Mattout, Anna et al. (2017) International Worm Meeting "Mechanism and function of genome organization in muscle development and integrity."

Kalck, Veronique, Harr, Jennifer C., Gonzalez-Sandoval, Adriana, Mattout, Anna, Gasser , Susan M.

[International Worm Meeting, 2017]

The loss of nuclear structural integrity leads to tissue-specific pathologies in a set of human diseases called laminopathies. These diseases, such as Emery Dreifuss Muscular Dystrophy (EDMD), are late-onset, tissue-specific, and degenerative. A C. elegans mutant that recreates the EDMD phenotypes arises from the introduction of a specific gain-of-function mutation into lamin (Y59C), which affects the proper release of a muscle-specific heterochromatic reporter from the nuclear periphery. We found that this muscle-specific misorganization of heterochromatin correlates with transcriptional defects and with perturbed locomotion and muscle integrity in C. elegans. To identify if chromatin misorganization is a cause or an effect of these physiological defects or if the alteration of gene expression is the primary cause, we took advantage of a cec-4 deletion mutant. The perinuclear C. elegans chromodomain protein-4 (CEC-4) anchors heterochromatin by binding H3K9 methylation. The mutant releases H3K9me containing heterochromatin from the nuclear periphery in embryos and does not alter transcription. In a strain that contains the cec-4 deletion, expresses the lamin Y59C mutant and harbors a muscle-specific heterochromatic reporter, there is a recovery of the proper positioning of the muscle-specific heterochromatic array in muscle. Additionally, we found that the cec-4 mutation rescues the impaired locomotion phenotype of the lamin mutant. This argues that the lamin-induced sequestration or retention of tissue specific genes in a heterochromatic compartment drives defects that can be overcome by reversing perinuclear sequestration. To identify the mechanism behind this phenomenon we are using in vivo biochemical tagging in the LMN-1 Y59C mutant and wild-type counterpart to identify perinuclear components that mediate sequestration of muscle-specific gene promoters. By identifying the proteins involved in this process, we can determine the mechanism in which misorganization of chromatin can lead to a loss of tissue integrity.

Jennifer Pilipiuk et al. (2006) European Worm Meeting "Analysis of Epithelial Genes during Postembryonic Development of C. elegans"

Olaf Bossinger, Jennifer Pilipiuk, Gisela Helbig

[European Worm Meeting, 2006]

Jennifer Pilipiuk, Gisela Helbig and Olaf Bossinger. We wish to understand how epithelial polarity and tissue integrity is maintained. Here, we consider the role of DLG-1 (Discs large), LET-413 (Scribble), ERM-1 (Ezrin-Radixin-Moesin), and the catenin-cadherin complex (HMP-1HMP-2HMR-1) during postembryonic development of C. elegans. In the course of embryogenesis these genes play an important role in the establishment and maintenance of epithelial polarity, the formation of the lumen, and cell-cell adhesion. In contrast, during larval and adult development only newly established epithelia (e.g. the spermatheka or the vulva) show severe defects after bacterial RNAi against DLG-1, LET-413 and ERM-1, while the depletion of the catenin-cadherin complex seems not to cause visible defects. Nevertheless, how polarity of already established epithelia (e.g. the intestine or the hypodermis) is maintained during postembryonic development remains elusive. In the case of DLG-1, our results suggest that a small amount of protein is sufficient, but cannot fall below a certain threshold without causing defects. Intestinal and hypodermal polarity during larval and adult development of C. elegans might also depend on other, so far unidentified proteins. A detailed analysis of DLG-1, LET-413 and ERM-1 phenotypes during postembryonic epithelial development in C. elegans will be presented and discussed.

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.

Jennifer Schisa et al. (2003) International Worm Meeting "Investigation of chemotaxis mutants in an undergraduate genetics laboratory"

Jennifer Schisa, Katie M Morrison

[International Worm Meeting, 2003]

A three-week series of C. elegans-based laboratories has been implemented in an introductory college genetics course (JS) that is based upon those used successfully in a high school program (KMM) and others (Jennifer Vowels, Debbie Birnby, Wendy Schackwitz, Jim Thomas). These labs are performed near the end of the semester, after students have been introduced to basic molecular biology concepts and at the same time that the lecture discusses the use of genetics to study behavior. The first session is a computer-based laboratory to introduce students to C. elegans and the vast array of information available at the C. elegans WWW server, Wormbase, and NCBI. Students are guided through questions and prompts to learn how to find different types of information such as: the literature published regarding chemotaxis in C. elegans, the names of researchers working on this problem, and the proteins that have been implicated in regulating chemotaxis. The second lab introduces students to the worm via three activities: 1) distinguishing males, adult hermaphrodites, and L4 worms; 2) identifying basic anatomical parts of the worm; and 3) sorting unc, dpy, and rol worms from a plate containing all three mutants. The first and third activity give students the opportunity to learn how to pick worms correctly, a skill they will need in the third lab. At the end of the second lab, students are assigned three unknowns (that they will identify in the third lab via chemotaxis assays) and pick worms to fresh plates in preparation for lab 3. The unknowns include two osm mutants, 1 che mutant, 1 che mutant marked with dpy, and wild-type. The third lab includes an attractance assay (based on Bargmann et al., 1993) and an avoidance assay, each performed with the three unknowns and wild-type controls. Students calculate avoidance and attractance indexes and perform statistical analyses to determine the identity of their unknowns. This series of laboratories could easily be expanded to incorporate student-directed projects, investigating other possible effectors of C. elegans behavior. Bargmann, C.I., Hartweig, E., and Horvitz, H.R. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74: 515-527.

