Zhao, P. et al. (2019) International Worm Meeting "Femtosecond laser microdissection isolates single C. elegans neurons for nerve regeneration RNA-seq studies."

Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, R.

[International Worm Meeting, 2019]

C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.

Carre-Pierrat M et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Caenorhabditis elegans as a screening tool to find active compounds and targets in rare neuromuscular diseases."

Carre-Pierrat M, Cisek J, Segalat L, Giacomotto J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Current high-throughput screening methods for drug discovery rely upon the existence of targets and therefore on the understanding of the pathogenesis of a disease. In the case of diseases where the pathogenesis is not yet known, drug discovery is only feasible on model organisms mimicking the disease. The nematode Caenorhabditis elegans, which combines genetic amenability, low cost and easy culture conditions, is compatible with large-scale screens. Here, we report the results of screens of chemical libraries on Caenorhabditis elegans models of the Duchenne Muscular Dystrophy, the Spinal Muscular Amyotrophy and the Schwartz-Jampel syndrome. We present the methodology used for each model to screen up to 7,000 compounds and the results of these screening campaigns. We further present the validation of our best hits in mammalian models and try to understand their mechanism of action.

David S Peal et al. (2001) International C. elegans Meeting "Chemical and Genetic Inhibition of Sulfation in C. elegans"

David S Peal, Laura L Kiessling

[International C. elegans Meeting, 2001]

In order to examine the process of sulfation in C. elegans, sulfation was inhibited chemically using sodium chlorate, and genetically using the process of RNA-mediated interference (RNAi). Sodium chlorate inhibition during early larval stages resulted in a dose-dependant developmental delay. BLAST searches of characterized sulfotransferases against the worm genome resulted in the identification of 4 putative sulfotransferases: C34F6.4 and F08B4.6 (previously identified: [1] and [2]), F40H3.5, and Y34B4A.e. RNAi of the putative N-deacetylase/N-sulfotransferase F08B4.6 resulted in "stacking" of eggs in the gonad, along with eggs laid at the 2- and 4-celled stage. RNAi of the putative hexuronic 2-O sulfotransferase C34F6.4 resulted in a shortened, bulbous gonad. These initial results indicate that sulfation may be important during development of C. elegans. [1] Shworak, NW, Liu, J, Fritze, LMS, Schwartz, JJ, Zhang, L, Logeart, D, Rosenberg, RD. JBC 272: 28008-19 (1997). [2] Kobayashi, M, Sugumaran, G, Liu, J, Shworak, NW, Silbert, JE, Rosenberg, RD. JBC 274: 10474-80 (1999).

Kiontke K et al. (2000) European Worm Meeting ""Saltational evolution" of phasmid position in nematodes"

Sudhaus W, Kiontke K, Fitch DHA

[European Worm Meeting, 2000]

Phasmids are structures in the tail region of secernentean nematodes. The two large groups within Nematoda, Secernentea and "Adenophorea", are distinguished by this character: "Adenophorea" lack phasmids. Phasmids are similarly built in C. elegans(Rhabditidae) Tylenchidae and Filariidae. They consist of 1-2 socket cells that contact the body epidermis, a glandular sheath cell, and one or two sensory processes projecting into a receptor cavity within the sheath cell. Through a pore in the socket cell, these recessed processes are exposed to the exterior. Phasmids are generally described for females of most secernentean species. However, especially in Rhabditidae, phasmids in males have rarely been reported. This is due to the fact that in males phasmids are easily confused with rays if they are integrated into the velum (Fitch & Emmons, 1995). With SEM and interference contrast LM, the pore in the phasmid socket cell is clearly visible, whereas in rays either one sensory process protrudes through an opening in the structural cell or this opening is very small. We studied 53 species of Rhabditidae including Heterorhabditis as well as Diplogastrina, Panagrolaimidae, Cephalobidae, Brevibuccidae, Myolaimus, Steinernema, and Strongylida with LM and SEM, and scanned the literature on animal parasitic Secernentea. Phasmids are present in males of all species. The rhabditid ancestor had 9 pairs of rays and one pair of phasmids instead of 10 pairs as rays as was previously assumed. Two alternative positions of the phasmids relative to the rays could be distinguished: an anterior position with 3-4 rays posterior to the phasmid, and a posterior position with all rays anterior to the phasmid as in C. elegans. There are never more than 4 rays posterior to the phasmid. Phasmids are anterior in Cephalobidae and Diplogastrina and posterior in Panagrolaimidae, in Steinernema, and in Strongylidae. Within Rhabditidae both character states occur. We mapped the phasmid position on a cladogram based on small subunit rDNA (Fitch et al. unpublished) and found that multiple changes between anterior and posterior phasmid position must have occurred during evolution. This could be explained in terms of the development of phasmid socket cells and the posterior three rays, which are all derived from the same blast cell (T) in the L1 larva. In C. elegans, the phasmid socket cells are descendants of the posterior daughter of the T cell. The polarity of the first division of the T cell might be reversed in species with anterior phasmids, such that the phasmid socket cells are now descendants of its anterior daughter (Fitch, 1997; Kiontke & Sudhaus in press). We have begun to test this hypothesis. References: Fitch, D.H.A. & Emmons S. (1995) Dev. Biol. 170: 564-582 Fitch, D.H.A. (1997) Syst. Biol. 56: 145-179. Kiontke, K. & Sudhaus, W. (in Press) J. Nemat. Morph. Syst.

