Aronoff R et al. (1996) West Coast Worm Meeting "PLASMID RESCUE FINALLY. IS SMG-4 A PROTEIN WITH AN RRM AND A KE RICH DOMAIN?"

Aronoff R, Hodgkin JA, Baran R

[West Coast Worm Meeting, 1996]

The seven smg genes mediate mRNA surveillance, a system that detects and destroys aberrant mRNA, in the nematode C. elegans. Previously cloned smg genes encode an RNA helicase, a protein kinase and several novel products, some of which contain motifs implicated in protein:protein interactions and can be shown to co-immunoprecipitate in vitro (Anderson lab, unpub'd results). Rescue of smg-4 mutants, assayed in an unc-54(r293);smg-4 (ma116) strain, was previously obtained with yeast artificial chromosomes, Y1D1 and Y42F5. Phage were isolated from a library generated from Y42F5 genomic DNA, and one clone, gmRB162, was found to rescue smg-4 mutant animals. Plasmid subclones were tested in further rescue assays, and the predicted gene sequence analyzed. The smallest rescuing subclone of 3.6Kbp, pgm185, could encode two potential open reading frames, which may be part of an operon. Subclones containing either ORF will not rescue. However, when only one or the other ORF is disrupted by site directed mutagenesis, only mutation of the downstream ORF will abrogate rescuing activity. So it seems the rescuing activity requires upstream sequences for its expression, and the downstream ORF mediates smg-4+ function. A 1.5Kb RNA is detected in both total and pA+ RNA, which could encode either predicted gene product. Restriction fragment length polymorphisms have been found associated with mutant smg-4 alleles by southern blotting. For instance, 2.5Kbp is deleted from this region in smg-4 (r1169), an allele generated with DEO by S. Kuchma. This 2.5Kb includes a domain which contains a good match to the octamer consensus sequence of the RNA Recognition Motif (RRM), an 80 a.a. RNA binding motif. In addition, the ORF has a lysine and glutamic acid (KE) rich domain which shares sequence identity with a few genes, of particular note, the yeast gene SPB. Analysis of cDNAs, sequencing of mutant alleles, and expression and more rescue studies are currently in progress.

Carroll AS et al. (1996) East Coast Worm Meeting "Stabilization of the DNA-bound SKN-1 basic region by an adjacent a-helical domain"

Ellenberger TE, Blackwell TK, Cheung JW, Wagner G, Liu XY, Carroll AS, Gunther CS, Gilbert DE

[East Coast Worm Meeting, 1996]

During the earliest stages of embryogenesis in C. elegans, maternally-expressed SKN-1 protein directs cells to adopt fates that lead to formation of the pharynx, the gut, and part of the body-wall muscle. At the C-terminus of SKN-1 is a basic region (BR) like those of the basic-"leucine zipper" (bZIP) proteins. However, SKN-1 lacks a ZIP segment, which in bZIP proteins is essential for DNA binding because it mediates their dimerization, and may stabilize and orient the BR as an a-helix in the major groove. In contrast, SKN-1 binds to DNA as a monomer with nanomolar affinity, to a specific sequence consisting of a bZIP half-site adjacent to an AT-rich region. At the C-terminus of its 85 amino acid DNA-binding domain (the Skn domain) is its BR, and at the N-terminus is a sequence related to the flexible N-terminal "arm" (HD arm) with which homeodomain proteins bind to DNA in the minor groove. We have undertaken a series of mutagenesis, spectroscopic, and biochemical "footprinting" experiments to investigate how SKN-1 binds to DNA at high affinity as a monomer. Our findings indicate that the Skn domain inserts the BR into the major groove of the bZIP half-site, and places the HD arm in the minor groove of the AT-rich sequence. The intervening residues, which are distinct from homeodomains and ZIP segments, are both necessary and sufficient to promote sequence-specific DNA binding by the SKN-1 BR. When the Skn domain is not bound to DNA, the BR is unstructured, and the intervening residues are predominantly a-helical. However, surprisingly, this helical region is not rigidly ordered in structure, but exists in a partially-ordered state termed a "molten globule." Upon DNA binding, the BR forms an a-helix, the HD arm binds in the minor groove, and the intervening residues form a helical structure which is stably ordered, and which itself stabilizes the bound BR helix. Stabilization of the BR does not appear to involve intramolecular interactions with its side chains, but is impaired by disruption of residues in the helix immediately N-terminal to it. These findings demonstrate that the BR is not "trapped" in the major groove by side chain interactions, and indicate instead that its stability requires direct extension of a continuous a-helix from the adjacent helical structure. The SKN-1 DNA binding domain is thus unique in two respects: it represents the only known example of monomeric binding by a BR, and is the only such domain which is capable of sequence-specific binding but does not have at least some portion that is rigidly ordered when not bound to DNA. These results further imply that stability deriving from helix extension is likely an important factor in DNA binding by other BR-containing proteins.

