Doyle TG et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON THE CHARACTERIZATION OF SEL-8"

Doyle TG, Greenwald IS

[International C. elegans Meeting, 1997]

Lin-12/Notch proteins mediate cell-cell interactions that specify cell fate. The isolation of extragenic suppressors and enhancers of lin-12 offers an approach to identifying factors that influence lin-12-mediated signalling. A single sel-8 allele was identified as a suppressor of lin-12(d) mutations (F. Tax, J. Thomas, E. Ferguson and H.R. Horvitz, 1997). We are performing a detailed genetic and molecular analysis of sel-8. The genetic analysis will involve characterizing the nature of the existing sel-8 allele, finding new alleles, and observing interactions between sel-8 and different alleles of lin-12 as well as other mutants. sel-8 maps to LG III; and we are presently using three-factor mapping to narrow down its location so it may cloned by complementation rescue. We hope to have progress on the cloning and the genetics of sel-8 by the meeting.

TG Doyle et al. (1999) International C. elegans Meeting "Genetic and molecular characterization of sel-8, a suppressor of lin-12 gain-of-function mutants"

I Greenwald, TG Doyle

[International C. elegans Meeting, 1999]

We have been studying sel-8 , a suppressor of lin-12(d) mutations that also shows genetic interactions with glp-1 (1). The one existing sel-8 allele, sa54 , suppresses lin-12(d) in an allele-nonspecific and dose-dependent way. In an otherwise wild-type background, sel-8(sa54) causes an incompletely penetrant early embryonic lethality at 15C. The lethality can be partially rescued by zygotic sel-8 activity. We used three-factor mapping to locate sel-8 in an interval of LG III between ben-1 and unc-93 that contains seven predicted ORFs. We sequenced these ORFs from a sel-8(sa54) mutant, and found a nonsense mutation in one ORF, C32A3.1. Over 95% of the other ORFs in this region have been sequenced and no other mutations have been found. The nonsense mutation cosegregates with the sel-8 suppression and cold-sensitive embryonic lethality phenotypes. In addition, RNAi using dsRNA from C32A3.1 produces embryonic lethality in 95% of F1s in an N2 background. Surviving F1s show defects characteristic of loss of lin-12 activity as well as germline proliferation defects reminiscent of loss of glp-1 activity. Based on the RNAi phenotype and on the presence a single nonsense mutation in sel-8(sa54) animals, we conclude that C32A3.1 is sel-8 . C32A3.1 is predicted to encode a glutamine-rich 468 amino acid protein with no similarity to any proteins in the database. It does not appear to contain a signal sequence or transmembrane domains. The nonsense mutation occurs relatively late in the sequence and this observation in combination with the RNAi data suggests that sel-8(sa54) is probably not a null allele. We are in the progress of analyzing sel-8 further genetically and molecularly, and we will report on our progress at the meeting. 1. Tax, F.E., J.H. Thomas, E.L. Ferguson, and H.R. Horvitz, Genetics 147:1675-1695 (1997).

Christina A Doyle et al. (2001) International C. elegans Meeting "Characterization of Human Early Onset Torsion Dystonia-Related Genes in C. elegans"

Robyn Lints, Scott W Emmons, Laurie Ozelius, Christina A Doyle

[International C. elegans Meeting, 2001]

Human Early Onset Torsion Dystonia is an autosomal dominant movement disorder characterized by sustained muscle contractions that cause involuntary twisting, repetitive movements, or abnormal postures. The disorder is caused by a three-base pair deletion in the coding region of the TOR1A gene that results in the loss of one of a pair of glutamic acid residues near the carboxy terminus of the protein. TorsinA shares functional domains with the AAA/HSP/Clp ATPase protein superfamily and is expressed in a variety of tissues and cell types, including neurons of the brain. However, neither the function of TorsinA, nor the mechanism by which deletions in TorsinA result in the disease state, is known. We are using C. elegans as a model for exploring the function of torsin. The C. elegans genome contains three genes predicted to encode tor sin-related proteins (TOR). One of these genes, F44G4.1, has been characterized by Basham et. al. (2000 West Coast Meeting abstract #55) and corresponds to ooc-5 . ooc-5 was identified in a screen for maternal-effect lethal mutants with altered embryonic cleavage patterns. ooc-5 mutants produce embryos of reduced size that fail to undergo P1 nuclear rotation. It is speculated that OOC-5 functions in P1 to effect the asymmetric localization of other PAR proteins. We are currently investigating the expression pattern and function of the two other torsin genes, tor-1 (Y37A1B.12) and tor-2 (Y37A1B.13). These genes lie together on linkage group IV (+12.3 map units), separated by only 400 bps. This suggests the possibility that they may either constitute an operon or may be coordinately regulated. Interestingly, two of the human torsin genes, TOR1A and TOR1B , have a similar organization. Through the use of GFP reporters we find that, as in humans, C. elegans tor-1 and tor-2 are expressed in a variety of cell types, including neurons. tor-1 expression is confined to neurons and muscles associated with feeding, defecation, and egg laying, namely M1, AVL, DVB, the anal depressor muscles, excretory canal and vulval muscles. tor-2 is expressed in a subset of these cells. This represents a further similarity to humans, as TOR1A and TOR1B also appear to have overlapping expression patterns. To investigate the function of tor-1 and tor-2 we are currently performing dsRNAi. In addition, we are constructing potential dominant negative versions of these genes based on the human dominant alleles to determine whether or not they can induce a mutant phenotype.

