Aslam S et al. (1998) European Worm Meeting "A SCREEN FOR GENES REDUNDANT WITH PES-1"

Aslam S, Hope IA, Mounsey A

[European Worm Meeting, 1998]

The pes-1 gene was identified in a promoter trapping screen for C. elegans genes expressed during early embryonic stages. The predicted PES-1 proteins have been found to have homology to the fork head family of transcription factors. This suggests an important role for pes-1 in the regulation of gene expression during early embryogenesis. Inactivation of this gene however does not cause developmental arrest or disrupt embryonic development in any evident way. pes-1 therefore appears to be non-essential or redundant in function. This could be due to another gene or other genes partially or fully substituting for the deletion of pes-1 as part of a redundant pathway. An initial genetic screen previously undertaken to identify genes redundant with pes-1 has now been expanded. This involved EMS mutagenesis of a strain of C. elegans with a pes-1 chromosomal deletion and an extrachromosomal array carrying a copy of an intact pes-1 gene and a rol-6 (su1006) marker gene. Mutants were identified which appeared to be dependant on the extrachromosomal pes-1 for survival. 28 mutants have now been generated which appear to cause arrest of embryonic development in a pes-1 deletion background because they no longer segregate non-roller progeny. Backcrossing of these mutants to a strain containing a chromosomal pes-1 deletion reveals that 16 are behaving as typical autosomal recessive mutations. The phenotype of these mutant strains is now being investigated and the mutations are being mapped using STS-mapping procedures.

Aslam S et al. (2000) European Worm Meeting "The forkhead transcription factors of C. elegans"

Aslam S, Hope IA, Mounsey A, Bauer PK

[European Worm Meeting, 2000]

Forkhead transcription factors are a large family of sequence-specific DNA binding proteins.These proteins have been identified in many species across diverse phyla, and have been implicated as regulators of a number of important developmental processes. The completion of the C. elegans genome sequencing project has allowed the identification of all the forkhead genes within the worm, and permits the simultaneous study of this family of proteins. There are 15 forkhead genes in C. elegans, of which five have been previously characterised, i.e. daf-16, lin-31, pes-1, pha-4, and unc-130. We have concentrated our studies on the other ten, that is : B0286.5, C25A1.2, C29F7.4, C29F7.5, C39H7.3, F26A1.2, F26B1.7, F40H3.4, K03C7.2, and T14G12.4. We have characterised these genes by generating lacZ and/or GFP expression patterns and RNA interference. (The results of our studies of T14G12.4 are discussed in the abstract of S. Aslam et al.) Unsurprisingly, the expression studies reveal that forkhead genes are expressed at various stages of development, and in a variety of tissues, including pharynx, neurons, and the developing gonad. Consistent with reverse genetic analyses of other gene families, only two of the forkhead genes (F26B1.7 and B0286.5) gave an obvious RNAi phenotype. F26B1.7 is expressed in the anterior of the embryo from an early stage (approximately 200 cells) until after elongation, and has a highly pleiotropic and variably penetrant RNAi phenotype. This phenotype includes larval lethality, aberrant oocyte and somatic gonad development, apparent absence of a uterus, and adult morbidity. B0286.5 is expressed in the developing somatic gonad (including the spermathecae), and consistent with this many worms show a sterile RNAi phenotype. In occasional animals, one arm of the gonad will produce fertilised embryos but few, or none, are laid and the uterus becomes bloated with eggs.

S Aslam et al. (2001) International C. elegans Meeting "Functional redundancy between pes-1 and fkh-2(T14G12.4)."

P Bauer, S Aslam, I A Hope, A Mounsey

[International C. elegans Meeting, 2001]

pes-1 is expressed in several lineages during early embryogenesis and encodes a fork head transcription factor. This suggests that pes-1 has an important role in the regulation of gene expression during early embryonic development. Inactivation of pes-1 however, either by RNAi or by a Tc1-mediated deletion, does not give rise to any obvious phenotype. We now know this is because of genetic redundancy and pes-1 functions redundantly with another C. elegans fork head gene, fkh-2 (T14G12.4). fkh-2 and pes-1 have strikingly similar expression patterns. fkh-2 : :gfp expression appears to start one round of cell divisions later than pes-1 :: gfp . The D lineage component for both of these expression patterns completely overlaps whereas partial overlap is seen in the AB lineage component of both of these expression patterns. Interestingly, double antibody staining suggests a reciprocal level of expression between fkh-2 and pes-1 within the AB lineage . This similar expression pattern suggested to us that fkh-2 function could overlap with that of pes-1 even though these fork head genes do not share particular sequence similarity. Inactivation of fkh-2 alone by RNAi, results in a subtle phenotype of restricted L1 movement. Disruption of both fkh-2 and pes-1 function, however, results in a penetrant lethal phenotype. 12% of progeny arrest during variable late stages of embryogenesis and a further 80% of progeny arrest at the first larval stage. Preliminary examination of the arrested larvae reveals anterior pharyngeal defects. A screen is being undertaken for a Tc3-mediated deletion mutant of fkh-2 . The double mutant phenotype for pes-1 and fkh-2 will then be studied in more detail.

