from 0.15 to 10 kb. The average deletion size was 2.2 kb; if the single 10-kb deletion is not included the average size was only 0.94 kb. Therefore, among mutations analyzed from the inclusive screen for both homozygotes and heterozygotes, 44% (8/18) were deletions of less than 10 kb in size. These data suggest that TMP produces small deletions at a higher frequency than any other mutagen in current use, which should be useful in generating mutations causing complete loss of function and likely to be associated with DNA polymorphisms.

Yandell MD et al. (1997) International C. elegans Meeting "SOME STATISTICS ON THE RAPID EVOLUTION OF C. elegans PROTEIN SEQUENCES"

Waterston RH, Korf I, Yandell MD

[International C. elegans Meeting, 1997]

One way to measure the relative rate at which a gene is evolving in two different organisms is to perform a relative rates test. This test uses the relative similarities of a group of orthologus sequences from three or more organisms to determine the relative amount of change in the sequences during the time elapsed since they last shared a common ancestor. C. elegans genes, for example, are often less similar to their orthologs in yeast, than are their counterparts from Drosophila and humans. Similar trends in the conservation of rRNA sequences have lead to the wide-spread belief that C. elegans belongs to a rapidly evolving lineage. There is as yet, however, little information available on how this excess divergence is distributed among different C. elegans proteins. Are all C. elegans proteins more or less equally divergent, or are some C. elegans proteins actually more conserved than their counterparts in other, more slowly evolving, animals. In an attempt to get at this and some related questions, we are examining the relative conservation of several gene families for which there exist orthologs, as determined by bootstrap analysis or synteny, in C. elegans, in a second triploblastic animal, and in a third, more distantly related, organism such as hydra or yeast. These data will then be broken up by gene class and function in the hope that such comparisons will reveal some interesting trends in the rates of evolution of different gene families in different organisms. Finally, EST-sequence data from the nematodes C. elegans, C. briggsae and P. pacificus will also be subjected to a similar analysis in order to examine the variation in the rate of sequence evolution within the nematodes.

Yandell MD et al. (1993) International C. elegans Meeting "Back through the eye of the needle: TGF-b's and reverse genetics."

Yandell MD, Edgar LG, Perry MD, Wood WB

[International C. elegans Meeting, 1993]

Suzuki Y et al. (1997) International C. elegans Meeting "MUTATION OF A C. ELEGANS BMP HOMOLOGUE (dbl-1) APPEARS TO AFFECT BODY SIZE AND MALE TAIL PATTERNING"

Culotti JG, Wood WB, Morris GA, Yandell MD, Roy PJ, Suzuki Y

[International C. elegans Meeting, 1997]

Bone morphogenetic proteins (BMPs) of the transforming growth factor-beta (TGF-beta) superfamily play important roles in development. To further explore their importance in C. elegans, we undertook to find a mutation in the molecularly identified C. elegans BMP-related gene dbl-1 V (decapentaplegic, BMP2,4-like)(1). Screening of a Tc1 insertion library for a mutant allele identified a mutant line that has a Tc1 in exon 4, C-terminal to the first cysteine in the predicted mature domain of the processed protein. The mutation is recessive, and the mutant animals are small (Sma) and male abnormal (Mab). The same phenotypes result from mutations in the daf-4, and sma-2, 3, 4 genes, which encode components of a TGF-beta signal transduction pathway(2). This suggests that dbl-1 may encode the previously unidentified ligand for this pathway. We are currently testing genetic interactions between dbl-1 and these genes. (1) Yandell et al. (1993) WBG 12(5):82; (1994) WBG 13(4):65; (1996) WBG 14(2):41 (2) Savage et al. (1996) Proc. Natl. Acad. Sci. USA 93:790-794

Baritaud, Mathieu et al. (2015) International Worm Meeting "Targeting mitochondria in muscular dystrophies through acting on ROS, mitohormesis and programmed cell death pathways ."