Shibata Y et al. (1997) International C. elegans Meeting "AN ANALYSIS OF A NEURONAL GENE IDENTIFIED BY PROMOTER TRAPPING"

Fujisawa H, Takagi S, Shibata Y

[International C. elegans Meeting, 1997]

By the promoter trapping using GFP, we screened genes which are predominantly expressed in the nervous system. We have isolated 15 clones. Among them, a clone 5E5 expressed GFP in a subset of neurons. To determine in detail the expression pattern of the gene corresponding to 5E5, transforming arrays of 5E5 were integrated into genome. The expression of GFP was detected in 15 circumpharyngeal neurons, 2 tail neurons, pharyngeal cells and several unidentified cells in the head. The expression was first detected at the threefold-stage. In juvenile larvae, additional expression of GFP was detected in cells posterior to the anus. Weak expression was also observed in HSN and vulval muscles in the L4 and adult stages. We have previously reported that the 5E5 insert hybridized with YAC Y53G9 on LGX as well as with cDNA, YK637, isolated by Dr. Yuji Kohara. We have determined the entire sequence of the cDNA. It encodes a polypeptide of 746 amino acid residues with 3 EGF motifs, an acidic region, and a putative transmembrane domain. To elucidate the function of the gene corresponding to 5E5, we are generating targeted mutants. One Tc1 insertion in the gene was identified, but the insert was positioned in an intron. We are trying to isolate deletants from the Tc1-inserted line.

Dan Bumbarger et al. (2001) International C. elegans Meeting "Electron Tomography at an Organismal Level, a Unique Application of a Powerful Tool"

James Baldwin, Dan Bumbarger

[International C. elegans Meeting, 2001]

It can be difficult to obtain three dimensional(3D) information with traditional transmission electron microscopy(TEM). Electron microscope tomography using thick sections represents an alternative to reconstructing 3D images from serial thin sections. In some circumstances this technique can provide better depth resolution, require less time and be less subject to certain types of artifacts. As a result, some types of questions that were possible but difficult to address with thin section are now more practical. These include taxonomic comparisons and the production of developmental time series at an ultrastructural level. The method has been utilized here to examine cuticular structures in Zeldia punctata (Rhabditida: Cephalobidae) and to compare the tomographic reconstruction with information obtained via thin section TEM. Limitations and possible methodological improvements are discussed. Electron tomography has been used primarily at a macromolecular to sub-cellular spatial scale. Due to their small size, nematodes provide a unique opportunity to apply this tool as a means of asking questions at an organismal level. Possible applications to Caenorhabditis elegansinclude the comparative characterization of cuticle mutations, elucidation of the connections between neurons and the incorporation of higher quality 3D information into ultrastructural descriptions of morphogenetic processes.

Benjamin Schlager et al. (2006) European Worm Meeting "An Ancestral Module Patterns a Derived Nematode Vulva Equivalence Group"

Ralf Sommer., Benjamin Schlager

[European Worm Meeting, 2006]