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.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven 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 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. 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 seven 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. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. 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 few years. There is no indication that we are even close to knowing all species in this genus.

Xiaodong Wang et al. (2003) International Worm Meeting "Evolution of a novel gene expression pattern in C. elegans"

Xiaodong Wang, Helen M Chamberlin

[International Worm Meeting, 2003]

Previous studies have shown that C. elegans ovo-related gene lin-48 expresses in a small number of cells including the excretory duct cell. In the related species C. briggsae, the expression is conserved in all cells except the excretory duct. This lin-48 expression difference affects excretory duct morphogenesis. In C. briggsae, as well as in C. elegans lin-48(sa496) mutants, the excretory duct is more anterior than in C. elegans wild type. This indicates that C. elegans lin-48 (Ce-lin-48) is involved in duct morphogenesis and positioning, but this gene function is absent in C. briggsae (1). We have made reporter transgenes composed of the lin-48 regulatory sequences from C. elegans or C. briggsae driving expression of green fluorescent protein (GFP). Tests of these clones in each species showed that only the Ce-lin-48 is expressed in excretory duct cell in C. elegans animal. These results indicate that there are differences in both cis-regulatory sequences and trans-acting proteins between the two species. By creating chimeric reporter transgenes including C. elegans and C. briggsae regulatory sequences, we have found that one difference between the two species is the presence of regulatory sequences in Ce-lin-48 that respond to the bZip protein CES-2 (1). The lin-48 gene expression differences between C. elegans and C. briggsae could result from loss of excretory duct expression in the C.briggsae lineage or acquired expression in the C. elegans lineage. To distinguish between these possibilities, we have analyzed three additional Caenorhabditis species (C. remanei, C. sp. CB5161 and C. sp. PS1010). We found these species have a duct morphology similar to C. briggsae indicating the C. elegans morphology is unique to this species. For comparison to C. elegans and C. briggsae, we have isolated the lin-48 gene from C. remanei and C. sp. CB5161. Alignment of the lin-48 regulatory sequences reveals that the sequences are more conserved among C. briggsae, C. remanei and C. sp. 5161. Several conserved domains are absent from C. elegans, whereas the previously identified CES-2 binding sites are absent from the other species. Currently, we are creating lin-48::gfp reporter transgenes for each species to observe the gene expression patterns. Further experiments with these transgenes will allow us to test whether the differences between C. elegans and the other species result from a loss of repressor elements or gain of activator elements in the C. elegans gene. (1)X. Wang and H. M. Chamberlin (2002) Genes & Development 16: 2345-2349.

Sugimoto, Asako et al. (2017) International Worm Meeting "Establishing genetic techniques to study Caenorhabditis sp. 34, a sister species of C. elegans."

Kanzaki, Natsumi, Hoshi, Yuki, Kumagai, Ryohei, Sugimoto, Asako, Kikuchi, Taisei, Namai, Satoshi, Tsuyama, Kenji

[International Worm Meeting, 2017]

Caenorhabditis sp. 34 is a sister species of C. elegans recently isolated from the syconia of the fig Ficus septica on Ishigaki Island, Japan (see abstract by T. Kikuchi, et al.). C. sp. 34 is gonochoric and shares typological key characters with other Elegans supergroup species, but strikingly, adults are nearly twice as long as C. elegans. The optimal culture temperature for C. sp. 34 is significantly higher (27 deg C) than that of C. elegans (20 deg C). Young adult males and females tend to form clumps, and Dauer larvae are rarely observed in laboratory culture conditions. Recently the C. sp. 34 genome assembly was produced into six chromosomes (see abstract by T. Kikuchi, et al.). The marked differences from C. elegans in morphology, behaviors and ecology, and the availability of the complete genome sequence make C. sp. 34 highly attractive for comparative and evolutionary studies. To make C. sp. 34 genetically tractable, we have been developing genetic and molecular techniques and tools. Stable transgenic lines of C. sp.34 could be obtained by microinjecting marker plasmids commonly used in C. elegans, although the efficiency was lower than that in C. elegans. Both soaking and feeding RNAi was as effective as in C. elegans. A panel of antibodies against C. elegans proteins successfully recognized expected structures in C. sp. 34 by immunofluorescence. Thus, many of the rich genetic and molecular resources for C. elegans can be directly used for C. sp. 34 studies. We well present some of the comparative analyses of gene functions regarding the body size, germ cell formation and sex determination.