Frisse LM et al. (1995) International C. elegans Meeting "EVALUATION OF A MOLECULAR DATABASE FOR NEMATODE TAXONOMY"

Frisse LM, Vida JT, Thomas WK

[International C. elegans Meeting, 1995]

A large number of nematode species remain to be identified. To overcome this problem a common set of taxonomic characters is needed. Nematode species exhibit great diversity at the molecular level. This molecular variation may be useful as a taxonomic tool. Recent technical advances, in particular the advent of the polymerase chain reaction and automated sequencing, have made the generation of sequences for large numbers of individuals a realistic goal. In an attempt to set up a molecular database, we have chosen the D3 expansion segment of the gene for the largest subunit of ribosomal RNA as our molecular species tag. This segment amplifies in a wide range of nematode taxa, can be sequenced from all developmental stages, and provides unambiguous character sets that require no subjective evaluation. When a complete sequence is obtained for each new sample, it is compared to existing sequences for identification. If the sequence represents a new sample it is added to the main database. The existing database contains over 80 sequences and is being evaluated for its usefulness as a taxonomic tool.

Frisse LM et al. (1997) International C. elegans Meeting "IDENTIFICATION OF MISMATCH REPAIR GENES IN C. ELEGANS"

Thomas WK, Lam KM, Vida JT, Frisse LM

[International C. elegans Meeting, 1997]

The MRS is critical for maintenance of genome stability and faithful genetic inheritance. Currently, Saccharomyces cerevisiae is the only eukaryotic system in which homologs of mutS and mutL have been studied thoroughly. MutS and MutL homologs of yeast MRS suppress error rates during replication and control recombination during meiosis. Deficiencies in MRS homologs of yeast display mismatch repair deficiency, microsatellite instability, and meiotic nondisjuction. We have identified four putative Caenorahbditis elegans MRS homologs by searching the C. elegans genome database using functionally characterized yeast MutS and MutL homologs as query sequences. The nematode homologs have been compared with homologs from bacteria, yeast, and other organisms in order to develop a phylogenetic hypothesis of relationships. Based on the relationships, we have identified and proposed functions for these genes. mlh-1, a homolog to yeast MLH1, and msh-2, a homolog to yeast MSH-2, may both be directly involved in mismatch repair. msh-4, a homolog to yeast MSH4, and msh-5, a homolog to yeast MSH5, are probably both involved in meitic recombination.

LM Frisse et al. (1999) International C. elegans Meeting "Origin and evolution of microsatellites in C. elegans"

M Lynch, WK Thomas, L Vassilieva, LM Frisse

[International C. elegans Meeting, 1999]

Microsatellites, small direct repeats, are a ubiquitous feature of eukaryotic genomes. The high mutation rate of microsatellite loci has made them useful in genetic mapping and evolutionary studies. Microsatellites have also been recognized as the causal agents of numerous human genetic diseases and are used as indicators for tumor formation. Despite their widespread use, little is known about the origin of microsatellites, factors that affect microsatellite frequency and distribution, and the rates and patterns of microsatellite mutation. Understanding the rates and patterns of microsatellite evolution is critical for their use in studies of evolution, genetic mapping and mutational mechanisms. The recent completion of the Caenorhabditis elegans genome allows the testing of many of the hypotheses concerning microsatellite evolution. We have identified 953 microsatellites with 2-5 bp repeats, which are at least 10 perfect repeat units in length. These loci have been physically mapped allowing us to investigate the frequency and distribution of microsatellites across an entire metazoan genome. The majority of the loci identified are dimers. There is no clear pattern for microsatellite distribution within the genome. Microsatellite frequencies are not consistent with base composition or dinucleotide sequence composition; however, there is some evidence that microsatellites may be seeded by a telomeric activity. In order to understand the rates and patterns of microsatellite mutations, we assayed 29 microsatellite loci in a set of 80 mutation accumulation lines of the nematode C. elegans propagated for 140 generations. Investigation of mutation rate included dimer loci over a wide range of allele sizes. This study clearly demonstrates that the mutation rate increases with increased repeat number and that the pattern of mutation is biased towards small additions.

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.

Vida JT et al. (1995) International C. elegans Meeting "MODE AND TEMPO OF MOLECULAR EVOLUTION IN FOUR CAENORHABDITIS SPECIES"

Nunn GB, Frisse LM, Vida JT, Thomas WK

[International C. elegans Meeting, 1995]

Up to this point, a major problem in investigating the molecular evolution of C. elegans and three of its commonly studied relatives (C. briggsae, C. remanei, and C. vulgaris), is the lack of a closely related taxa to use as an outgroup. We have completely sequenced a 2.2kb cDNA encoding fragment ofthe large subunit of RNA polymerase II, as well as the genes for the large subunit (26s) of ribosomal RNA and the rnitochondrial cytochrome oxidase II for C. elegans, C. briggsae, C. remanei, C. vulgaris, and an undescribed new species (PS1010). Analysis of the data indicates PS1010 is a closely related sister taxa to the Caenorhabditis. Outgroup rooting of the Caenorhabditis with PS1010 irnproves the resolution of the phylogenetic relationships among the Caenorhabditis species. In addition, rooting with the closely related PS1010 improves the ability to compare rates and patterns of evolution in the three genes sequenced.