Benjamin Schlager and Ralf Sommer. Nematode vulva formation provides a paradigm to study the evolution of pattern formation and cell fate specification. The Caenorhabditis elegans vulva is generated from three of six equipotent cells that form the so-called vulva equivalence group. During evolution, the size of the vulva equivalence group has changed: Panagrellus redivivus has eight, C. elegans six and Pristionchus pacificus only three cells that are competent to form vulval tissue. In P. pacificus, the reduction of the vulva equivalence group is achieved by programmed cell death of individual precursor cells. We have identified the genes controlling this cell death event and the molecular mechanism of the reduction of the vulva equivalence group. Mutations in Ppa-hairy, a gene which is unknown from C. elegans, result in the survival of two precursor cells, which expands the vulva equivalence group. Mutations in Ppa-groucho cause a similar phenotype. Ppa-HAIRY and Ppa-GROUCHO form a molecular module that represses the Hox gene Ppa-lin-39 and thereby, reduces the size of the vulva equivalence group. The C. elegans genome does not encode a similar hairy-like gene and no typical HAIRY/GROUCHO module exists. Instead, the size of the C. elegans vulva equivalence group is regulated by the EGF and WNT signaling pathways.. Therefore the derived vulva equivalence group of P. pacificus is patterned by an ancestral hairy/groucho module.. I will also present a highly speculative model of what kind of adaptive values might be associated with the Pristionchus specific reduction of the size of the vulva equivalence group.

Alan M Zahler et al. (2003) International Worm Meeting "Sup-39 is a U1 snRNA gene which acts as an informational suppressor of a splice donor mutation."

A Brock Roller, Alan M Zahler, John D Tuttle

[International Worm Meeting, 2003]

sup-39 mutations are dominant allele-specific suppressors of the uncoordination defect of unc-73(e936) in C. elegans (1). The unc-73(e936) allele suppressed by sup-39 is a point mutation in the splice donor for intron 16 which changes the canonical G at the first nucleotide of the intron to a T. Given the allele-specific suppression of a splice site defect, we set out to determine whether sup-39 mutations alter the splicing of e936 mutant pre-mRNAs. The e936 mutation activates the use of three different cryptic 5 splice sites, two of which lead to a change in reading frame. The third cryptic site, found in 10% of the stable messages, is unusual; the wild type 5 splice site is used even though it defines an intron beginning with a T. In the presence of a sup-39 mutation, the same three e936 cryptic sites are used, but the levels of the different sites in stable mRNA change; the wild type site represents 35% of stable e936 messages and a concomitant increase in full length UNC-73 protein is detected. We also detect this same effect of sup-39 on chimeric fusion protein constructs. Our initial characterization of the effects of sup-39 on e936 cryptic splicing has been published (2). We have recently cloned sup-39 by SNP mapping. sup-39 is a U1 snRNA gene, one of 12 in the C. elegans genome. Both known alleles possess the same mutation, which is a compensatory mutation in the 5 end of this U1 snRNA to match the mutant splice donor in e936. This is the first example of an informational suppressor (allele-specific gene non-specific suppressor) identified in an snRNA gene by forward genetics. We propose that this U1 snRNA mutation increases the spliceosomes affinity for the mutant 5 splice site. The pre-mRNA sequence requirements for splicing of introns beginning with T will be discussed. We are currently mapping syr-1, a recessive synthetic roller isolated by Jeff Ways group which rolls only in the presence of sup-39 mutations. We hope that identification of a second gene regulated by sup-39 will yield additional clues about U1 snRNA function in recognition of unusual 5 splice sites. 1. Run, J. Q. et al. (1996). Genetics 143: 225-36; 2. Roller, A.B. et al. (2000) Genetics 154: 1169-79.

Quintin S et al. (1997) International C. elegans Meeting "Does lin-26 act as an activator or as a repressor?"

Camut E, Quintin S, Labouesse M

[International C. elegans Meeting, 1997]

lin-26 has been shown to be required to specify and/or maintain the fates of all non-neuronal ectodermal cells. The LIN-26 protein has two C2H2 motifs related to TFIIIA zinc-fingers and three potential transactivation domains suggesting that it could be a transcription factor. If this is the case, then LIN-26 could either activate genes that specify hypodermal and glial-like cell fate or repress genes required for neuronal fate. In order to resolve this problem, we ectopically expressed the LIN-26 protein. We injected a construct containing the lin-26 cDNA under the control of a heat shock (H.S.) promoter (pPD 49.78) into wild type animals. The heat-shocked transgenic embryos show a striking phenotype compared to control animals (carrying the same construct with stop codons inserted at the beginning of the lin-26 coding region). When the H.S. is applied immediately after egg-laying, many embryos arrest with about 250 cells, most of which express LIN-26 and the hypodermal marker MH27, suggesting that cells have adopted some hypodermal features. When H.S. is applied slightly later generation of the pharynx is frequently affected and embryos do not elongate properly. To further characterise the effects of ectopic LIN-26 expression, we generated an integrated line. We also drove LIN-26 expression in early touch neurons using a mec-3 promoter (pPD 57.56). Preliminary results show that ectopic expression of LIN-26 does not seem to have an effect on neuronal differentiation in these cells. Altogether, these data suggest that lin-26 can trigger hypodermal fate in undifferentiated cells but not in cells that have already been commited.