S Aslam et al. (1999) International C. elegans Meeting "Identification of fork head transcription factors expressed during C. elegans embryogenesis."

P Bauer, A Mounsey, S Aslam, IA Hope, C Royall

[International C. elegans Meeting, 1999]

The pes-1 gene was identified in a promoter trapping screen for C. elegans genes expressed in specific cell lineages during early embryogenesis. The predicted PES-1 proteins have homology to the fork head family of transcription factors which includes members from vertebrates, Drosophila and yeast. Most amino acid identity is in a 60 amino acid domain which has been shown to have DNA-binding activity. This suggests that the fork head gene family encodes transcription factors involved in the developmental control of other genes. We have sought to identify fork head transcription factors which could function to control events in early C. elegans embryogenesis by identifying those expressed at this stage of development. The C. elegans genome sequencing project has provided the entire complement of fork head genes in the genome. Eleven novel fork head genes have been revealed in addition to the four previously identified. Reporter gene constructs have been made for ten of these eleven genes, five of which show embryonic expression or an embryonic component in their expression pattern. These embryonic expression patterns are now being characterized in terms of the cell lineage. We plan to examine the function of the embryonically expressed fork head genes using RNA interference.

Saikat Mukhopadhyay et al. (2004) East Coast Worm Meeting "Identification of genes regulating chemosensory neuron-specific morphologies"

Anne Lanjuin, Saikat Mukhopadhyay, Piali Sengupta

[East Coast Worm Meeting, 2004]

Amphid chemosensory neurons sense the external environment via their non-motile cilia, which are specialized for the particular neuron type. For example, AWB has characteristic forked cilia, while AWC has fan-like cilia. How are these structures specified and maintained? FKH-2, a forkhead domain transcription factor is expressed widely in the embryo (Molin et al., 2000) but expression is restricted to the AWB, ASK and ASI amphid neurons in larval stages and adults. In fkh-2 mutants, the ciliary structures of the AWB and AWC neurons but not other neuron types are affected. Dendritic development of AWB and AWC neurons is also affected in fkh-2 mutants, and this effect is manifested as stunting of the dendritic processes as early as the first larval stages. FKH-2 expression in the amphid neurons in early larval stages is regulated by DAF-19, an RFX-type transcription factor regulating general cilia development in all sensory neurons. However, the expression of fkh-2 is partly restored in daf-19 dauers and adults. It is possible that daf-19 could be regulating fkh-2 to determine cell-specific cilia morphology in AWB in addition to its general function in cilia formation. FKH-2 also acts in parallel with the OTX family homeodomain protein CEH-37 to specify the subtype identities of the AWB and ADF sensory neurons. Currently we are screening for additional mutants exhibiting altered cilia morphology specifically in the AWB neurons in order to characterize cell-specific factors necessary for their specialized cilia structure. We are also investigating the role of neuronal activity in the maintenance of ciliary structure. References: Molin L., Mounsey. A., Aslam, S., Bauer, P., Young, J., James, M., Sharma-Oates, A., Hope, I. (2000) Development 127, 4825-4835.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Mounsey A et al. (2000) European Worm Meeting "Redundancy between pes-1 and T14G12.4, a C. elegans homologue of Drosophila slp-2"

Aslam S, Hope IA, Molin LF, Mounsey A, Bauer PK

[European Worm Meeting, 2000]

The C. elegans pes-1 gene was identified in a promoter trapping screen for genes expressed in specific cells in the early embryo and was found to encode a member of the fork head family of transcription factors. These observations suggested that pes-1 functions to control gene expression during early embryogenesis. However, neither a putative null mutation of this gene, generated by a Tc1-mediated deletion, nor inactivation by RNA interference give rise to any apparent phenotype. Thus embryogenesis is not dependent upon pes-1. A novel fork head gene, T14G12.4, shares a redundant function with pes-1. T14G12.4 is the C. elegans orthologue of the Drosophilasegmentation gene slp-2 and has a strikingly similar expression pattern to that of pes-1. This similar expression pattern (determined in our analysis of this gene family, see the abstract of Mounsey et. al.), suggested to us that T14G12.4 function could overlap with that of pes-1, even though these fork head genes do not share particular sequence similarity. Whereas only a subtle phenotype of restricted L1 larval movement is observed for dsRNA-mediated inactivation of T14G12.4 alone, disruption of function for both this gene and pes-1 results in developmental arrest of 80% of progeny at the first larval stage. Homologues of pes-1 and T14G12.4 have been identified in the related species, C. briggsae, and are also redundant (see the abstract of Molin et. al.). Closer analysis of these arrested larvae reveals defects in the anterior pharynx. A possible feeding deficiency, due to these defects, could give rise to the developmental arrest observed. Cells of the AB lineage, in which pes-1 is expressed, do give rise to the anterior pharynx. Furthermore, glp-1 is necessary, both for the correct specification of the anterior pharynx, and for pes-1 expression in the AB lineage. These results provide an example of how expression pattern data can be used to identify genes with overlapping functions. Such information may be crucial for a complete understanding of developmental processes.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.