Kim S et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The N-Terminal Extension of a Half-Molecule ABC Transporter, HMT-1, is Required for its Protein-Protein Interactions and Function in Cadmium Detoxification"

Selote D, Grant B, Sharma A, Kim S, Vatamaniuk O, Shi A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

We showed recently that a half-molecule ATP-binding cassette (ABC) transporter HMT-1(heavy metal tolerance factor 1, alias ABCB6, according to the human ABC transporters nomenclature) is acutely required for detoxification of different heavy metals and is expressed in intestinal cells, head and tail neurons and coelomocytes (Vatamaniuk et al 2005; Schwartz et al 2010). HMT-1 can be distinguished from other ABC transporters by its unique structural architecture. Unlike canonical, ""full-molecule"" ABC transporters, consisting of two transmembrane domains (TMD) and two ATP-binding domains (NBD), HMT-1 is a half-transporter that in addition to one TMD and one NBD contains a hydrophobic N-terminal extension (NTE). These structural features distinguish HMT-1 from other family members and allow identifying homologs in genomes of other species including Drosophila and humans. However, functional ABC transporters must be comprised of at least four-domains (two TMD and two NDBs), suggesting that HMT-1 must form homo- and/or hetero-oligomeric complexes. Whether HMT-1 oligomerizes, the functional significance of NTE and HMT-1 subcellular localization are not known. Using confocal microscopy, gel-filtration chromatography in combination with mating-based split-ubiquitin yeast two-hybrid system (mbSUS) and functional in vivo assays, we showed that HMT-1 localizes to recycling endosomes in C. elegans intestinal cells, exists as oligomer and at least homomerizes. We also showed that NTE is essential, but is not sufficient for HMT-1 homomerization and its ability to confer Cd tolerance. The role of NTE and its sub domains with respect to endomembranes trafficking will be discussed.

David Harris et al. (2005) International Worm Meeting "A New Genetic Screen for Hermaphrodites with Inappropriately Surviving CEM Neurons"

David Harris, Bob Horvitz

[International Worm Meeting, 2005]

Two sexually dimorphic cell deaths occur during C. elegans development. The HSN neurons survive in hermaphrodites, in which they are required for egg laying, but die in males. The CEM neurons survive in males, in which they may play a role in chemotaxis towards hermaphrodites, but die in hermaphrodites. Genetic studies have identified one gene, ceh-30, that plays a key role in regulating the CEM neuron sexually dimorphic survival. ceh-30 is required for CEM survival in males, and ceh-30 gain-of-function mutations cause CEM survival in hermaphrodites (see abstract by Schwartz and Horvitz).We have begun a screen to identify new genes that regulate the hermaphrodite-specific programmed cell death of the CEM neurons. Previous screens for CEM survival in hermaphrodites recovered numerous mutations that masculinized hermaphrodites. The CEM neurons of these animals survived because they adopted the male CEM survival fate, not because of a defect in programmed cell death. As ceh-30 appears to act downstream of the sex-determination genes, mutations that masculinize ceh-30 loss-of-function (lf) mutant hermaphrodites should not cause CEM survival. By screening for CEM survival in a ceh-30(lf) mutant background we should be able to eliminate the recovery of a large number of mutations in genes functioning in sex determination.To date, we have recovered at least 27 independent mutations that may disrupt the execution of programmed cell death or prevent proper CEM neuron cell fate. We are currently performing complementation tests between these isolates and alleles of the programmed cell death genes egl-1, ced-4, and ced-3, which are known to cause CEM neuron survival in hermaphrodites.

Vuong, Thanh TK et al. (2013) International Worm Meeting "Mechanical forces in C. elegans embryo elongation."

