Thomas R. Burglin (2006) WormBook "Homologs of the Hh signalling network in C. elegans."

Thomas R. Burglin

[WormBook, 2006]

In Drosophila and vertebrates, Hedgehog (Hh) signalling is mediated by a cascade of genes, which play essential roles in cell proliferation and survival, and in patterning of the embryo, limb buds and organs. In C. elegans, this pathway has undergone considerable evolutionary divergence; genes encoding homologues of key pathway members, including Hh, Smoothened, Cos2, Fused and Suppressor of Fused, are absent. Surprisingly, over sixty proteins (i.e. WRT, GRD, GRL, and QUA), encoded by a set of genes collectively referred to as the Hh-related genes, and two co-orthologs ( PTC-1 ,-3) of fly Patched, a Hh receptor, are present in C. elegans. Several of the Hh-related proteins are bipartite and all can potentially generate peptides with signalling activity, although none of these peptides shares obvious sequence similarity with Hh. In addition, the ptc -related ( ptr ) genes, which are present in a single copy in Drosophila and vertebrates and encode proteins closely related to Patched, have undergone an expansion in number in nematodes. A number of functions, including roles in molting, have been attributed to the C. elegans Hh-related, PTC and PTR proteins; most of these functions involve processes that are associated with the trafficking of proteins, sterols or sterol-modified proteins. Genes encoding other components of the Hh signalling pathway are also found in C. elegans, but their functions remain to be elucidated.

Freeman MN et al. (1998) "Establishment of quality assurance procedures for aquatic toxicity testing with the nematode Caenorhabditis elegans"

Williams PL, Marse TJ, Freeman MN

[1998]

In this study initial data were generated to develop laboratory control charts for aquatic toxicity testing using the nematode Caenorhabditis elegans. Tests were performed using two reference toxicants: CdCl2 and CuCl2. All tests were performed for 24 h without a food source and for 48 h with a food source in a commonly used nematode aquatic medium. Each test was replicated 6 times with each replicate having 6 wells per concentration with 10 +/- 1 worms per well. Probit analysis was used to estimate LC50 values for each test. The data were used to construct a mean laboratory control chart for each reference toxicant. The coefficient of variation (CV) for three of the four reference toxicant tests was less than 20%, which demonstrates an excellent degree of reproducibility. These CV values are well within suggested standards for determination of organism sensitivity and overall test system credibility. A standardized procedure for performing 24 h and 48 h aquatic toxicity studies with C. elegans is

Haeussler S et al. (2022) Methods Mol Biol "Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans."

Conradt B, Haeussler S

[Methods Mol Biol, 2022]

The nematode Caenorhabditis elegans is a powerful model to study cellular stress responses. Due to its transparency and ease of genetic manipulation, C. elegans is especially suitable for fluorescence microscopy. As a result, studies of C. elegans using different fluorescent reporters have led to the discovery of key players of cellular stress response pathways, including the mitochondrial unfolded protein response (UPR^mt). UPR^mt is a protective retrograde signaling pathway that ensures mitochondrial homeostasis. The nuclear genes hsp-6 and hsp-60 encode mitochondrial chaperones and are highly expressed upon UPR^mt induction. The transcriptional reporters of these genes, hsp-6::gfp and hsp-60::gfp, have been instrumental for monitoring this pathway in live animals. Additional tools for studying UPR^mt include fusion proteins of ATFS-1 and DVE-1, ATFS-1::GFP and DVE-1::GFP, key players of the UPR^mt pathway. In this protocol, we discuss advantages and limitations of currently available methods and reporters, and we provide detailed instructions on how to image and quantify reporter expression.

Barbara Conradt (2005) WormBook "Programmed cell death."

BARBARA CONRADT

[WormBook, 2005]

Programmed cell death is an integral component of C. elegans development. Genetic studies in C. elegans have led to the identification of more than two dozen genes that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular and biochemical studies have revealed the underlying conserved mechanisms that control these three phases of programmed cell death. In particular, an interplay of transcriptional regulatory cascades and networks involving CES-1 , CES-2 , HLH-1 / HLH-2 , TRA-1 , and other transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 in cells destined to die. A protein interaction cascade involving EGL-1 , CED-9 , CED-4 and CED-3 results in the activation of the key cell death protease CED-3 . The activation of CED-3 initiates the cell disassembly process and nuclear DNA fragmentation, which is mediated by the release of apoptogenic mitochondrial factors ( CPS-6 and WAH-1 ) and which involves multiple endo- and exo-nucleases such as NUC-1 and seven CRN nucleases. The recognition and removal of the dying cell is mediated by two partially redundant signaling pathways involving CED-1 , CED-6 and CED-7 in one pathway and CED-2 , CED-5 , CED-10 , CED-12 and PSR-1 in the other pathway. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in multicellular organisms.

