-
Xu, Chuan, Barrett, Alec, Varol, Erdem, Cook, Steven J, Basavaraju, Manasa, Yemini, Eviatar, Hammarlund, Marc, Miller III, David M, Weinreb, Alexis, Abrams, Alexander, Rafi, Ibnul, Santpere, Gabriel, Vidal, Berta, Sestan, Nenad, Oikonomou, Panos, McWhirter, Rebecca, Cros, Cyril, Glenwinkel, Lori, Tavazoie, Saeed, Taylor, Seth R, Hobert, Oliver, Reilly, Molly B, Poff, Abigail
[
International Worm Meeting,
2021]
Neurons are the fundamental structural and functional units of the nervous system. Although all neurons share many common features, they also display remarkably diverse morphological and functional characteristics. To uncover the underlying genetic programs that specify individual neuron identities, the CeNGEN consortium produced scRNA-Seq profiles of > 100,000 cells from the L4 stage C. elegans hermaphrodite, including all neuron classes and several non-neuronal cells (e.g., glia, muscle, hypodermis, reproductive tissues). In addition, we identified distinct subclasses for 10 of the 118 anatomically-defined classes. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of ~23 neuropeptide genes and ~36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to identify cis-regulatory elements for neuron-specific gene expression. Because our scRNA-Seq data match the single cell resolution of the wiring diagram, we also sought to correlate expression of cell adhesion proteins with neuron-specific fasciculation and connectivity in the nerve ring. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system. These data are available at cengen.org and can be interrogated with the web application CengenApp at cengen.shinyapps.io/CengenApp.
-
[
Dev Cell,
2013]
Genomes are constantly challenged by invaders, so determining what belongs is crucial. Small RNAs silence alien DNA, but Conine etal. (2013), Seth etal. (2013), and Wedeles etal. (2013) now report in Cell and Developmental Cell that these tiny transcripts can also license trusted DNA for expression.
-
Barrett, Alec, Taylor, Seth R., Weinreb, Alexis, Miller III, David M., Varol, Erdem, Sestan, Nenad, Hobert, Oliver, McWhirter, Rebecca, Li, Mingfeng, Hammarlund, Marc
[
International Worm Meeting,
2021]
Advances in RNA-seq for bulk and single cell (sc) approaches have produced increasingly fine dissections of the C. elegans transcriptome. Although both techniques can yield transcriptomes for individual cell types, each comes with strengths and weaknesses. scRNA-Seq affords high resolution, but suffers from dropout, leading to false negatives. Bulk sequencing detects more genes, but suffers from contaminating cell types, resulting in false positives. In this work we integrated these orthogonal approaches to improve the accuracy of both methods. We used bulk samples collected for specific neuron types and sc datasets for all C. elegans neurons and additional non-neuronal cells (1). We used sc data to estimate contamination in each bulk sample, and developed novel methods for removing these gene counts. In one approach we used linear histogram matching to scale sc counts, and subtracted putative contamination using data from non-neuronal clusters. In another approach we used bootstrapping to estimate gene level contributions from target and contaminating tissues in sc data and apply them to bulk counts, providing a bootstrap sample distribution of corrected expression data. We assessed these approaches in two ways: 1) Measuring improvements in calling genes with known expression in all neurons; 2) Examining effects on eliminating genes expressed exclusively in contaminating tissues. We found that our analysis reduced false positives, while maintaining robust true positive detection, thus offering a unique strategy for utilizing complementary bulk and sc RNA-Seq data sets to enhance the accuracy of cell-specific expression profiling data. 1. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. bioRxiv. 2020:2020.12.15.422897.
-
[
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.
-
[
STAR Protoc,
2021]
Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from <i>C.elegans</i>, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth etal. (2019).
-
[
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.
-
[
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.
-
[
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.
-
[
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.
-
[
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.