Doyle, James Julian et al. (2017) International Worm Meeting "C. elegans model of spinal muscular atrophy."

Vrancx, Celine, Doyle, James Julian, Maios, Claudia, Parker, Alex, Patten, Kessen

[International Worm Meeting, 2017]

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder which is the leading genetic cause of infant death. SMA is characterized by a progressive degeneration of lower motor neurons resulting in muscle atrophy, and ultimately death. At present, there are no clinically approved treatments for SMA, therefore, a better understanding of the molecular mechanisms involved in the regulation of the degenerative process is essential for developing new therapeutic treatments. Here, we investigate the effects of a point mutation in the C. elegans smn-1 gene, orthologous to the human disease-causing SMN1 (Survival Motor Neuron 1) and SMN2 genes, which models the same mutation that leads to SMA in humans. We observe that smn-1 mutant animals recapitulate key aspects of the human disease, including accelerated paralysis and death. Furthermore, we show that interference of ER stress, which is elevated during the course of the disease, exerts a protective effect on mutant animals. Accordingly, our results indicate that our novel SMA model is a relevant tool to study this disease, and can be useful to discover new therapeutic treatments.

Doyle, James-Julian et al. (2015) International Worm Meeting "C. elegans model of PGRN-deficient FTLD."

Parker, Alex, Bennett, Hugh, Doyle, James-Julian, Bateman, Andrew

[International Worm Meeting, 2015]

Frontotemporal lobar degeneration (FTLD) is a devastating neurodegenerative disease, characterized by the progressive atrophy of the frontal and temporal lobes of the brain. Advances in genetic testing have identified many of its underlying genetic causes. Among the genes identified are mutations in the GRN gene encoding for Progranulin (PGRN). To date over 70 different mutations have been identified, most resulting in a haploinsufficiency of the protein. Recent studies have shown that PGRN has neuroprotective properties, protecting against mutant TDP-43 and polyglutamine toxicity, and plays a significant role in cell survival and stress resistance. Nonetheless, its normal function in the brain, as well as its pathogenic mechanism, remain poorly understood. In order to understand the mechanism of PGRN-deficient FTLD, our lab has turned to the C. elegans nematode as a model. Given that C. elegans have an orthologue of the human GRN gene, pgrn-1, we used the pgrn-1(tm985) deletion mutant to model the loss-of-function. We began by characterizing the C. elegans pgrn-1(tm985) phenotype. This revealed that the worms present a motility defect when assessed in liquid culture. In order to better understand the action of PGRN-1 in the worms, we used RNAi knockdown technology to supress gene expression in the worms' muscle, intestinal, and neuronal cells. Furthermore, we have expressed GFP in the mutant worms' GABAergic motor neurons to assess neuronal health. Finally, we have taken advantage of the worm as a genetic tool to study the interactions between pgrn-1 and the worm orthologues of other known FTLD-causative genes. Based on our findings thus far, we will utilize the worm motility defect to our advantage by performing a high-throughput drug screen to identify potential therapeutics. Together, our findings will increase current understandings on the cause of PGRN-deficient FTLD.

Doyle, James Julian et al. (2017) International Worm Meeting "C. elegans chemical genetic screen to counter Progranulin deficiency."