Cressman CP et al. (1997) "Reference toxicants for toxicity testing using Caenorhabditis elegans in aquatic media."

Cressman CP, Williams PL

[1997]

Caenorhabditis elegans aquatic toxicity assays were standardized with five common reference toxicants: CdCl2, NaCl, KCl, sodium lauryl sulfate (SLS), and sodium pentachlorophenate (PCP). Aquatic toxicity testing was conducted in 3 media: a standard C. elegans medium; EPA moderately hard reconstituted water; and EPA moderately hard mineral water. Test duration in each medium was 24h without a food source, and 24h and 48h with Escherichia coli strain OP50 as a food source. Each test was replicated three times with each replicate having 6 wells per concentration, 10 worms per well. LC50 values were calculated using probit analysis. The average LC50s for each set of replicants were compared to assess sensitivity and reproducibility of the data, identifying expected variation between replicate tests. These reference toxicants increase the database for C. elegans and provide a benchmark for further application.

Siddiqui SS (1989) "Axonal outgrowth and process placement of sensory lumbar neurons in the nematode Caenorhabditis elegans."

Siddiqui SS

[1989]

One of the most remarkable features of the brain is the degree of precision with which neurons are interconnected. As the neural circuitry of the nematode Caenorhabditis elegans has been completely established through serial section electron micrographs, and because the nematode is amenable to genetic analysis, it is an excellent system to study the genetic basis of axonal outgrowth and process placement in the formation of a neural network. Here, I will describe the identification of 11 genes which affect the axonal outgrowth and guidance of five pairs of lumbar neurons in C. elegans. A pair of bilaterally symmetric luumbar ganglia are situated in the tail of C. elegans, each consisting of 12 neurons. We have examined the morphology and pattern of axonal processes of three embryonic (PHA, PHB, and PLM) and two postembryonic (PHC and PVN) lumbar neurons on the wild type and existing uncoordinated (unc) mutants, immunocytochemically. We have earlier shown that antibodies against horseradish peroxidase (HRP) stain PHA, PHB, PHC and PVN neurons, and monoclonal antibodies specific to different tubulin isotypes stain the mechanosensory neuron PLM. A monoclonal antibody TY21, raised against C. elegans crude homogenate is specific for phasmid neurons PHA, and PHB. In the wild type, PHA, PHB, PHC, and PVN send anteriorly directed processes ventrally via the lumbar commissures to targets in the ventral cord; whereas, PLM sends a process anteriorly in a subventral position, sending a branch ventrally to the ventral cord near the vulva. PHA, PHN, PHC, and PLM are bipolar neurons, extend a backward process into the tail spike. Mutations in nine genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98) result in abnormal axonal outgrowth and process placement of PHC, PCN and PLM neurons. Four (unc-6, unc-33, unc-44, and unc-51) of the nine genes identified above, and unc-76 were previously shown to affect the growth of PHA and PHB neurons. Mutants in unc-53, which are apparently normal in the growth of PHA, PHB, PHC, and PVN neurons, and unc-76, harbor defects in PLM axonal outgrowth and guidance. In summary, we have identified a set of 11 genes, which exert overlapping, but distinct effects on the axonal outgrowth and process placement of embryonic (PHA, PHB, and PLM), and postembryonic (PHC and

Bjorkgren I et al. (2017) "Fertility and TRP Channels"

Lishko PV, Bjorkgren I

[2017]

Since their discovery in late 1970, transient receptor potential (TRP) channels have been implicated in a variety of cellular and physiological functions (Minke, 2010). The superfamily of TRP channels consists of nearly 30 members that are organized into seven major subgroups based on their specific function and sequence similarities (Owsianik et al., 2006; Ramsey et al., 2006). With the exception of TRPN channels that are only found in invertebrates and fish, mammalian genomes contain representatives of all six subfamilies: (1) TRPV (vanilloid); (2) TRPC (canonical); (3) TRPM (melastatin); (4) TRPA (ankyrin); (5) TRPML (mucolipin); and (6) TRPP (polycystin). TRP channels play crucial regulatory roles in many physiological processes, including those associated with reproductive tissues. As calcium-permeable cation channels that respond to a variety of signals (Clapham et al., 2003; Wu et al., 2010), TRP channels exert their role as sensory detectors in both male and female gametes, and play regulatory functions in germ cell development and maturation. Recent evidence obtained from Caenorhabditis elegans studies point to the importance of these proteins during fertilization where certain sperm TRP channels could migrate from a spermatozoon into an egg to ensure successful fertilization and embryo development. In this chapter we discuss how TRP channels can regulate both female and male fertility in different species and their specific roles.

