Rudel D et al. (2001) Genetics "Conservation of glp-1 regulation and function in nematodes."

Kimble JE, Rudel D

[Genetics, 2001]

The Caenorhabditis elegans (Ce) glp-1 gene encodes a Notch-like receptor. We have cloned glp-1 from C. briggsae (Cb) and C. remanei (Cr), two Caenorhabditis species that have diverged from C. elegans by roughly 20-40 million years. By sequence analysis, we find that the Cb-GLP-1 and Cr-GLP-1 proteins have retained the same motif architecture as Ce-GLP-1, including number of domains. In addition, two regions (CC-linker and regions flanking the ANK repeats) are as highly conserved as regions previously recognized as essential for signaling (e.g., ANK repeats). Phylogenetic analysis of glp-1 sequences suggests a C. briggsae/C. remanei clade with C. elegans as a sister taxon. Using RNAi to test biological functions, we find that Ce-glp-1, Cb-glp-1, and Cr-glp-1 are all required for proliferation of germline stem cells and for specifying blastomere fates in the embryo. In addition, certain biological roles of Cb-glp-1, e.g., in the vulva, have diverged from those of Ce-glp-1 and Cr-glp-1, suggesting a change in either regulation or function of the Cb-glp-1 gene during evolution. Finally, the regulation of glp-1 mRNA, previously analyzed for Ce-glp-1, is conserved in Cb-glp-1, and we identify conserved 3' UTR sequences that may serve as regulatory elements.

Rudel D et al. (1997) International C. elegans Meeting "FUNCTION AND REGULATION OF GLP-1: AN EVOLUTIONARY APPROACH"

Kimble JE, Friedman LC, Durkin E, Rudel D

[International C. elegans Meeting, 1997]

The GLP-1 receptor regulates inductive interactions in the germ line and early embryo of Caenorhabditis elegans. We have begun to investigate the evolutionary conservation of both function and regulation of GLP-1. As a first step, we have isolated a glp-1 homolog from Caenorhabditis briggsae, which we call Cb-GLP-1. Three lines of evidence support the identity of this homolog as GLP-1 rather than LIN-12. First, the protein coding region is more similar to GLP-1 than LIN-12. Second, injection of antisense RNA prepared against this homolog into C. briggsae results in phenotypes similar to those of glp-1 loss-of-function mutations in C. elegans . Both embryonic lethality and sterility are observed among progeny of injected mothers. Dead embryos lack an anterior pharynx and fail to undergo morphogenesis, while sterile progeny possess only a few sperm. Third, antibodies raised against C. briggsae GLP-1 reveal a pattern similar to that of C. elegans GLP-1. Cb-GLP-1 antibodies stain the mitotic region of the germ line, and AB, but not P1 descendants in early embryos. Comparison of the 3'UTRs of glp-1 from C. elegans and C. briggsae reveals conserved sequences that may be cis-acting elements involved in translational regulation.

Rudel, D. et al. (2017) International Worm Meeting "Nickel, a common environmental pollutant, is an animal genotoxin and mutagen."

Campbell, H., Lockley, J., Schaaf, N., Hvastkovs, E., Atkinson, J., Rudel, D.

[International Worm Meeting, 2017]

Natural nickel (Ni) exposure occurs through volcanism and weathering of rocks. However, Ni's anthropogenic exposure has increased and overwhelmed natural exposure. Ni is a component in metal alloys and car exhaust. Ni is also an essential metal in plants and a component of cigarette smoke. Ni is highly allergenic and genotoxic, inducing carcinomas in industrial workers. Despite known Ni-induced DNA damage in prokaryotes, Ni-induced DNA damage has not been demonstrated in animals. Our data from genetic screens using Ni treatments on N2 animals and programmed cell death (PCD) mutant animals indicate Ni induces the apoptosis of germ cells in Caenorhabditis elegans. We have quantified Ni-induced germcell PCDs using an apoptotic gfp expression marker and dyes. Ni-induced PCDs rely upon genome surveillance as removal of p53 reduces these Ni-induced PCDs. In a PCD mutant background, animals treated with Ni acquire mutations as demonstrated through the generation of heritable morphological phenotypes, i.e. Dpy, Unc, Bli, Rol, Muv, and Vul. Previously Ni's mutagenicity may have been masked through apoptosis of affected cells. Two models are proposed for Ni action. Direct-action implies Ni or Ni-generated reactive oxygen species (ROS) damages the DNA in an autonomous fashion. Indirect-action implies that Ni interacts with DNA packaging and repair proteins making DNA more susceptible to endogenous direct-acting mutagens. Genetic screens involving Ni-alone, ethyl methanesulfonte (EMS)-alone, and joint treatments were performed. Ni does not substantially enhance the mutagenicity of the direct-acting mutagen EMS. The joint treatment's effect appears additive in terms of number of mutations generated. Thus Ni putatively acts directly. Using an electrochemical assay, DNA harvested from Ni-treated worms shows damage in comparison to DNA from un-treated controls. Chemical reconstitution experiments using Ni, hydrogen peroxide and genomic DNA suggests Ni generates ROS as a part of its mechanism. We are identifying chemical changes to DNA upon Ni exposure. Preliminary mass spectrometry (MS) analysis of hydrolyzed Ni-damaged DNA in comparison to untreated DNA from the N2 strain shows accentuated molecular species and the generation of new m/z species corresponding to chemically modified DNA bases. Lastly we have compared Ni-induced DNA damage from strains isolated from highly volcanic regions (high Ni) to common laboratory stocks isolated from non-volcanic regions (low Ni) for both C. elegans and P. pacificus. Volcanic strains do not accrue as much damage as non-volcanic upon Ni exposure. This indicates compensatory genomic differences between strains based upon ecology and life history.

