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Background: In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-beta pathway, that controls post-embryonic growth and male tail development., Results: Here we characterize a novel gene affecting body size. lon-8 encodes a secreted product of the hypodermis that is highly conserved in Rhabditid nematodes. lon-8 regulates larval elongation as well as male tail development. In both processes, lon-8 appears to function independently of the Sma/Mab pathway. Rather, lon-8 genetically interacts with dpy-11 and dpy-18, which encode cuticle collagen modifying enzymes., Conclusion: The novel gene lon-8 encodes a secreted product of the hypodermis that controls body size and male ray morphology in C. elegans. lon-8 genetically interacts with enzymes that affect the composition of the cuticle.

Although it is well established that reactive oxygen species (ROS) can function as intracellular messengers, the mechanism of ROS dependent signaling is largely unknown (Rhee et al.,2005). In a recent paper in Nature Cell Biology, Funato et al. (2006) demonstrate that ROS can modulate signaling by the Wnt/beta-catenin pathway. This work provides interesting new insight into cross-talk between redox and Wnt/beta-catenin signaling in normal physiology and cancer.

Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. Here, we show that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the Caenorhabditis elegans Wnt ortholog EGL-20. The retromer functions in EGL-20-producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells.

beta-Catenin is a multifunctional protein that mediates Wnt signaling by binding to members of the T cell factor (TCF) family of transcription factors. Here, we report an evolutionarily conserved interaction of beta-catenin with FOXO transcription factors, which are regulated by insulin and oxidative stress signaling. beta-Catenin binds directly to FOXO and enhances FOXO transcriptional activity in mammalian cells. In Caenorhabditis elegans, loss of the beta-catenin BAR-1 reduces the activity of the FOXO ortholog DAF-16 in dauer formation and life span. Association of beta-catenin with FOXO was enhanced in cells exposed to oxidative stress. Furthermore, BAR-1 was required for the oxidative stress-induced expression of the DAF-16 target gene sod-3 and for resistance to oxidative damage. These results demonstrate a role for beta-catenin in regulating FOXO function that is particularly important under conditions of oxidative stress.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

Wnt glycoproteins are signaling molecules that control a wide range of developmental processes in organisms ranging from the simple metazoan Hydra to vertebrates. Wnt signaling also plays a key role in the development of the nematode C. elegans, and is involved in cell fate specification and determination of cell polarity and cell migration. Surprisingly, the first genetic studies of Wnt signaling in C. elegans revealed major differences with the established (canonical) Wnt signaling pathways of Drosophila and vertebrates. Thus, the Wnt-dependent induction of endoderm in the early embryo and the specification of several asymmetric cell divisions during larval development are mediated by as yet novel Wnt signaling pathways that repress, rather than activate the TCF/LEF-1 transcription factor POP-1. Recently, however, it has been shown that, in addition to these divergent Wnt pathways, C. elegans also has a canonical Wnt pathway that converts POP-1 into an activator and controls the expression of several homeobox genes. Interestingly, these different Wnt pathways use distinct beta-catenins to control POP-1 function: the endoderm induction pathway requires the beta-catenin WRM-1 and parallel input from a mitogen-activated kinase (MAPK) pathway to downregulate POP-1, whereas the canonical Wnt pathway employs the beta-catenin BAR-1 to activate Wnt target gene expression., (Copyright 2002 Wiley Periodicals, Inc.)

Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3 beta, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/beta-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway, pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/beta-catenin signaling in C. elegans.

During Caenorhabditis elegans vulval development, activation of receptor tyrosine kinase/Ras and Notch signaling pathways causes three vulval precursor cells (VPCs) to adopt induced cell fates. A Wnt signaling pathway also acts in cell fate specification by the VPCs, via regulation of the Hox gene lin-39. We show here that either mutation of pry-1 or expression of an activated BAR-1 beta-catenin protein causes an Overinduced phenotype, in which greater than three VPCs adopt induced cell fates. This indicates that pry-1, which encodes a C. elegans axin homolog, acts as a negative regulator of Wnt signaling in the VPCs. Loss of activity of the APC homolog apr-1 increases the penetrance of this Overinduced phenotype, suggesting that APR-1 may play a negative role in Wnt signaling in this process in C. elegans similar to APC proteins in other systems. The Overinduced phenotype is suppressed by reduction of function of the genes pop-1 TCF and lin-39 Hox. Surprisingly, the Overinduced phenotype caused by hyperactivated Wnt signaling is not dependent on signaling through the Ras pathway. These data suggest that hyperactivation of Wnt signaling is sufficient to cause VPCs to adopt induced fates and that a canonical Wnt pathway may play an important role during C. elegans vulval induction.