Parker, Alex, Maios, Claudia, Vrancx, Celine, Doyle, James Julian, Bateman, Andrew, Bennett, Hugh

[International Worm Meeting, 2017]

Progranulin (PGRN) is a widely-studied neurotrophic factor known for its ability to reverse toxicity induced by amyloid beta , TDP-43, and Huntingtin, and is an attractive therapeutic target for its role as a neuronal survival factor. Mutations in the encoding gene result in either frontotemporal dementia or neuronal ceroid lipofuscinosis. Despite the important role of this protein in the maintenance of neuronal health, there are no approved therapeutics capable of modulating the downstream molecular action of PGRN. Since performing large-scale chemical screens in vivo are costly and unfeasible in rodents, screening in C. elegans is an attractive approach to address this issue. Characterization of pgrn-1 mutant worms indicated they displayed an age-dependent paralysis, an effect which can be accelerated when they are placed in liquid culture allowing for large scale, rapid screening. We screened ~4000 commercially-available small molecules in liquid culture for their ability to rescue pgrn-1(-)-induced motility defects, and validated them by their ability to also rescue age-dependent paralysis. In total, we have identified 12 compounds as hits capable of specifically restoring pgrn-1 deficiency, and we will further assess the preclinical efficiency of these compounds in zebrafish and cell culture models. This screen represents the first step in a collaborative effort to translate compounds from the bench to the clinics.

Deng, L. et al. (2017) International Worm Meeting "The role of cross-inhibition in C. elegans locomotion."

Haspel, G., Deng, L., Doyle, C., Marfil, V.

[International Worm Meeting, 2017]

To achieve translocation, many locomotor systems produce alternation of antagonist muscle, including axial bending and limb alternation. Cross-inhibition is believed to be key to antagonistic alternation so that when a muscle is activated by the neural circuit, cross-inhibitory elements inhibit a counteracting muscle. Nineteen GABAergic motoneurons in C. elegans have been suggested to provide cross inhibition, based on their morphology and inhibitory effect on muscles. They each receive synaptic input from excitatory motoneurons and provide inhibition to its opposing motoneurons and muscles. However, the activity of D motoneurons during locomotion has not been reported. The locomotion behavior of mutants that are deficient in GABA transmission has been described and their phenotype was generally termed Shrinker. While their forward locomotion was described as normal, shrinker mutants contract their anterior section instead of undulating backward in response to noxious stimuli. However, we show that the deficiencies are not related to the direction of locomotion but rather to its speed and that while cross-inhibition is not necessary for slow antagonistic alternation, it plays a crucial role in fast alternation. We use a combination of behavior analysis, optogenetics, and calcium imaging to clarify the role of the inhibitory D motoneurons. GABA transmission mutants moved at lower frequency and slower translocation speed in both forward and backward directions. Moreover, we found that both forward and backward undulation frequencies of wild type animals each fall into slow and fast distributions, and that the frequencies of mutants overlap with the low frequency distribution of wild type. When mutant animals were exposed to noxious stimuli that would have induced fast forward or backward undulation in wild type animals, they contracted their posterior or anterior sections, respectively. Similar changes in locomotion behavior occurred when D motoneurons were acutely inactivated with optogenetics. We recorded the neuronal activity of D motoneurons, as well as the activity of excitatory motoneurons and muscle cells, during restricted locomotion using microfluidic channels that mimic natural shape of natural undulation so that all elements can be correlated in the same framework of undulatory phase. We manipulate undulation frequency by changing the ambient viscosity. We hypothesize that cross inhibition sharpens the coordination of fast antagonistic alternation.

Zhang H et al. (2003) Dev Cell "Global regulation of Hox gene expression in C. elegans by a SAM domain protein."

Lints R, Emmons S, Zhang H, Teng Y, Haber D, Doyle C, Azevedo RBR

[Dev Cell, 2003]

Polycomb group (PcG)-mediated repression of C. elegrans Hox genes has not been demonstrated, and genes homologous to components of one of the PcG complexes (PRC1) have not been identified in the C. elegans genome. We find that a mechanism of general Hox gene repression exists in C. elegans, carried out in part by SOP-2, a protein related to, but not orthologous with, any PcG protein. sop-2 mutations lead to widespread ectopic expression of Hox genes and homeotic transformations. SOP-2 contains a SAM domain, a self-associating protein domain found in other repressors, including a core component of PRC1 and ETS transcription factors. Phylogenetic analysis indicates that this domain is more closely related to those of the ETS family than to those of PcG proteins. The results suggest that global repression of Hox genes has been taken over by a different branch of the SAM domain family during the evolution of nematodes.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.