Palmer RE et al. (1994) Worm Breeder's Gazette "Use of an Expression Library to Clone Genes by Complementation"

Palmer RE, Sternberg PW

[Worm Breeder's Gazette, 1994]

Use of an expression library to clone genes by complemcntation. RE. Palmer and P.W. Sternberg, HHMI and Division of Biology, Caltech, Pasadena, CA 91125

Vieux, Karl-Frederic et al. (2021) International Worm Meeting "Screening by deep sequencing reveals mediators of miRNA tailing in C. elegans"

McJunkin, Katherine, Prothro, Katherine, Veksler-Lublinsky, Isana, Palmer, Cameron, Vieux, Karl-Frederic

[International Worm Meeting, 2021]

Most micro-RNAs (miRNA) result from the processing of longer primary transcripts transcribed from miRNA genes by the RNA polymerase Pol II. These transcripts harbor hairpin structures which are recognized and processed twice to yield mature miRNAs: (1) once in the nucleus by Drosha, with the support of Pasha (PASH -1 in C. elegans) and (2) again in the cytoplasm by Dicer. The resulting ~22nt mature miRNAs are effective regulators of gene expression. When loaded onto the RNA-induced silencing complex (RISC), mature miRNAs direct RISC activity to transcripts with complementary sequence elements to repress their expression. In turn, their levels must also be regulated. Despite this, the processes responsible for miRNA turnover remain poorly understood. In instances where they are turned over, miRNAs are frequently modified by the addition of untemplated nucleotides to their 3' end, but the role of this tailing is often unclear. Here we characterized the prevalence and functional consequences of microRNA tailing in vivo, using the C. elegans model. Our data showed that miRNA tailing in C. elegans consists mostly of mono-uridylation of mature miRNA species, with rarer mono-adenylation - likely added to microRNA precursors. Through a targeted RNAi screen, we discovered that the TUT4/TUT7 gene family member CID-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2 is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We also examined the role of tailing in turnover in the absence of ongoing miRNA production, using a temperature sensitive pash-1 strain. At permissive temperatures, PASH-1 is functional, and biogenesis is uninterrupted. When upshifted to 25°C, PASH-1 activity is abrogated, and mature miRNA production ceases. We determined the half-lives of miRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median=20.7h), as observed in other systems. Despite an increased proportion of tailed species in older microRNAs, we detected no changes to microRNA abundance or decay dynamics upon disrupting tailing. Thus, tailing is not a global regulator of miRNA decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this work lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

Hudson ML et al. (1998) Worm Breeder's Gazette "The Physiological Roles of Soluble Guanylate Cyclase in Caenorhabditis elegans"

O'Shea M, Hudson ML

[Worm Breeder's Gazette, 1998]

Cyclic GMP (cGMP) is an important intracellular signaling molecule in both vertebrates and invertebrates mediating a number of important physiological roles such as cardiovascular tone, neuronal signalling, platelet function and phototransduction (Hobbs, 1997). cGMP is synthesized from GTP by the enzyme guanylate cyclase (GC) which has two main classes; membrane bound or particulate GC (mGC) and the soluble, cytosolic form (sGC). The former generally mediate the action of signaling peptides such as atrial natriuretic peptide whereas sGC is the primary target of nitric oxide (NO), a gaseous intracellular messenger (Palmer et al, 1987). Around thirty GCs have been identified by the C. elegans genomic sequencing program to date (Yu et al,1997), with about five probable sGCs and the remainder being putative mGCs (most closely related to the mammalian natriuretic peptide receptors). We are interested in identifying and characterising the sGCs in C. elegans in terms of their expression, activation and physiological roles. We have already cloned and sequenced a cDNA corresponding to the putative sGC C06B3.8, now identified by Yu et al as gcy-32. By using a combination of RT-PCR and PCR amplification of a cDNA library, the clone was found to use two different polyadenylation signals and also shows a different splicing form to that predicted by the Genefinder software. It is not yet apparent whether this cDNA is trans-spliced. In other systems, sGC has been shown to function as a heterodimeric protein and work is under way to identify which genes are coexpressed in particular cells, using a combination of GFP fusion constructs and immunocytochemistry. A radioimmunoassay has been developed to detect cGMP generation in extracts of nematode tissue. Interestingly, worm cytosolic extract appears to be insensitive to NO stimulation. This is consistent with work carried out in our lab which has failed to demonstrate Ca2+ stimulated turnover of 3H-arginine to 3H-citrulline; a standard assay for NO production by the enzyme nitric oxide synthase (NOS). Also, no genes have yet been identified by the worm sequencing project which show homology to NOS. This has lead us to the intriguing possibility that C. elegans does not possess a classical NO-cGMP signaling pathway. Our ultimate goal is to prove or disprove this theory and to shed light on the physiological and behavioral role of the sGCs that have been found in the worm genome. References. Hobbs, A. J. (1997) Trends Pharmacol. Sci. 18, 484-491 Palmer, R. M. J., Ferridge, A. G. and Moncada, S. (1987) Nature 327, 524-526 Yu, S., Avery, L., Baude, E. and Garbers, D. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3384-3387