In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signalling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologues have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homologue POP-1 mediate Wnt signalling by a mechanism that has challenged current views of the Wnt pathway. Here we show that BAR-1 is the only beta-catenin homologue that interacts directly with POP-1. BAR-1 mediates Wnt signalling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. We conclude that a canonical Wnt pathway exists in C. elegans. Furthermore, our analysis shows that the functions of C. elegans beta-catenins in adhesion and in signalling are performed by separate proteins.

Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal-transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. We characterized a Caenorhabditis elegans G(s) gene, gsa-1, which encodes a G(s) alpha-subunit (G alpha(s)) that is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of G alpha(s) expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for G alpha(s) in the regulation of these behaviors. Expression of a constitutively active form of G alpha(s) from an inducible promotor results in hypercontraction of body-wall muscle cells and vacuolization and degeneration of neurons within hours of induction. Neurons that are susceptible to the degeneration induced by activated G alpha(s) are predominantly motoneurons located within the ventral nerve cord. Phenotypic analysis shows that the induced neural degeneration is not the result of programmed cell death but is probably caused by the activation of ion channels. A genetic suppressor of activated G alpha(s) was isolated that identifies a putative downstream target of G(s) signaling.

Acceleration of lymphomagenesis in oncogene-bearing transgenic mice by slow-transforming retroviruses has proven a valuable tool in identifying cooperating oncogenes. We have modified this protocol to search for genes that can collaborate effectively with the transgene in later stages of tumor development. Propagation of tumors induced by Moloney murine leukemia virus (M-MuLV) in E mu-Pim1 or H2-K-myc transgenic mice by transplantation to syngeneic hosts permitted proviral tagging of 'progression' genes. Molecular cloning of common proviral insertion sites that were detected preferentially in transplanted tumors led to the identification of a novel gene, designated Frat1. The initial selection for integrations near Frat1 occurs in primary tumor cells that have already acquired proviruses in other common insertion sites, yielding primary lymphomas that contain only a minor fraction of tumor cells with an activated Frat1 allele. Transplantation of such primary lymphomas allows for a further expansion of tumor cell clones carrying a proviral insertion near Frat1, resulting in detectable Frat1 rearrangements in 17% of the transplanted E mu-Pim1 tumors and 30% of the transplanted H2-K-myc tumors, respectively. We have cloned and sequenced both the mouse Frat1 gene and its human counterpart. The proteins encoded by Frat1 and FRAT1 are highly homologous and their functions are thus far unknown. Tumor cell lines with high expression of Myc and Pim1 acquired an additional selective advantage in vivo upon infection with a Frat1-IRES-lacZ retrovirus, thus underscoring the role of Frat1 in tumor progression, and the ability of Frat1 to collaborate with Pim1 and Myc in lymphomagenesis.

We present an approach to map large numbers of Tc1 transposon insertions in the genome of Caenorhabditis elegans. Strains have been described that contain up to 500 polymorphic Tc1 insertions. From these we have cloned and shotgun sequenced over 2000 Tc1 flanks, resulting in an estimated set of 400 or more distinct Tc1 insertion alleles. Alignment of these sequences revealed a weak Tc1 insertion site consensus sequence that was symmetric around the invariant TA target site and reads CAYATATRTG. The Tc1 flanking sequences were compared with 40 Mbp of a C. elegans genome sequence. We found 151 insertions within the sequenced area, a density of approximately 1 Tc1 insertion in every 265 kb. As the rest of the C. elegans genome sequence is obtained, remaining Tc1 alleles will fall into place. These mapped Tc1 insertions can serve two functions: (i) insertions in or near genes can be used to isolate deletion derivatives that have that gene mutated; and (ii) they represent a dense collection of polymorphic sequence-tagged sites. We demonstrate a strategy to use these Tc1 sequence-tagged sites in fine-mapping mutations.

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) Go from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 caused a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated Go alpha subunit (G alpha o) in transgenic nematodes resulted in lethargic movement, delayed egg laying, and reduced mating efficiency. Induced expression of activated G alpha o in adults was sufficient to cause these phenotypes, indicating that G alpha o mediates behavior through its role in neuronal function and the functioning of specialized muscles.

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.