Labouesse, Michel, Vuong, Thanh TK

[International Worm Meeting, 2013]

Embryo development requires precise coordination of mechanical forces and their failure can lead to diseases. During the morphogenesis of C. elegans embryo, the cooperation of epidermal acto-myosin network and muscle contractions is essential. The acto-myosin activity in the epidermis, which has been shown to be more important in lateral than in dorsal-ventral cells, squeeze the embryo and make it elongate [1]. Muscle contractions become active around 1.7-1.8 fold stage. They have been showed to induce a mechano-transduction pathway [2], which is important for elongation. However, it is unclear how the contractions along the anterior-posterior axis help to increase the length of the embryo. Our project aims to elucidate the mechanical role of muscle contractions and its coordination with acto-myosin forces. The experiments are designed following a working model where muscle contractions induce a change in the elasticity of the embryo. We are using a laser nano-dissection technique to investigate cortical tension and elasticity of epidermal cells before and after the onset of muscle contractions. In parallel, we are evaluating the relative changes of acto-myosin forces with a FRET sensor [3] inserted in HMP-1 - a component of the adherens junctions. I will present our observations and preliminary results of the epidermal cortex nano-dissection experiments and measures of acto-myosin forces exerted on adherens junctions. References 1.Gally C, Wissler F, Zahreddine H, Quintin S, Landmann F, Labouesse M. Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles. Development. 2009 Sep;136(18):3109-19. Epub 2009 Aug 12. 2.Zhang H, Landmann F, Zahreddine H, Rodriguez D, Koch M, Labouesse M. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature. 2011 Mar 3;471(7336):99-103. 3.Grashoff C, Hoffman B, Brenner M, Zhou R, Parsons M, Yang M, McLean M, Sligar S, Chen C, Ha T, Schwartz M. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010 July 8; 466(7303): 263-266.

Sharma A K et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Systematic Studies of ABC Transporters Function in Cadmium Detoxification"

Merchant S S, Danku J, Douglass S, Vatamaniuk O, Chen A G, Salt D E, Pellegrini M, Sharma A K, Kim S

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Understanding the cellular mechanisms of heavy metal (e.g. cadmium [Cd], mercury [Hg] and lead [Pb]) detoxification is critical for the cure of heavy metal-caused diseases, such as neurodegenerative conditions, dysfunction of vital organs, and cancer. Among the major contributors to heavy metal detoxification are members of one of the largest,structurally conserved and functionally diverse family of integral membrane proteins, ATP-binding cassette (ABC) transporters. Canonical,""full-molecule"", ABC transporters consist of two transmembrane domains (TMD) and two ATP-binding domains (NBD). ""Half-molecule"" transporters contain a single TMD and NBD. The C. elegans genome encodes 61 ABC proteins, of which 25 are half-transporters. Full-molecule, MRP-1, PGP-1 and PGP-3 have been shown to detoxify Cd and As (Broeks et al 1996). We showed that a half-transporter, HMT-1, is acutely required for detoxification of Cd, As and Cu and is expressed in intestinal cells, head and tail neurons and coelomocytes (Vatamaniuk et al 2005; Schwartz et al 2010). Which other family members contribute to heavy metal detoxification and how ABC transporter-mediated metal detoxification pathways are related, are not known. Here we used knock-out mutant alleles and RNAi knockdown worms to systematically test the role of each family member in heavy metal detoxification. These screens identified 13 ABC transporters (4 half-transporters and 9 full-transporters), in addition to HMT-1, MRP-1, PGP-1 and PGP-3, that contribute to Cd detoxification. Effects of Cd on their transcript abundance and alternate transcript forms were then studied by q-RT-PCR or RNA-Seq technology. To distinguish Cd-specific from oxidative stress-induced changes in selected ABC transporter gene expression, the effect of Cd vs H2O2 was compared. Finally, the effect of mutations in selected ABC transporters on the ability of worms to accumulate Cd as well as other mineral elements was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The results of these studies as well as relationship between different ABC-transporter metal detoxification pathways will be discussed.

Cha, S. et al. (2015) International Worm Meeting "An annotated genome of the root-knot nematode, Meloidogyne chitwoodi."

Cha, S., Bird, D.

