Patterson GI et al. (1995) International C. elegans Meeting "REGULATION OF DAUER DEVELOPMENT BY A TGF-BETA PATHWAY"

Patterson GI, Ruvkun GB

[International C. elegans Meeting, 1995]

During L1 and L2 identified neurons sense environmental conditions (food, pheromone) and transduce a signal to the rest of the animal to promote dauer or non-dauer development. Genes required for this signal transduction have been identified; daf-7, daf-1, and daf-4 have been shown by the Riddle lab to be homologous to TGF-beta and its receptors. The study of dauer formation provides an excellent system to identify genes that regulate or are regulated by TGF- beta receptors, and to understand a process in which neuronal signalling causes changes in development. Previous screens for recessive suppressors of daf-7 (a TGF-b homologue) identified two genes (daf-3 and daf-5), but would have missed any lethals or redundant genes. We performed an F1 screen for dominant mutations that suppress the dauer constitutive phenotype of daf-7, with the hope of identifying new genes or constitutively active DAF-7 receptors. The screen was done in a smg-1 background to increase the likelihood of getting dominant mutations. We screened 1,000,000 genomes and identified one dominant mutation, a smg-1-depen-dent dominant negative mutant on chromosome II. Tests to de-termine whether this is an allele of daf-5 are in progress. This allele should be helpful in understanding the mechanism of action of the gene, because it is likely to encode a truncated protein that interferes with the wild-type protein We have done a mutator screen to identify re- cessive suppressors of daf-7, with the intention of cloning genes that act downstream of the growth factor and receptors. We isolated 7 suppressors, and complementation testing of five of these sup-pressors indicates that three are alleles of daf-5 and two are alle-les of daf-3. This result is expected, because previous EMS screens in the Riddle and Thomas labs turned up daf-3 and daf-5 most frequently. We are using these alleles to clone these two genes, which may encode regulators of the TGF-beta pathway, or genes that act downstream to transduce the signal to enter dauer.

Thatcher JD et al. (1997) International C. elegans Meeting "CONTROL OF PHARYNGEAL GENE EXPRESSION BY THE DAUER FORMATION PATHWAY"

Thatcher JD, Okkema PG, Hahn C

[International C. elegans Meeting, 1997]

Gene expression in the pharynx is regulated by organ-specific signals, which in the myo-2 enhancer target a short DNA sequence termed the C sub-element. To begin to understand these signals, we performed a one hybrid screen in yeast to identify C. elegans cDNAs encoding factors that bind the C sub-element (see abstract by Thatcher and Okkema). One of the clones identified in this screen is from the daf-3 gene, which is required for dauer formation. To understand the role of daf-3 in controlling pharyngeal gene expression, C sub-element activity was examined in daf-3 mutants, as well as in other daf mutants. Surprisingly, these results indicate daf-3 is a negative regulator of C sub-element activity. Although expression of a C sub-element driven reporter in a daf-3 null mutant was similar to wild type, expression was reduced by a mutation in daf-4, a negative regulator of daf-3. Furthermore, a daf-3 mutation suppresses the daf-4 phenotype in daf-4; daf-3 double mutants, demonstrating that the daf-4 repression requires wild type daf-3. daf-3 encodes a mothers against dpp (SMAD) homolog (see abstract by Patterson and Ruvkun). SMAD proteins are downstream components of TGFb like signaling pathways, but are not known to bind DNA directly. Attempts to demonstrate binding of DAF-3 to the C sub-element in vitro have not yet been successful, so it remains unclear how daf-3 controls C sub-element activity. These results indicate the C sub-element is targeted by the TGFb like signaling pathway controlling dauer formation, and suggest that intercellular signaling is involved in regulation of pharyngeal gene expression.

Muneesh Tewari et al. (2001) International C. elegans Meeting "Searching for new proteins involved in dauer formation by two-hybrid protein interaction mapping."