Brendan Galvin et al. (2001) International C. elegans Meeting "A ced-4 SUPPRESSION SCREEN TO IDENTIFY NEW GENES INVOLVED IN PROGRAMMED CELL DEATH"

Brendan Galvin, Bob Horvitz, Scott Cameron

[International C. elegans Meeting, 2001]

Many genes involved in programmed cell death in C. elegans have been identified by screening for mutations that allow survival of cells that are normally destined to die. The opposite approach, screening for mutations that result in an increase in the number of cell deaths, has been comparatively unexplored, in part because of the lack of efficient methods to identify such mutations. A lin-11::gfp reporter (generated by Scott Cameron) expresses in the Pn.aap cells of the ventral cord. In wild-type animals, the six Pn.aap cells of the P3-P8 lineages survive, differentiate into VC neurons, and express lin-11::gfp . In strong cell-death defective mutants, all 12 Pn.aap cells survive and express GFP. In the lin-11::gfp reporter strain the amount of cell death can be quantified by scoring the number of fluorescent nuclei in the ventral cord using a fluorescence-equipped dissecting microscope. Using the lin-11::gfp reporter, I have performed a screen for suppressors of a partial loss-of-function ced-4 mutant by looking for mutants with a reduction in the number of GFP-positive Pn.aap cells. To date approximately 40,500 mutagenized genomes have been screened; 5,000 were screened clonally and 35,500 non-clonally. Two strong suppressors were obtained that reduce the number of GFP-positive Pn.aap cells. The first suppressor, identified in the clonal screen, is recessive and is recessively sterile. The second suppressor, identified in the non-clonal screen, is dominant and is recessively sterile. Further characterization of these mutants will be described.

S Cameron et al. (1999) International C. elegans Meeting "A screen for mutations that affect programmed cell death in the ventral cord"

S Cameron, N Tsung, B Horvitz

[International C. elegans Meeting, 1999]

We have developed an efficient screen for mutations that affect the survival or death of specific ventral cord cells during normal development. A lin-11::gfp reporter construct is expressed in the six VC motor neurons that arise from the P3-P8 lineages as Pn.aap cells. The lineally equivalent Pn.aap cells from the W, P1, P2 and P9-12 lineages die by programmed cell death. In ced-3 animals, in which cell death is prevented, all the "undead" Pn.aap nuclei express GFP and fluoresce brightly. We are using this reporter to screen for mutations that result in the survival of Pn.aap cells in lineages in which they normally die, or the death of Pn.aap cells in lineages in which they normally survive. Hermaphrodites are mutagenized with EMS, and the F2 progeny are screened using a stereomicroscope for animals with an abnormal number or pattern of fluorescing nuclei. To date, 20,000 haploid genomes have been screened, and 70 mutants isolated. Mutants with abnormal survival of Pn.aap cells include new loss-of-function mutations in ced-3 , ced-4 , and egl-1 . New pag-3 alleles have been recovered (see abstract by Cameron et al. ). Mutants with abnormal death of Pn.aap cells include alleles of at least one gene not previously identified as affecting Pn.aap survival. As the Pn.aap cells are sexually dimorphic, weak, self-fertile Tra mutations are also recovered. Mutants isolated in this screen will be characterized with the goal of identifying the factors that determine whether individual Pn.aap cells survive or die.

EJ Aamodt et al. (1999) International C. elegans Meeting "Conservation of sequence and intron/exon structure between the homologous pag-3 genes from C. elegans and C. briggsae"

L Shen, EJ Aamodt, J McDermott, B Rose

[International C. elegans Meeting, 1999]