Thomas WK et al. (1995) International C. elegans Meeting "AN EVOLUTIONARY FRAMEWORK FOR SOME RELATIVES OF C. ELEGANS"

Frisse LM, Baldwin JG, Thomas WK, Vida JT

[International C. elegans Meeting, 1995]

The evolutionary interpretation of developmental differences requires a framework in the form of a phylogenetic hypothesis. Such a hypothesis allows for the assignment of direction of change and the understanding of phenomena such as shared characteristics. In conjunction with the comparative developmental studies of the nematode vulva by Somrner, Stemberg, and others; we have begun a phylogenetic analysis of key nematode species representing cntical changes in vulval development. The species include Mesorhabdidtis sp., Teratorhabditis palmarum, Cruznema tripartitum, C. elegans and an undescribed new species (PS-1010). Current phylogenetic hypotheses on the relationship among major lineages in the Rhabditidae are based on relatively few useful morphological characteristics. In order to develop a phylogenetic hypothesis independant of the developmental comparsions and based on a large number of characters, we have used coding portions of the largest subunit of RNA polyrnerase II and the large subunit of rRNA. Three observations are particularly noteworthy. 1) The branching order among the five taxa is resolved significantly. 2) A morphologically distinct taxa, PS-1010, is the sister taxa to the Caenorhabditis. 3) The divergence levels among nematode taxa from the same superfamily are as high as those found between major metazoan lineages.

Lam KM et al. (1997) International C. elegans Meeting "THE ROLE OF MALES IN C. ELEGANS"

Frisse LM, Vida JT, Shores ML, Raw HL, Lam KM, Thomas WK

[International C. elegans Meeting, 1997]

Wild type strains of Caenorhabditis elegans are primarily self-fertilizing hermaphrodites but also produce functional males by meiotic non-disjunction. While it is presumed that males occur in nature, we have no evidence that males "perform" in nature. The goal of this study is to investigate whether or not C. elegans males participate in effective outbreeding during the evolution of this species or whether C. elegans evolves as a set of non-interbreeding clonal lineages. To evaluate the evolutionary contributions of outcrossing by males, we first developed an evolutionary history of C. elegans mitochondrial DNA, which represents a maternal clonal history. We then compared the mitochondrial phylogeny with one based on nuclear markers, including Tc1 and microsatellite loci. If inheritance was strictly clonal, the resulting phylogenies should be identical. However, if males play an effective role in genome evolution, the mitochondrial and nuclear phylogenies should be different. Analysis of 28 independently isolated C. elegans strains resulted in mitochondrial and nuclear phylogenies that were almost completely congruent. Nevertheless, strains with differences in phylogenetic placement based on mitochondrial and nuclear variation were identified. These exceptions represent evidence that males play a role in the evolution of C. elegans.

Blaxter ML et al. (1998) European Worm Meeting "The phylogenetics of nematodes"

Blaxter ML, Vanfleteren JR, De Ley P, Thomas K, Dorris M, Frisse LM

[European Worm Meeting, 1998]

We are continuing in our quest to define the evolutionary history of the phylum Nematoda using molecular phylogenetic methods. The published analysis (Blaxter et al, Nature 392:71-75 (1998)) has now been complemented with over 100 additional sequenced taxa, arising from our work and that of others (Kampfer et al, Invert. Biol. 117:29-36 (1998) and Aleshin et al, Russ. J. Nematol. in press (1998)). Our analysis continues to support the division of the Nematoda into five major clades, and does not support the division of the phylum into two classes (Adenophorea and Secernentea). The origin of the Secernentea has been more closely defined as residing within the Chromadorida, although a separate chromadorid radiation is now evident. Caenorhabditis remains a close relative of the parasitic strongyles. We are extending the dataset using sequence data derived from fixed museum specimens. Formalin fixation preserves nematode structures very well, but is not good for DNA preservation. We have been able to extract PCR-amplifiable DNA from single 12-year fixed specimens of Strongyloides species using an amino acid titration method. Fragments up to 600 bp are recoverable and sequencable. For specimens preserved in alcohol, even at low temperatures, it has been much more difficult to obtain amplifiable DNAs, but we now have a rehydration-extraction method which works on larger samples. Current projects are focussing on the phylogenetics of the genus Strongyloides, parasites of vertebrate guts which have a facultative free-living generation. This group has turned out to be entertainingly complex, with the species divided into two distinct clades separated by freeliving and parasitic taxa from other genera. We find no correlation between host and parasite phylogenies, suggesting that horizontal transfer between hosts has been common in these parasites. We are examining in detail the coevolution of filarial nematodes and an endosymbiotic Wolbachia-like bacterium. In this case, we have strong evidence for vertical transmission of the endosymbiont within the filarial lineage. We are also providing a phylogenetic framework for comparative EST-based genome projects on additional filarial and other nematode species.

Blaxter ML et al. (1997) International C. elegans Meeting "PUTTING C. ELEGANS IN ITS PLACE: A MOLECULAR PHYLOGENY OF THE PHYLUM NEMATODA"

Dorris M, Thomas WK, Scheldeman P, Garey JR, Vanfleteren JR, Liu LX, DeLey P, Vida JT, Blaxter ML, Frisse LM

[International C. elegans Meeting, 1997]

The Phylum Nematoda includes four of every five metazoan animals on the planet. Nematodes are important because of their diversity, ecological significance and impact as parasites of plants, humans and other animals. Morphological studies have failed to provide a unifying phylogenetic framework due to a lack of informative fossils and the limitations of light-microscopy. We present here a phylogenetic analysis using small subunit (SSU) ribosomal DNA sequences from a wide range of nematode taxa. For the first time animal-parasitic, plant-parasitic and free-living taxa can be compared using the same metric. We pinpoint the multiple origins of parasitism within the phylum, identify candidate research models from free-living sister taxa of parasitic clades, and place into context the metazoan research model Caenorhabditis elegans. Our results also suggest that convergent evolution has occurred more frequently in nematodes than hitherto assumed, and that classification will need significant changes.

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.