[International Worm Meeting, 2015]

Plant-parasitic nematodes are responsible for annual crop losses in excess of USD123 billion worldwide. Most important are the root-knot nematodes (RKN: Meloidogyne spp.), which establish an intimate association with their host. As a genus, Meloidogyne has a host-range that spans the tracheophyta, although individual isolates are more restricted. In cool climates, M. hapla and M. chitwoodi predominate and are a significant problem on potato in Europe and the US. Because of its genetic tractability, we selected M. hapla for sequencing, and an annotated public release (HapPep1) was made in 2008 (Opperman et al., 2008). Since then we have undertaken on-going curation, and the current release (HapPep5) includes ~2xE9 RNA-Seq reads (Guo et al., 2014). We have also sequenced two additional strains of M. hapla (VW8 and LM) but they have not yet been released. Because M. hapla and M. chitwoodi are sympatric, they presumably have similar gene compliments. To test this, we sequenced the M. chitwoodi genome.Genomic DNA was isolated from nematodes collected in a potato field in Washington State, and confirmed by Axel Elling to be M. chitwoodi. Using an Illumina MiSeq II we obtained 20,079,197 short (~300 bp) paired-end reads. The assembly parameters were empirically optimized, and 4,735 contigs assembled; N50 is 82 kbp. The longest is ~758 kbp with coverage of 33 reads per bp. Average coverage genome-wide is 289 reads. At the protein level, CEGMA identified 245 (98.79%) of the core proteins, pointing to near 100% genome coverage. When CEGMA proteins as a query were blasted against the assembled contigs as a database, it was observed that one protein had hits with more than two contigs. Using CEGMA (and other) proteins, as well as M. chitwoodi ESTs, we trained AUGUSTUS for gene prediction. GO categorization was performed using InterProScan. Analysis of these data is in progress, with an emphasis on genes encoding replicas of plant peptide hormones (xenomones). Because of a potential role in speciation of the closely-related parasites, we have, in collaboration with David Lunt (University of Hull), examined the numbers and distribution of transposable elements: not surprising, M. chitwoodi is much more similar to M. hapla than it is to M. incognita.Operman et al., 2008. Proc Nat Acad Sci 105: 14802 -14807.Guo et al. 2014. Worm 3:e29158.

Xie, Yusu et al. (2013) International Worm Meeting "DAF-19 acts as a negative regulator to modulate environment-dependent GABA phenotypes."

Xie, Yusu, Sze, Ji Ying, Moussaif, Mustapha

[International Worm Meeting, 2013]

Our lab is interested in the regulation of neurotransmitter biosynthesis under aversive conditions. Using GFP reporters, we found that the expression of GABA synthesis gene unc-25/glutamic acid decarboxylase was increased, while acetylcholine (ACh) synthesis gene cha-1/choline acetyltransferase was reduced in dauer animals. By contrast, we observed that in starved L1 animals, the expression of both unc-25::gfp and cha-1::gfp was decreased, suggesting that GABA biosynthesis is differentially regulated under distinct aversive conditions. A previous report indicated that the RFX transcription factor DAF-19 is expressed in the locomotory neurons and daf-19 mutants exhibited enhanced resistance to aldicarb (Senti and Swoboda, 2008). We found that daf-19::gfp levels in the locomotory neurons was reduced in wild-type dauers. Since either reduced ACh neurotransmission or increased GABA neurotransmission could lead to enhanced resistance to aldicarb (Loria et al., 2004; Vashlishan et al., 2008), we analyzed unc-25::gfp and cha-1::gfp in daf-19 mutants. We observed that cha-1::gfp was reduced in daf-19 mutants. Furthermore, we found that the expression of unc-25 and unc-47/vesicular GABA transporter was significantly increased in both daf-19(m86) and daf-19(yz70) mutants. While several previous studies have shown that DAF-19 acts as a transcriptional activator (Swoboda et al., 2000; Wang et al., 2010; Xie et al., 2013), our results suggest that DAF-19 may also act as a negative regulator of GABA neurotransmission under optimal growth conditions. Enhanced GABA neurotransmission could represent a genetic program that inhibit un-necessary locomotion under aversive growth conditions and may contribute to the characteristic relax appearance of dauers. References: Loria PM, Hodgkin J and Hobert O, 2004. J. Neurosci. 24:2191-2201.Senti G and Swoboda P, 2008. Mol Biol Cell 19(12):5517-28.Swoboda P, Haskell T, Adler HT and Thomas JH, 2000. Mol Cell 5:411-421.Vashlishan AB, Madison JM, Dybbs M, Bai J, Sieburth D, Ch'ng Q, Tavazoie M and Kaplan JM, 2008. Neuron 58:346-361.Wang J, Schwartz HT, Barr MM, 2010. Genetics 186:1295-1307.Xie Y, Moussaif M, Choi S, Xu L and Sze JY, 2013. PLoS Genet 9(3): e1003324.