Ewald, Collin et al. (2009) International Worm Meeting "GENETIC INTERACTIONS BETWEEN apl-1, A GENE ENCODING AN AMYLOID PRECURSOR-RELATED PROTEIN, AND daf-16, A REGULATOR OF LIFESPAN."

Li, Chris, Ewald, Collin

[International Worm Meeting, 2009]

Alzheimer''s Disease (AD) is an age-dependent, neurodegenerative disease. Aggregation of the b-amyloid peptide is toxic to neurons and may be the main cause of neurodegeneration in AD. The b-amyloid peptide is a cleavage product of the amyloid precursor protein (APP). Although the processing of APP has been elucidated, the physiological function of APP remains unclear. In C. elegans, APL-1 has high sequence similarity to human APP, but does not contain the b-amyloid peptide. Similarly to APP, APL-1 is also cleaved to produce a large extracellular fragment and a small intracellular stub. Null mutations of apl-1 lead to an L1 lethality, which can be rescued by neuronal expression of only the extracellular fragment of APL-1 (APL-1EXT), suggesting the importance of apl-1 for development and viability. Duplication of the human APP locus can lead to familial AD. Hence, we examined how APL-1 overexpression can affect later stages. Overexpressing APL-1 resulted in a shortened body length and decreased mean lifespan in a dose-dependent manner. Overexpression of only the extracellular domain of APL-1 was also sufficient to decrease lifespan and body length. Since APL-1 is expressed in neurons and other cell types, we hypothesized that APL-1 overexpression in neurons was responsible for the decreased lifespan. Surprisingly, driving APL-1 expression with a predominantly neuronal promotor (Psnb-1::apl-1) increased the mean lifespan by 20%. Overexpressing only the extracellular domain of APL-1 under the control of the snb-1 promotor (Psnb-1::APL-1EXT) was also sufficient to enhance lifespan. To determine whether this lifespan effect is due exclusively to neuronal overexpression, we drove APL-1 expression with the rab-3 promotor (Prab-3::apl-1), which only expresses in neurons. Although the Prab-3::apl-1 construct is sufficient to rescue the apl-1 L1 lethality, these animals do not have an enhanced lifespan. Hence, the APL-1-dependent lifespan effect must be due to overexpression in a different tissue. To further characterize the tissue-specific lifespan effect of APL-1, we tested whether APL-1 overexpression effects were dependent on daf-16, a transcription factor regulating lifespan in a tissue-specific manner. The increased lifespan of animals carrying Psnb-1::apl-1 was abolished in a daf-16 null background, suggesting the requirement of DAF-16 for lifespan extension. Furthermore, while overexpression of APL-1 decreases body length, the normal length of the animal can be restored in a daf-16 null mutant background. Hence, DAF-16 is required for two APL-1 overexpression phenotypes, the tissue-specific lifespan effects and the short body length, suggesting a genetic interaction between daf-16 and apl-1.

Hongkyun Kim et al. (2004) West Coast Worm Meeting "Role of an acetylcholine transporter in a C. elegans model of muscular dystrophy."

Janet E Richmond, Steven L McIntire, Matthew J Rogers, Hongkyun Kim

[West Coast Worm Meeting, 2004]