The pag-3 gene encodes a zinc finger protein involved in controlling neuron specific gene expression. pag-3 mutants were first isolated based on misexpression of touch receptor genes in the BDU interneurons, the lineal sisters to the ALM touch neurons. pag-3 mutants are lethargic, kink when trying to move backward (reverse kinker Unc) and have lineage defects in the P-neuroblast lineages (see abstract by Cameron et al., this meeting). We identified a fosmid containing the C. briggsae homologue of the C. elegans pag-3 gene and the Washington University Genome Sequencing Group sequenced it. When transformed into a C. elegans pag-3 mutant, the C. briggsae pag-3 gene rescued the pag-3 reverse kinker and lethargic phenotypes. The C. elegans pag-3 gene fused to lacZ was expressed in the same pattern in C. elegans and C. briggsae . Unlike many gene homologues compared between C. elegans and C. briggsae , extensive sequence conservation was found in the noncoding regions upstream of the pag-3 exons, in several of the introns and in the downstream noncoding region. Furthermore, the splice acceptor and splice donor sites were conserved and the size of the introns and exons was surprisingly similar. Based on the genomic sequence, the sequence of the C. briggsae PAG-3 protein was predicted. The predicted protein sequence was 85% identical to the sequence of C. elegans PAG-3. These results showed the evolutionary conservation of the pag-3 gene sequence, its expression and function. Because so much of the non-coding regions of pag-3 are conserved, the control of pag-3 may be quite complex, involving the binding of many transacting factors.

PW Reddien et al. (1999) International C. elegans Meeting "The engulfment of dying cells contributes to the killing process of programmed cell death"

HR Horvitz, JS Cameron, PW Reddien

[International C. elegans Meeting, 1999]

The engulfment of a cell corpse by a neighboring cell typically occurs rapidly after the generation of a cell programmed to die. This engulfment can initiate before the completion of cell division, as shown by electron microscopy (1). Because cell corpses are generated in mutants defective in engulfment, the proposed function of engulfment has long been solely the removal of unwanted apoptotic cell bodies. We have discovered, however, that in addition to functioning in cell-corpse removal, engulfment assists in the killing of dying cells. Animals with strong loss-of-function mutations in the killer gene ced-3 lack most if not all programmed cell deaths. However, weaker ced-3 mutants exist that lack only a small percentage of programmed cell deaths. We found that such a partial block in the execution of programmed cell death is enhanced by mutations in genes that are involved in the engulfment process ( ced-1 , ced-2 , ced-5 , ced-6 , ced-7 , and ced-10 ). Furthermore, mutations in these engulfment genes alone are sufficient to result in a low-penetrance survival of some cells that normally die in the ventral cord. Lineage analysis shows that cells that fail to die initially show some morphological characteristics of programmed cell deaths but ultimately appear morphologically indistinguishable, using Nomarski optics, from living cells. Surviving Pn.aap cells in these ventral cord lineages are capable of expressing the cell-type specific reporter lin-11::gfp (see Cameron, Tsung, and Horvitz, this meeting), suggesting that they are capable of differentiating. We are analyzing the relative contributions of the different engulfment genes to cell killing, how the engulfment role in killing interacts genetically with ced-8 and ced-9 , and the contribution of the killing role of engulfment to the death of different cell types. 1. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78 , 542-576.

Erich M Schwarz et al. (2003) International Worm Meeting "Comparative analysis of cis-regulatory sequences using four Caenorhabditis species."

Nora Mullaney, Erich M Schwarz, Tristan De Buysscher, Paul W Sternberg, Barbara J Wold, John A DeModena, Eunpyo Moon, Hiroaki Shizuya

[International Worm Meeting, 2003]

Comparing homologous cis-regulatory DNA sequences from three or more genomes has advantages over pairwise comparison of only two. Cis-regulatory sequences are short (6-20 bp) and tolerate substantial variation. Purely random pairing of unrelated 100-bp DNA segments is expected to yield two perfect 6 bp matches. Alignment of a third or fourth sequence should greatly lower the frequency of false positive regions, allowing small but real cis-regulatory sequences to be efficiently detected. This increased resolution should also allow direct comparison between phylogenetically conserved sequences and statistically overrepresented sequences, which may yield complementary views of regulatory elements. In the Caenorhabditis genus, C. remanei appears to be most closely related to C. briggsae; two other species, CB5161 and PS1010, comprise the two closest known and culturable Caenorhabditis species outside the elegans-briggsae group (Fitch, 2000). CB5161 is closest to C. elegans, and PS1010 the next most divergent; these two species thus provide an evolutionarily graded series. We have constructed fosmid libraries from CB5161 and PS1010, and begun sequencing individual fosmids for comparative analysis of genes involved in vulval or sensory neuron development. At the same time, we have devised the Mussa software package to adapt the algorithms of Davidson and coworkers (Brown et al., 2002) to multiple sequence analysis. At this writing, we have sequence data from the egl-30, lin-11, and mab-5 loci of both CB5161 and PS1010. Initial results of sequencing and comparative sequence analysis will be presented. References: Brown, C.T., Rust, A.G., Clarke, P.J., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., and Bolouri, H. (2002). New computational approaches for analysis of cis-regulatory networks. Dev. Biol. 246, 86-102; Fitch, D.H.A. (2000). Evolution of Rhabditidae and the male tail. J. Nematol. 32, 235-244.