Kretz-Remy, Carole, Pierson, Laura, Baritaud, Mathieu, Walter, Ludivine, Mariol, Marie-Christine, Chazaud, Benedicte, Gieseler, Kathrin, Martin, Edwige

[International Worm Meeting, 2015]

Muscular dystrophies are characterized by muscle weakness and degeneration induced by genetic mutation of muscle structure or signaling components. The development of treatments against dystrophies is challenging, in part due to various primary genetic defects affecting different cellular functions. Nonetheless, many studies observed mitochondrial dysfonctions in muscle pathologies. Moreover, using a worm model of Duchenne Muscular Dystrophy (DMD) our team clearly demonstrated an implication of the mitochondrial key effectors Dynamin Related Protein-1, (DRP-1) and cytochrome c in the early process of muscle degeneration (MD). We are now focusing on Reactive Oxygen Species (ROS) and Programmed Cell Death (PCD) pathways to identify common mitochondrial disorders that contribute to MD in muscular dystrophies. We established different C. elegans models that mimic human muscular dystrophies such as Duchenne and Becker dystrophies, Limb-Girdle, Emery-Dreifuss or congenital muscular dystrophy. We revealed in the C. elegans model for DMD that MD can be reduced by treatments with the mitohormetic compound Paraquat (0,1mM) or the antioxidant compounds NAC and vitamin C. This observation suggested that ROS pathways mediated by mitochondria could be targets to reduce MD. In addition, our preliminary results indicate that these treatments are also efficient against MD in other C. elegans models for human muscular dystrophies. Moreover, using a gain of function mutation in the anti-apoptotic effector ced-9 gene (ortholog of Bcl-2) and RNA interference of key molecular actors of PCD linked to mitochondria: ced-3, ced-4, ced-13 and egl-1, we investigated whether specific mitochondrial PCD pathways are involved in MD. To validate our data obtained in C. elegans, we use human immortalized myoblasts from healthy or DMD patients. Preliminary data indicated that DMD myoblasts proliferate less and have a reduced fusion capacity during myogenic differentiation than myoblasts from healthy patients. Ongoing investigations examine mitochondrial pathways identified in C. elegans by challenging myoblasts for proliferation, cell fusion and oxidative stress sensibility.Our original approach using both worm models and human myoblasts, will allow us to establish whether manipulating mitochondrial pathways, particularly ROS signaling and PCD pathways can reduce progression of MD in muscular dystrophies.

Hongkyun Kim et al. (2002) West Coast Worm Meeting "A Genetic Study of Muscular Dystrophy in C. elegans"

Hongkyun Kim, Steven L McIntire

[West Coast Worm Meeting, 2002]

Muscular dystrophy (MD) is a common genetic disorder characterized by progressive muscle weakness and wasting. Mutations in dystrophin were identified as one cause of this disease in humans. Mutations in the C. elegans homologue of the dystrophin gene, dys-1, cause exaggerated bending of the anterior body during locomotion and muscle degeneration in a sensitized genetic background (1, 2).

Brewer, Jacob et al. (2017) International Worm Meeting "The HSN command neuron co-releases serotonin and NLP-3 neuropeptides to activate egg laying."

Brewer, Jacob, Koelle, Michael

[International Worm Meeting, 2017]

Major depression (MD), one of the most common mental disorders, is highly heritable and is commonly treated with selective serotonin reuptake inhibitors (SSRIs), which increase serotonin signaling in the brain. Yet, genome-wide association studies have failed to definitively implicate genetic defects in serotonin signaling as the cause of MD.1 We are studying the mechanism by which serotonin activates egg laying in C. elegans as a model for understanding the role of serotonin signaling in the activity of neural circuits. The C. elegans egg-laying system is well-suited for studying how serotonin functions in a circuit. The serotonergic HSN motor neurons initiate ~2.5 minute egg-laying active phases that occur every ~20 minutes. The evidence for this is that rhythmic HSN Ca2+ activity measured using a GCaMP reporter always accompanies egg-laying active phases, and optogenetic activation of HSN neurons is sufficient to stimulate activity of the egg-laying circuit.2 Interestingly, while egg laying requires the serotonergic HSNs3 and can be hyperactivated with an SSRI4, mutations knocking out serotonin biosynthesis have only weak effects on egg laying. This puzzling result draws striking parallels with the role of serotonin signaling in MD. We found that the HSNs release a neuropeptide in addition to serotonin to stimulate egg laying. The nlp-3 neuropeptide gene as well as the enzymes necessary for serotonin biosynthesis and release are all expressed in the HSNs. Knocking out either nlp-3 or serotonin biosynthesis caused mild egg-laying defects, but knocking out both caused a strong egg-laying defect that phenocopies that of animals lacking HSNs. Further, we optogenetically stimulated the HSNs and observed that the resulting egg-laying behavior is profoundly dependent on nlp-3, and is completely silenced in animals lacking both NLP-3 neuropeptides and serotonin. We propose that the HSN releases both serotonin and NLP-3 neuropeptides, which act cooperatively in a partially redundant fashion to stimulate egg laying, explaining the absence of strong egg-laying defects in mutants lacking serotonin. Serotonin receptors are found only on the muscles within this circuit, and sensitize them to contract in response to acetylcholine.2 We are currently analyzing the Ca2+ activity in the circuit to determine roles for NLP-3 neuropeptides in circuit activity. Co-release of serotonin and neuropeptides also occurs in the mammalian brain, and by analogy with the worm egg-laying circuit may be an important feature in explaining the absence of a strong genetic link between MD and serotonin. 1Flint and Kindler. Neuron. 2014. 2Collins, et al. eLife. 2016. 3Trent, et al. Genetics. 1983. 4Weinshenker, et al. J. Neurosci. 1995.