Rudel D et al. (2000) Midwest Worm Meeting "Conservation of glp-1 regulation and function in C. briggsae and C. remanei"

Rudel D, Kimble JE

[Midwest Worm Meeting, 2000]

The C. elegans (Ce) glp-1 gene encodes a Notch-like transmembrane receptor that regulates cell fates and patterning of the germline and early embryo. We have begun to analyze glp-1 in related Caenorhabditis species. Specifically, we cloned glp-1 from C. briggsae (Cb) and C. remanei (Cr), analyzed their phenotypes by RNA mediated interference and examined the glp-1 gene products in C. briggsae germ lines and embryos. Comparison of Ce-GLP-1, Cb-GLP-1, and Cr-GLP-1 proteins shows that both homologs have retained all known functional domains. Amino acid identity ranges from 56%-65% between homologs over the entire length of the GLP-1 protein. Identity is relatively uniform throughout GLP-1. Blocks of identity within the ankyrin repeats and comparison with a solved X-ray structure reveal putative sites of protein-protein interactions. Additionally, amino acid alignments suggest a cryptic ankyrin repeat on either side of the six recognized repeats. The biological roles of glp-1 in C. briggsae and C. remanei are similar to those in C. elegans. Specifically, Cb-glp-1 RNAi and Cr-glp-1 RNAi embryos die with no anterior pharynx, as do Ce-glp-1 mutant embryos. Furthermore, escapers that survive embryogenesis can exhibit a germ line typical of Ce-glp-1 mutants. The regulation of maternal glp1 mRNA appears to be conserved in both C. briggsae and C. remanei. Cb-GLP-1 is restricted to AB descendants in C. briggsae early embryos, while the Cb-glp-1 mRNA is uniform in these embryos. Additionally, the 3UTRs of Cb-glp-1 and Cr-glp-1 harbor a highly conserved (80% identity) region corresponding to the part of the 3UTR that is critical for translational regulation in Ce-glp-1. The coding region, by contrast, is only 55%-65% identical. Lastly, we have cloned portions of lin-12, a second Notch-like receptor, from C. briggsae and C. remanei. In contrast to glp-1, lin-12 is more critical for post-embryonic somatic cell fate decisions. We hypothesize that glp-1 and lin-12 duplicated and adopted their distinct roles prior to the radiation of Caenorhabditis.

Rudel D et al. (2013) PLoS One "Assaying environmental nickel toxicity using model nematodes."

Rudel D, Huffnagle IM, Douglas CD, Ingersoll CG, Besser JM

[PLoS One, 2013]

Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 g Ni/g dry weight of sediment and 50-800 g NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

Rudel D et al. (2002) Evol Dev "Evolution of discrete Notch-like receptors from a distant gene duplication in Caenorhabditis."

Rudel D, Kimble J

[Evol Dev, 2002]

Caenorhabditis elegans possesses two Notch-like receptors, LIN-12 and GLP-1, which have both overlapping and individual biological functions. We examined the lin-12 and glp-1 genes in closely related nematodes to learn about their evolution. Here we report molecular and functional analyses of lin-12 orthologs from two related nematodes, C. briggsae (Cb) and C. remanei (Cr). In addition, we compare these lin-12 findings with similar studies of Cb-glp-1 and Cr-glp-1 orthologs. Cb-LIN-12 and Cr-LIN-12 retain the same number and order of motifs as Ce-LIN-12. Intriguingly, we find that LIN-12 conservation differs from that of GLP-1 in two respects. First, individual motifs are conserved to a different degree for the two receptors. For example, the transmembrane domain is 16-32% identical among LIN-12 orthologs but 65-70% identical among GLP-1 orthologs. Second, certain amino acids are conserved in a receptor-specific manner, a phenomenon most prevalent in the CC-linker. We suggest that LIN-12 and GLP-1 have been molded by selective constraints that are receptor specific and that the two proteins may not be entirely interchangeable. To analyze the functions of the lin-12 orthologs, we used RNA-mediated interference (RNAi). Cb-lin-12(RNAi) or Cr-lin-12(RNAi) progeny are nearly 100% Lag, a larval lethality typical of C. elegans lin-12 glp-1 double mutants, but not the primary defect observed in Ce-lin-12 null mutants or Ce-lin-12(RNAi). Therefore, LIN-12 functions are similar, but not identical, among the Caenorhabditis species. We suggest that ancestral functions may have been divided between LIN-12 and GLP-1 receptors in a process contributing to the retention of both genes after gene duplication (i.e., subfunctionalization).

