Kohler R et al. (1998) European Worm Meeting "MAPPING THE CEH-13 ENHANCER"

Tobler H, Marty T, Kohler R, Mueller F, Affolter M

[European Worm Meeting, 1998]

Metazoan development is in part regulated by a group of homeobox containing genes arrayed in an evolutionarily conserved cluster. ceh-13, a member of the C. elegans HOM-C cluster, is the orthologue of the Drosophila labial gene. Antibody staining and reporter gene expression experiments have revealed that CEH-13 has a highly complex and dynamic expression pattern. In the course of these studies we identified an 8 kb upstream enhancer/promoter region that was sufficient to confer essentially wild-type expression to GFP reporter gene constructs. Based on this data, we initiated a detailed in vivo analysis of the enhancer region of the ceh-13 gene. The final goal of these studies is to uncover the mode of transcriptional regulation of ceh-13 and the identification of protein factors that modulate its expression by binding directly to its enhancer. To date we have identified a 700bp cis-acting element that is responsable for the early expression of CEH-13 in the ABxxxp and in the Ep intestinal precursor cell and an enhancer fragment that participates in the activation of the gene in the seam cells. Furthermore, we tested the ceh-13 enhancer for activity in Drosophila . To our surprise, we found that a reporter gene construct containing a large fragment of the ceh-13 enhancer that drives a LacZ reporter gene, essentially mimics endogenous labial expression. Sequence analysis of the worm enhancer identified several conserved enhancer elements. Among those we found were a LAB/EXD binding element, a CRE (cAMP-responsive element)-site and a potential LEF-1 (lymphocyte enhancer binding factor 1) binding site. So far we have found that the LAB/EXD "autoregulatory" element seems to play an important role in the activation of the gene around comma-stage, which is when ceh-13 mutant phenotyp is first manifested. Moreover we have indication that the putative LEF-1 binding site might play a role in ceh-13 transcriptional activation in the Ep lineage. The latter result is in agreement with a role of the wingless pathway in the determination of the gut fate in C. elegans.

Kohler R et al. (1997) International C. elegans Meeting "MAPPING THE CEH-13 ENHANCER"

Kohler R, Tobler H, Brunschwig K, Fleischmann M, Mueller F

[International C. elegans Meeting, 1997]

Metazoan development is in part regulated by a group of homeobox containing genes arrayed in an evolutionarily conserved cluster. ceh-13, a member of the C. elegans HOM-C cluster, is the orthologue of the Drosophila labial gene. Antibody staining and reporter gene expression experiments have revealed that CEH-13 has a highly complex and dynamic expression pattern (see also posters by Fleischmann M. and Brunschwig K.). Additionally, these studies identified an 8 kb upstream enhancer/promoter region that was sufficient to confer essentially wild type expression to GFP reporter gene constructs. Different biochemical and genetic aspects of the regulation of Hox/HOM-C expression have been described in various species. Nevertheless a complete picture of Hox/HOM-C transcriptional control awaits to be put forward. Furthermore, recent studies indicated that the control of these genes in C. elegans may be significantly different from what is known from other species. For those reasons, we initiated a detailed in vivo analysis of the ceh-13 enhancer region by means of expression monitoring of various GFP reporter gene constructs in transgenic animals. So far these studies have shown that different, dissectable enhancer elements are present in the ceh-13 regulatory region. These elements appear to independently direct different aspects of CEH-13 expression. A preliminary analysis of different enhancer elements and their significance in the context of C. elegans development will be presented.

Schierenberg E "Laser-induced cell fusion."