Monica C. Vella et al. (2005) WormBook "C. elegans microRNAs."

Monica C. Vella, Frank J. Slack

[WormBook, 2005]

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7 . Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3'' untranslated region (UTR) to repress LIN-14 , while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3'' UTR to down-regulate LIN-41 . Two other miRNAs, lsy-6 and mir-273 , control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

Gregory SG et al. "Contig assembly by fingerprinting."

Coulson AR, Soderlund CA, Gregory SG

A previous chapter in this series (1) described, primarily, the physical mapping of the 100 Mb Caenorhabditis elegans genome by fingerprinting of cosmid clones, and the linking of the contigs thus derived by YAC hybridization. At that time, the primary function of the map was to enhance the molecular genetics of the organism by facilitating the cloning of known genes, and to serve as an archive for genomic information. However, a clonal physical map - even with good alignment to the genetic map - carries only a tiny proportion of the information present in the genome. Consequently, the current objective of the C. elegans genome project (2) is to establish of the entire genomic sequence. The bacterial clone map, although incomplete by virtue of the uncloneability of regions of the genome in cosmid vectors (a factor which we shall discuss later in this chapter), has proved a sound basis for the systematic sequence analysis. The sevenfold cosmid coverage has a resolution sufficient to enable the selection of a subset of cosmids for sequencing such that, on average, each clone contributes 30 kb of unique sequence to the whole. Sequencing projects based on bacterial clone maps (3-5) of a number of other genomes of a range of sizes are also well advanced, in particular Saccharomyces cerevisiae (15 Mb; complete), Schizosaccharomyces pombe (15Mb), and Drosohpila melanogaster (150 Mb). Although it has recently been demonstrated that small bacterial genomes can be sequenced by direct shotgun sequence analysis of the entire genome with no prior mapping (6), the ability to interrelate and map clone sets, whether derived by random selection of in a directed manner, is still the most convenient route to the sequence analysis of larger genomes.

Anderson P (1995) Methods Cell Biol "Mutagenesis."

Anderson P

[Methods Cell Biol, 1995]

Choosing the right mutagen means selecting the right combination of mutagen efficiency and mutagen specificity. For mutagen efficiency, nothing beats EMS. It is extremely potent, it is easy to use, and its mutational specificity is well documented. If mutations other than G/C-->A/T transitions are desired, mutagens other than EMS must be used. Based on initial observations, ENU appears to be as efficient as EMS. Work with other organisms predicts that ENU will yield a wider variety of transitions and transversions than EMS. If this proves to be true, ENU will become an important mutagen for routine genetic analysis. For investigators wanting large multigene deletions, gamma irradiation, UV irradiation, formaldehyde, and DEO are the mutagens of choice. Gamma irradiation yields the highest frequency of events by far, but may also yield more complex rearrangements. Based on limited information, UV irradiation, formaldehyde, and DEO appear to be effective deletion mutagens. Of the three, UV appears to be the most efficient. For investigators wanting small intragenic deletions, TMP appears most effective. TMP is not very potent, but a large proportion of TMP-induced unc-22 mutations are small deletions. Hopefully this will be true of all genes. For investigators wanting other types of genome rearrangements (e.g., translocations, crossover suppressors), gamma irradiation (or possibly X irradiation) is effective. For transposon insertions, mut-2 (especially strain TR679) provides the highest possible frequency of events. Because mut-2 activates several families of transposons, it yields insertions in genes that are poor targets for Tc1. Manipulating a strain with such high frequencies of spontaneous mutations, however, can be problematical (see above). For Tc1-specific events, mut-6 (strain RW7097) is the best choice. It provides frequencies comparable to those of Bergerac, but its Tc1 copy number is much lower. A reasonable strategy for spontaneous mutagenesis is to use TR679 only if mutants are not obtained in strains with lower levels of activity (e.g., MT3126 or RW7097).