Rudel, D. et al. (2011) International Worm Meeting "Changes in the Wnt and Netrin pathways that may give rise to new gonad morphologies."

Sommer, R.J., Wyatt, B., Tian, H., Rudel, D.

[International Worm Meeting, 2011]

How new morphologies are generated has been a subject of speculation for centuries by such likes as Charles Darwin and Jean-Baptiste Lamarck. Changes in the shape of organs are an essential component to the development of new body plans, new species, and the capacity to take advantage of new ecological niches. However molecular investigations into the advent of novelties are in their infancy. Here, we show that within Diplogastridae, the hermaphrodite gonad has a novel path of gonad arm extension and makes a unique ventral migration in comparison to many other representative nematodes including C. elegans. This novel ventral migration can be prevented in Pristionchus pacificus by laser ablation or genetic ablation of the vulva. Ablation of all three vulval precursor cells (VPCs) results in all gonad arms failing to migrate ventrally. Wnt signaling plays a putative direct role in instructing the distal tip cells (DTCs) to migrate from the dorsal to the ventral side of the animal as the extending arms come back to the anterior-posterior center of the animal. In P. pacificus hermaphrodite animals where the Wnt pathway members bar-1, mom-2 and cwn-2 are mutated, gonad arms fail to extend ventrally. Ppa-BAR-1 is expressed in the nuclei of DTCs consistent with a signaling role for BAR-1 in these cells. Ppa-MOM-2 is expressed in the developing vulva at the appropriate stage suggesting it is part of a complex Wnt signal from the vulva to the DTCs. Taken together it is likely that cooption of Wnt signaling members has allowed for the advent of new organ morphology. We hypothesize that the Wnt signal is permissive and acts through the Netrin cell guidance system in P. pacificus; this hypothesis is based upon the observation that members of this pathway are necessary for all post-embryonic dorsal-ventral migrations in C. elegans. We are currently investigating the expression of Netrin receptors in the DTCs of P. pacificus hermaphrodites. Intriguingly analysis of the P. pacificus genome and the available expressed sequence tags (ESTs), suggest the composition of the Netrin pathway and its molecular functional organization operate differently in P. pacificus than C. elegans. This system provides in roads towards investigating the acquisition of new signaling functions and altered molecular networks and their roles in generating new organ shapes and body plans.

Rudel D et al. (2005) Dev Biol "Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad."

Rudel D, Riebesell M, Sommer RJ

[Dev Biol, 2005]

The nematode gonad is an exemplary system for the study of organogenesis and fundamental problems in developmental and cellular biology. Nematode gonads vary dramatically across species (Chitwood, B.G., Chitwood, M.B., 1950. Introduction to Nematology." University Park Press, Baltimore; Felix, M.A., Sternberg, P.W., 1996. Symmetry breakage in the development of one-armed gonads in nematodes. Development 122, 2129-2142). As such, comparative developmental biology of gonadogenesis offers the potential to investigate changes in developmental and cellular processes that result in novel organ morphologies and thus may give insights into how these changes can affect animal bauplane. Pristionchus pacificus is a free-living nematode that diverged from the model nematode Caenorhabditis elegans around 200-300 million years ago. The morphology and development of P. pacificus is highly homologous to that of C. elegans. However, many differences in morphology and the underlying molecular signaling networks are easy to identify, making P. pacificus ideal for a comparative approach. Here, we report a detailed description of the P. pacificus hermaphrodite gonad using electron and fluorescent microscopy that will provide a basis for both phenotypic studies of genetic mutations and in vivo molecular studies of cloned genes involved in P. pacificus gonad development. We report that the morphology of the P. pacificus gonad is distinct from that of C. elegans. Among these differences are germ line patterning differences, heterochronic differences, novel gonadal arm-migrations, novel cellular composition of some somatic tissues (e.g., the number of cells that comprise the sheath and different spermathecal regions are different), the absence of a somatic tissue (e.g., the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, we report a set of cell ablations in P. pacificus that indicate extensive cell communication between the somatic gonadal tissues and the germ line. Individual ablation experiments in P. pacificus show significant differences in the effects of individual somatic tissues on germ line patterning in comparison to C. elegans.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.