Schierenberg E

In recent years a number of different techniques have been developed to fuse cells of various origin in order to obtain hybrids with qualities different from each of the original cells. In many cases, one wants to fuse large numbers of cells (e.g. to generate hybridomas), and fusion occurs more or less randomly (e.g. Kohler and Milstein, 1975). With other methods, individual cells have been fused under controlled conditions (e.g., McGrath and Solter, 1984). So far a prerequisite for fusing two preselected cells has been that they must be isolated away

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

Fleischmann M et al. (1997) International C. elegans Meeting "SEARCHING FOR REGULATORS OF ceh-13 EXPRESSION"

Tobler H, Kohler R, Fleischmann M, Mueller F, Brunschwig K

[International C. elegans Meeting, 1997]

ceh-13 is the homologue of labial in D. melanogaster, a homeotic cluster gene important for specifying positional information along the anterior-posterior body axis. Despite the vast conservation of homeotic cluster genes in metazoans, there seems to be a remarkable difference in how these genes are activated in C. elegans compared to D. melanogaster. In C. elegans cell-autonomous and lineage-dependent factors direct expression into the right regions of the embryos (Refs 1,2), whereas in D. melanogaster early onset of transcription is accomplished by gradients of regulatory proteins. Among the homeotic cluster genes, ceh-13 is the first to be expressed, namely at the 26-cell stage in the Ep cell and later in all ABxxxp cells. In order to identify genes regulating this early expression, a screen for maternal-effect lethal mutations affecting ceh-13::gfp expression was initiated. Mutants which show strongly reduced expression are currently being backcrossed and analyzed with a 4D microscope. In addition, in order to find factors which bind to CEH-13 and which might assist in the autoregulation of the ceh-13 gene (as well as in the regulation of target genes), a two-hybrid screen was initiated. Since dpp is known to influence labial expression in D. melanogaster, we tested whether this pathway is also used for the regulation of ceh-13 in C. elegans. Indeed, we found that ceh-13 expression in four cells in the adult male tail is abolished in sma-2, sma-3, and sma-4 mutants, which are defective in the dpp pathway (Ref 3). 1 Wittmann, C. et al., submitted. 2 Cowing, D. and Kenyon, C. (1996). Nature 382, 353-356. 3 Savage, C. et al. (1996). Proc. Natl. Acad. Sci. USA 93, 790-794.

Lu WX et al. (1990) J Biol Chem "Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans."

Bagchi S, Lu WX, Rubin CS, Gross RE

[J Biol Chem, 1990]

The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.

Winge P et al. (1994) Worm Breeder's Gazette "R-ras 1 and R-ras 2 (TC21) Homologs."

Gobel V, Friend S, Winge P, Fleming JT

[Worm Breeder's Gazette, 1994]

R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with bc1-2, the human homolog to ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.

Wang B et al. (2021) Nat Commun "Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence."

Jo J, Wang B, Price-Whelan A, Brown LM, Sieben C, Vasquez-Rifo A, Lin YC, McDonald ST, Dietrich LEP

[Nat Commun, 2021]

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.

Buyannemekh K et al. (2024) Dev Biol "Left/right asymmetrically expressed ephrin and Flamingo proteins regulate lateralized axon growth in C. elegans."

Buyannemekh K, Bertrand V, Villoutreix P

[Dev Biol, 2024]

While the nervous system of bilaterian animals is mainly left-right (L-R) symmetric at the anatomical level, some molecular and functional L-R asymmetries exist. However, the extent of these molecular asymmetries and their functional consequences remain poorly characterized. C. elegans allows to study L-R asymmetries in the nervous system with single-neuron resolution. We have previously shown that a neural bHLH transcription factor, HLH-16/Olig, is L-R asymmetrically expressed in the AIY neuron lineage and regulates AIY axon projections in a L-R asymmetric manner. Here, by combining a candidate approach and single-cell RNA sequencing data analysis, we identify the ephrin protein EFN-2 and the Flamingo protein FMI-1 as downstream targets of HLH-16 that are L-R asymmetrically expressed in the AIY lineage. We show that EFN-2 and FMI-1 collaborate in the L-R asymmetric regulation of axonal growth. EFN-2 may act via a non-canonical receptor of the L1CAM family, SAX-7. Our study reveals novel molecular L-R asymmetries in the C. elegans nervous system and their functional consequences.

Pohl C (2011) Commun Integr Biol "Left-right patterning in the C. elegans embryo: Unique mechanisms and common principles."

Pohl C

[Commun Integr Biol, 2011]

The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.