Kristopher Kelley et al. (2007) International Worm Meeting "The nuclear receptor NHR-6 regulates cell proliferation and cell differentiation during development of the spermatheca and spermatheca-uterine valve."

Kristopher Kelley, Chris R. Gissendanner, Riddhi Bhatt

[International Worm Meeting, 2007]

Members of the NR4A group of nuclear receptors are critical transcriptional regulators of cell proliferation and differentiation during vertebrate organogenesis. In C. elegans, the NR4A ortholog, NHR-6, also has an essential function in organ development. Specifically, nhr-6 is required for the development of the spermatheca, an organ that serves as the site of sperm storage and fertilization. nhr-6 expression begins in spermatheca progenitor cells and continues until development of the organ is complete. The spermatheca is specified properly in nhr-6 mutants. However, the spermathecae of nhr-6 mutants exhibit severe developmental defects including abnormal expression of differentiation markers and decreased organ size. We have determined that nhr-6 is required for proliferation of the spermatheca lineage, and mutant spermathecae consistently display ~1/3 the normal number of cells. In addition, nhr-6 is also required for formation of the spermatheca-uterine valve. nhr-6 is expressed in the spermatheca-uterine junction core cells (sujc) but not in the cells that form the valve syncytium. Cell marker analysis reveals abnormal differentiation of the sujc cells in nhr-6 mutants. Temporal RNAi experiments have revealed potential distinct functions for NHR-6 during early and late spermatheca development. The regulatory elements that drive expression of nhr-6 in the spermatheca and spermatheca-uterine valve reside in a 1.8 kb intron that lies upstream of the sixth exon of nhr-6. Constructs containing only this intron and downstream nhr-6 coding sequence are sufficient to fully rescue nhr-6 mutants. Transcription factor binding site searches in this 1.8 kb intron reveal the presence of several putative homeobox transcription factor binding sites. Several of these sites are similar to sites recognized by the Nkx6.1 transcription factors. The Nkx6.1 homolog in C. elegans, COG-1, is expressed during development of the sujc and is required for the formation of the sujc (Palmer et al, 2002). Therefore, we are also investigating a role for the homeobox transcription factor COG-1 in the regulation of nhr-6 expression during development of the spermatheca and spermatheca-uterine valve.

Palmer RE et al. (1994) Worm Breeder's Gazette "Identification of An Egg-Laying Defective Mutant With An Abnormal Contact Between the Anchor Cell and the Vulval Precursor Cells"

Palmer RE, Tietze K, Sternberg PW

[Worm Breeder's Gazette, 1994]

Identification of an egg-laying defective mutant with an abnormal contact between the anchor cell and the vulval precursor cells. R.E. Palmer, K. Tietze, and P.W. Sternberg, HHMI and Division of Biology, Caltech, Pasadena, CA 91125 The development of the hermaphrodite vulva involves many previously characterized cell-cell interactions. One important interaction is between the anchor cell of the somatic gonad and the vulval precursor cells (VPCs). During the late L2 larval stage the anchor cell has been shown to necessary for the induction of the VPCs. Using Nomarski optics we have shown that during the L3 larval stage another cell interaction occurs between the anchor cell and the VPCs. After the second round of cell division of the P6.p VPC (four cell stage) the anchor cell begins to invaginate between the two innermost granddaughters of P6.p (P6.pap and P6.ppa). This process occurs just prior to the invagination of these cells. Throughout the L3 larval stage the anchor cell remains positioned between these cells. We have identified an egg-laying defective mutant, sy275, with an abnormal contact between the anchor cell and P6.pap and P6.ppa. In this mutant the anchor cell fails to invaginate between the P6.pap and P6.ppa and is often observe on top of the two cells. In addition to the anchor cell contact abnormality hermaphrodites have other slight defects. (1) The timing of the last round of cell divisions of the P6.p lineage is delayed. (2) In a small fraction of animals there is a third distal tip cell. As adults, the hermaphrodites are completely egg-laying defective and have a slightly protruding vulva. Using criteria outlined in Trent et al. (Genetics 104; 619 647) sy275 has no hermaphrodite specific neuron defect and behaves as a class A egg-laying defective mutant. sy275 is recessive and both males and hermaphrodites are mating competent. Linkage analyses place sy275 on LGII. Three factor mapping indicates that sy275 is close to unc-52 on the right arm of LGII.