Suzuki Y et al. (1998) East Coast Worm Meeting "A C. elegans BMP2,4 HOMOLOGUE (dbl-1) FUNCTIONS IN BODY SIZE DETERMINATION AND MALE TAIL PATTERNING"

Suzuki Y, Ross R, Padgett RW, Mueller F, Morris GA, Roy PJ, Yandell MD, Fleischmann M, Krishna S, Savage C, Wood WB

[East Coast Worm Meeting, 1998]

We used degenerate PCR to clone a C. elegans decapentaplegic (dpp)/BMP2,4 homologue designated dbl-1 (dpp, BMP2,4-like). Loss-of-function (lf) mutations in dbl-1 obtained by both reverse and forward genetics result in animals with reduced body size and defective rays and spicules in the male. Overexpression of dbl-1 causes increased body size (Lon phenotype) and complementary male tail defects. Defects similar to the dbl-1(lf) phenotype result from mutations in genes that encode receptors (DAF-4, SMA-6) and Smad gene products (SMA-2, -3, -4) thought to transduce signals from TGF-beta-family ligands, suggesting that dbl-1 encodes a previously unidentified ligand for this pathway. In support of this view, genetic tests indicate that DBL-1 acts upstream of the known receptors and Smad transducing factors. dbl-1 reporter genes are expressed primarily in neurons, most notably of the ventral and dorsal nerve cords. The effects of dbl-1(lf) mutations on the rays of the male tail appear to be dorsal-to-ventral transformations of tail ray morphologies. Because the labial homologue ceh-13 is expressed in the male tail during its morphogenesis, and because Decapentaplegic (Dpp) in Drosophila activates labial in the midgut, we tested for a similar interaction in C. elegans and showed that expression of a ceh-13 reporter in the male tail is greatly reduced in a dbl-1(lf) mutant background. These results suggest that 1) BMP signaling from the central nervous system along the length of the animal might affect determination of body size, 2) DBL-1 acts as a dorsalizing factor in male tail patterning, and 3) control of a labial-like Hox gene by a Dpp-like signal may be a conserved interaction in development.

Katherine Schouest et al. (2007) International Worm Meeting "Development of a C. elegans model system for genetic and molecular dissection of epigenetic mechanisms underlying trinucleotide expansion disorders."

Charles Spillane, Katherine Schouest, Avril Coghlan

[International Worm Meeting, 2007]

Expansion of nucleotide repeats is now recognized as a cause of at least 20 different developmental and degenerative disorders in humans. Nucleotide-repeat expansion diseases can be classified into 3 main categories. The first disease category consists of loss-of-function recessive mutations due to disruption of gene expression (e.g. Friedrichs ataxia). The second category involves the expansion of nucleotide repeats (e.g. CAG) within exons, which are then translated into poly-amino acid (e.g. polyglutamine) stretches within the corresponding protein (e.g. Huntingtons Disease: HD). The third category involves expansions within non-coding regions of a gene, such as intronic or untranslated regions (e.g. Myotonic Dystrophy: MD). Model organisms are essential for the rapid advancement of genetic research on human diseases. C. elegans has already proven to be a powerful biomedical model in dissecting principles underlying diseases like HD or MD, as well as in revealing potential targets for therapeutic treatment. We are using C. elegans as a model organism to investigate epigenetic phenomena associated with nucleotide repeat disorders and to identify modifiers that regulate the size and rate of the expansions. To identify candidate genes for our research we have screened the C. elegans genome for trinucleotide repeats that have at least 12 trinucleotide repeats and a high level of purity, as these are most likely to undergo expansion or contraction. We have so far identified 20 such repeats located in C. elegans exons and 17 such repeats located within introns. To select one or more variable repeats for further analysis, the polymorphism of these repeats are currently being examined over 48 wild isolates of C. elegans. Once variable repeats have been identified, these alleles will be monitored through multiple generations in control animals and animals depleted of candidate modifiers via RNAi. The project will ultimately facilitate the understanding of the epigenetic phenomena that can cause nucleotide expansion disorders.