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.

Lawrence Schriefer et al. (2001) International C. elegans Meeting "Defining Regulatory Regions In Nematode Muscle Genes By Computational And Transgenic Studies."

Robert Waterston, Gary Stormo, Lawrence Schriefer, Debraj GuhaThakurta, Michelle Hresko

[International C. elegans Meeting, 2001]

We have initiated experiments designed to understand the regulatory regions of C. elegans genes using known muscle genes of C. elegans as a model. Two approaches are being used to pursue this goal. The first approach is to computationally compare the muscle genes from C. elegans to the orthologous muscle sequences from C. briggsae . This comparison is useful because the patterns of gene regulation and regulatory elements are often conserved across species. The C. briggsae orthologue are found by making a probe from the C. elegans muscle gene and probing the C. briggsae fosmid filter available from Incyte. The most promising positive clones are determined by fingerprinting and these are sequenced by the Genome Sequencing Center. To compare the orthologous sequences from C. elegans and C. briggsae , we will use pairwise alignment methods like BlastZ(4) or Bayes aligner(5) to identify regions of interest. Local multiple alignment programs can then be used to search for common regulatory elements in these regions. Since the local multiple alignment methods work best with sequences which are only 1000-2000 nucleotides long, phylogenetic footprinting will be useful in identifying shorter regions from much longer regions(10,000-20,000 nucleotides). The second approach is to use a combination of computational methods to identify potential muscle specific regulatory elements from the known set of C. elegans muscle genes. Local multiple sequence alignment methods like Consensus(1), Ann-Spec(2) and Co-Bind(3) are being used to identify these potential regulatory elements. Using the above method we have already identified several potential regulatory elements which show high degree of specificity for the muscle genes. The regulatory elements that these computational methods predict can then be used to screen the C. elegans genome for new genes that are expressed in muscle cells. To test our results we have developed a method to examine the expression patterns of genes in C. elegans using gfp promoter fusions. We are including in our promoter fusions 6,000 nucleotides upstream of the start methionine, all of the first exon and all the first intron. In our initial experiments, known muscle genes tested in this manner show muscle-like expression. We can now use this method to test the requirement for regulatory regions predicted by the computational work to determine if they convey muscle specific expression. In addition, we can use this method to test genes we predict to be, but not previously known to be, expressed in muscle. Furthermore, we are developing these methods to allow for the rapid production of these promoter fusions so that ultimately, a genome wide program to categorize all C. elegans genes by gfp and automated lineaging can be done. 1. Hertz, G.Z., and Stormo, G.D. (1999) Bioinformatics, vol. 15, pp. 563-577 2. Workman, C.T., and Stormo, G.D. (2000) Pacific Symposium on Biocomputing, vol 5, pp. 464-475 3. GuhaThakurta, D., and Stormo, G.D. (2001) Bioinformatics, in press. 4. Schwartz, S. et.al. (2000) Genome Research, vol. 10, pp. 577-586. 5. Zhu, J., Liu, J.S., and Lawrence, C.E. (1998) vol. 14, pp. 25-39.

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.

AJ Wright et al. (1999) International C. elegans Meeting "Functions of C. elegans cell fate determining genes in C. briggsae"

AJ Wright, CP Hunter

[International C. elegans Meeting, 1999]

We are investigating the conservation of function of SKN-1 and PAL-1 between C. elegans and C. briggsae . In C. elegans , skn-1 is required to specify the EMS fate and pal-1 is required to specify the C and D fates. We have identified C. briggsae skn-1 and pal-1 homologues and are using RNA mediated interference (RNAi) to inhibit their function in C. briggsae . To assist in our phenotypic characterization we are identifying tissue specific antibodies that cross react with C. briggsae and ablating early blastomeres to ensure that C. briggsae early development does not differ substantially from that of C. elegans . Our preliminary results are encouraging. We have identified several monoclonal antibodies that cross react with C. briggsae antigens. These include 3NB12 which recognizes pharynx muscle, 5-6 which recognizes body wall muscle, J126 which recognizes intestinal cells and intestinal valve cells, and MH-27 which recognizes adherence junctions. Ablation experiments on early C. briggsae embryos have not yet indicated any obvious differences in cell fate between C. briggsae and C. elegans . Likewise blastomere interactions important in the early C. elegans embryo, like that of MS signaling to ABa at the 12 cell stage to promote pharynx formation in ABa, also appear to be required. C. briggsae pal-1 (RNAi) produces dead embryos with an overall gross morphology different from that of C. elegans pal-1 (RNAi) embryos. Preliminary characterization indicates that while C. elegans pal-1 function is restricted to specifying C and D blastomere fates, C. briggsae pal-1 function may be more broadly required. Future work will focus on investigating the C. briggsae pal-1 (RNAi) phenotype more thoroughly and cloning and performing similar experiments on the SKN-1 homologue.

Schartner, Caitlin M et al. (2011) International Worm Meeting "Comparison of Dosage Compensation Between Caenorhabditis briggsae and Caenorhabditis elegans."

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

[International Worm Meeting, 2011]

Dosage compensation is a mechanism that equalizes gene expression from the X chromosomes between heterogametic sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both hermaphrodite X chromosomes to repress transcription by approximately half, to equal the level expression from the single male X. Although C. elegans and C. briggsae diverged 15-30 million years ago, our analysis has shown that dosage compensation complex (DCC) subunits are conserved between species. Each C. elegans DCC component has a homolog in C. briggsae, and the DCC components DPY-27, MIX-1, and SDC-2 have been shown to have similar functions in C. briggsae dosage compensation. However, while DCC components appear conserved, DCC binding sites appear diverged. The C. elegans consensus motif (MEX, motif enriched on X) pivotal for C. elegans DCC recruitment to X is only enriched 0.6-2-fold on C. briggsae X compared to autosomes, in contrast to the 3.8-24-fold enrichment on the C. elegans X chromosome. Furthermore, we characterized the recruitment potential of several C. elegans recruitment sites and their C. briggsae homologous regions in both species. No C. elegans or C. briggsae sequences tested were able to recruit the DCC in C. briggsae to the same degree as in C. elegans. This suggests that the cis-acting DNA recruitment sites in C. briggsae have diverged. Ongoing ChIP-seq experiments to define the C. briggsae DCC binding sites will reveal the degree of divergence. The identification of DNA binding sequences in C. briggsae will set the stage to allow us to investigate the molecular co-evolution of the DNA sequence motif and the DNA-binding domain of the DCC.