Muneesh Tewari, Patrick Hu, Marc Vidal, Gary Ruvkun, Svetlana Busiguina

[International C. elegans Meeting, 2001]

This project aims to identify new proteins involved in dauer formation by large-scale yeast two-hybrid screens using all the known dauer gene products as baits. Open reading frames encoding each of 34 proteins implicated in dauer formation (daORFs) were cloned into yeast two-hybrid bait (DB-daORFs) and prey (AD-daORFs) vectors by Gateway recombinational cloning. First all pairwise DB-daORF/AD-daORF combinations were tested. This matrix experiment recapitulated an interaction between the DAF-3 (an ortholog of SMAD4) and DAF-5 (a relative of the SNO oncoprotein; Garth Patterson, personal communication) gene products. Individual yeast two-hybrid screens of a C. elegans AD-cDNA library were then carried out with each DB-daORF. This large-scale mapping experiment identified a total of 58 putative interactors. Among others, an interaction between AGE-1 (an ortholog of PI-3-kinase) and the worm ortholog of the p85 adaptor protein was detected. Most of the interactors identified correspond to previously uncharacterized gene products. They will be subjected to functional analysis by systematic RNA-mediated interference to test their involvement in dauer formation.

King KV et al. (1998) Midwest Worm Meeting "DAF-8 is a SMAD protein required in amphid chemosensory neurons to promote growth."

King KV, Riddle DL, Estevez AOZ

[Midwest Worm Meeting, 1998]

Non-dauer development in C. elegans involves activation of a signal transduction pathway analogous to Transforming Growth Factor-beta (TGF-beta) signaling pathways in other organisms. Previous genetic analysis indicates that daf-8 and daf-14 are partially redundant positive regulators of non-dauer development, whereas daf-3 promotes dauer formation. The daf-8 (King et al., unpublished), daf-3 (Patterson et al., 1997) and daf-14 (J. Thomas, personal communication) genes each encode SMAD proteins. SMAD proteins mediate transcriptional regulation of TGF-beta-responsive genes in response to phosphorylation by TGF-beta receptors. Mutations in daf-8 result in constitutive dauer larva formation, and some embryonic and larval lethality. One mutation truncates the protein in the putative DNA binding domain and eight other daf-8 mutations change residues in the putative transcriptional activation domain. Expression of a daf-8::GFP fusion gene is restricted to one pair of amphid chemosensory neurons in the head and is down-regulated in dauer larvae. This implies that dauer formation is sensitive to the levels of daf-8. This limited expression pattern suggests that DAF-8s function in regulating dauer formation may be different from DAF-14.

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.

Ayesha N Shajahan et al. (2001) International C. elegans Meeting "Regulation of endocytosis of albumin in the intestinal epithelial cells of C.elegans by TGFbeta-like signaling"

Ayesha N Shajahan, Shahid S Siddiqui

[International C. elegans Meeting, 2001]

In C. elegans , TGF b -like signaling pathways regulate a range of developmental processes such as dauer formation (Estevez et al., 1993), body size growth and male tail formation (Morita et al., 1999; Suzuki et al., 1999), and axon guidance (Colavita et al., 1998). Recently, Siddiqui et al. have shown that daf-4 (TGF b -like-RII) and daf-1 (TGF b -like-RI) (at 25 o C) mutants show poor uptake of FITC-BSA (fluorescein-5-isothiocyanate isomer- bovine serum albumin) in the intestinal cells when compared to wild-type animals. Our aim is to identify the genes in the TGF b -like pathways that affect endocytosis of albumin in the intestinal epithelial cells. We will examine the uptake of FITC-BSA in the mutants of the TGF b -like pathway in comparison to its uptake in wild type. daf-4::gfp (Patterson et al., 1997) and daf-1::gfp (Gunther et al., 2000) are expressed in the intestinal cells. Expression and regulation of other TGF b -like receptors, Smad proteins and the downstream trancription factors in the TGF b -like signaling pathway will be examined in the intestinal cells of wild type and various daf mutants, in the presence and absence of albumin, by immunofluorescence and immunoprecipitation assays. This approach will determine the role of TGF b -like signaling in endocytosis of albumin, and also uncover any possible cross talk between such pathways in the process.

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.

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.