Duchenne muscular dystrophy (DMD) is one of the most common genetic diseases in humans with an incidence of 1 in 3,500 male births. Duchenne and other muscular dystrophies are characterized by progressive muscle weakness and wasting. In the 1980s, dystrophin was identified as the gene defective in DMD. It is now known that dystrophin is a component of the dystrophin-glycoprotein complex (DGC), which is found in the plasma membrane of muscle cells (including neuromuscular junctions). Genetic defects in components of the DGC result in many different forms of muscular dystrophy. The DGC links the cytoskeleton to the extracellular matrix and is thought to be important for maintaining muscle structural stability and organizing signaling molecules. The exact role of the DGC in the pathogenesis of disease has, however, remained uncertain. C. elegans possesses most of the DGC components found in mammals, albeit with less diversity. Segalat and colleagues have shown that mutations in C. elegans DGC genes (such as dys-1 , a dystrophin homologue) lead to an exaggerated bending of the anterior body and head during forward movement, hypersensitivity to aldicarb and acetylcholine, and muscle degeneration in a sensitized ( hlh-1 ) genetic background. Previously, we identified twelve mutants with phenotypes that are indistinguishable from those of the DGC mutants, including five alleles ( eg28 , eg114 , eg115 , eg121 , eg137 ) of a novel gene. We cloned the novel gene based on the locomotory phenotype. The gene encodes snf-6 , a member of the sodium-dependent neurotransmitter transporter family. Tissue-specific rescue experiments indicate that snf-6 functions in muscle cells. In an in vitro uptake assay using radio-labeled substrates we found that SNF-6 transports acetylcholine and choline in a saturable and sodium-dependent manner. Consistent with these results, GFP-tagged SNF-6 expressed under the control of a muscle-specific myo-3 promoter was observed in the end of muscle arms where neuromuscular junctions are located. Whole-cell voltage-clamped current recordings from body wall muscles suggest that SNF-6 clears acetylcholine at neuromuscular junctions. Evoked responses after a single stimulation are the same in snf-6 and wild-type animals; however, evoked responses after repetitive stimulation are significantly greater in snf-6 than in wild-type animals. These results are consistent with a role for SNF-6 mediated acetylcholine uptake during periods of increased synaptic activity. Several lines of evidence indicate that SNF-6 interacts with the DGC. First, co-immunoprecipitation experiments demonstrate that SNF-6 interacts in vitro with the DGC through the PDZ domain of STN-1, a b 1-syntrophin homologue. The PDZ interacting sequence in SNF-6 is required for complete rescue of the snf-6 phenotype. Second, mutations in DGC genes cause a progressive loss of SNF-6 at neuromuscular junctions. Third, an equivalent degree of muscle degeneration occurs in snf-6 and DGC mutants (in the same hlh-1 background). Our studies in C. elegans suggest that improper clearing of acetylcholine and prolonged excitation of muscles may have a role in the pathogenesis of muscular dystrophies.

Santella, A. et al. (2019) International Worm Meeting "Toward an EM Time Series: Automated Cell Identification in a Developmental Context."

Santella, A., Kolotueva*, I., Bao, Z.

[International Worm Meeting, 2019]

Though automation makes EM data more accessible, navigation and identification of structures remains a bottleneck for interpretation and analysis. To assist with this problem we present a robust, general method for assigning single cell identities from a template to a sample in the presence of both differences in the cells present and changes in spatial configuration, validating this in the context of the C. elegans embryo. We introduce relative neighbor graph constraints to model the invariant spatial structure of a labeled samples and use this to assign a global quality score to an answer based on its internal consistency with expected cell-cell contacts. This score is used in a novel gradient descent optimization of the template sample which removes cells whose presence cause neighbor constraint violations and are therefore hypothesized to be missing in the unlabeled sample. Our final answer is produced by an instance-based learning like approach where the sample is independently matched against each example in the ensemble of reference data sets and a consensus identity is assigned. We apply this method to identifying all cells in Electron Micrographs of two C. elegans embryos at ~300min and 350min p.f.c. Imaging with Focused Ion Beam SEM and a serial array method provides an undistorted image of the worm simplifying the problem of alignment. Time lapse fluorescence microscopy provides the reference atlas data set of cell positions. Three embryos, underlying data for the WormGUIDES atlas, have identities established by lineaging between the four-cell stage and twitching. This data and our method were used to assign cell identities to all nuclei in the two unknown data sets. To validate automated results cell identities were independently and manually assigned to a subset of cells in the two EM data sets based on position and cell morphology. Predicted identities were 88% and 91% accurate in the early and late data sets on the cases confirmed by morphology suggesting cell identities can be reliably assigned based on position alone. *Irina Kolotueva co-first author and co-PI