Kuroyanagi H et al. (2014) Worm "Comprehensive analysis of mutually exclusive alternative splicing in C. elegans."

Takei S, Kuroyanagi H, Suzuki Y

[Worm, 2014]

Mutually exclusive selection of one exon in a cluster of exons is a rare form of alternative pre-mRNA splicing, yet suggests strict regulation. However, the repertoires of regulation mechanisms for the mutually exclusive (ME) splicing in vivo are still unknown. Here, we experimentally explore putative ME exons in C. elegans to demonstrate that 29 ME exon clusters in 27 genes are actually selected in a mutually exclusive manner. Twenty-two of the clusters consist of homologous ME exons. Five clusters have too short intervening introns to be excised between the ME exons. Fidelity of ME splicing relies at least in part on nonsense-mediated mRNA decay for 14 clusters. These results thus characterize all the repertoires of ME splicing in this organism.

Zuckerman BM et al. (1991) International C. elegans Meeting "A MICROBIAL EGG-LAYING INHIBITING FACTOR FOR CAENORHABDITIS ELEGANS."

Zuckerman BM, Willett JW, Dicklow MB

[International C. elegans Meeting, 1991]

A microbial exometabolite (ME) has been isolated which inhibits egg- laying in Caenorhabditis elegans. Nematodes exposed to active concentrations of the factor, and maintained at 22C in axenic culture ( liver extract medium), mature and grow normally. Eggs are fertilized and larvae develop and move within eggs as in untreated nematodes. However, eggs are not laid, though occasionally hatch occurs within the gonad. In shake culture of the microbe, active yellow-pigmented ME appears at 3-4 days. A concentration of 8 parts ME: 1 part liver extract completely inhibits egg-laying. Concentrations of 4 ME: 1 liver extract and 2 ME: 1 liver extract, inhibit egg-laying about 50 and 25%, respectively. Sections from nematodes exposed to extracts of a microbe showing similar properties examined by transmission electron microscopy indicated that the constrictor and dilator muscles associated with vulval function in egg-laying appear normal. ME is thermostable (at 100C for 5 min.), and the active fraction does not pass through 6,000-8,000 MW dialysis tubing. About one-half of the activity is lost when ME is dialyzed through 12,000 14,000 MW membrane tubing, suggesting the presence of at least two active fractions. Trypsinization obliterated activity. Thus far, the double control experiment with trypsin inhibitor has not yielded definitive results. SDS gel electrophoresis of boiled ME, with molecules below 8,000 MW removed by dialysis, revealed several strong protein bands, one or more of which may be ME. ME purification and characterization studies continue.

Sulston JE (2003) Biosci Rep "C. elegans: the cell lineage and beyond."

Sulston JE

[Biosci Rep, 2003]

Thank you so very much for inviting me to be here. It gives me a mingled sense of humility at how much I owe to others, and of joy that the collective work on the worm has been recognized in this way.

Sulston JE (2003) Chembiochem "Caenorhabditis elegans: the cell lineage and beyond (Nobel lecture)."

Sulston JE

[Chembiochem, 2003]

Thank you so very much for inviting me to be here. It gives me a mingled sense of humility at how much I owe to others, and of joy that the collective work on the worm has been recognised in this way.

White JG (2000) Int J Dev Biol "Worm tales."

White JG

[Int J Dev Biol, 2000]

1969 was a landmark year. But for me it was not Neil Armstrong's giant leap or Woodstock heralding the beginning of the end of the sixties that sticks in my mind. It was a visit I made to Cambridge to meet a "bloke who is starting a new project to study some sort of worm", as my head of department at the Medical Research Council's National Institute of Medical Research informed me...

Reuben M et al. (2002) Dev Biol "Germline X chromosomes exhibit contrasting patterns of histone H3 methylation in Caenorhabditis elegans."

Reuben M, Lin R

[Dev Biol, 2002]

In mammals, one of the two somatic X chromosomes in the female is inactivated, thereby equalizing X chromosome-derived transcription in the two sexes, a process known as dosage compensation. In the germline, however, the situation is quite different. Both X chromosomes are transcriptionally active during female oogenesis, whereas the X and Y chromosomes are transcriptionally silent during male spermatogenesis. Previous studies suggest that Caenorhabditis elegans germline X chromosomes might have different transcriptional activity in the two sexes in a manner similar to that in mammals. Using antibodies specific to H3 methylated at either lysine 4 or lysine 9, we show that the pattern of site-specific H3 methylation is different between X chromosomes and autosomes as well as between germline X chromosomes from the two sexes in C. elegans. We show that the pachytene germline X chromosomes in both sexes lack Me(K4)H3 when compared with autosomes, consistent with their being transcriptionally inactive. This transcriptional inactivity of germline X chromosomes is apparently transient in hermaphrodites because both X chromosomes stain brightly for Me(K4)H3 after germ nuclei exit pachytene. The male single X chromosome, on the other hand, remains devoid of Me(K4)H3 staining throughout the germline. Instead, the male germline X chromosome exhibits a high level of Me(K9)H3 that is not detected on any other chromosomes in either sex, consistent with stable silencing of this chromosome. Using mutants defective in the sex determination pathway, we show that X-chromosomal Me(K9)H3 staining is determined by the sexual phenotype, and not karyotype, of the animal. We detect a similar high level of Me(K9)H3 in male mouse XY bodies, suggesting an evolutionarily conserved mechanism for silencing the X chromosome specifically in the male germline.

Goff SP (1999) Cancer Research "Genetic control of programmed cell death in the nematode Caenorhabditis elegans - Introduction."

Goff SP

[Cancer Research, 1999]

It is an honor and a great pleasure to introduce Dr. Robert Horvitz to you as the 1998 recipient of the Alfred Sloan Prize of the General Motors Cancer Research Foundation. Let me begin by telling you a little bit about Bob's

Alter JR et al. (1998) East Coast Worm Meeting "Analysis of Protein Expression in a C. elegans Model of Huntington's Disease"

Hart AC, Faber PW, MacDonald ME, Alter JR

[East Coast Worm Meeting, 1998]

We have developed a C. elegans model of Huntington's Disease (HD) and other neurodegenerative CAG repeat diseases. HD is an autosomal dominant disorder caused by the expansion of an exonic CAG repeat in the human huntingtin gene. The 350 kDa ubiquitously expressed huntingtin protein is known to be essential, yet its function remains elusive. While unaffected individuals have a range of ~20-34 CAG repeats, affected individuals have greater than 35. The mechanism by which the corresponding expanded polyglutamine tract causes neurodegeneration and eventual cell death is unknown. Recent evidence from tissue culture experiments, transgenic mouse models and human patient data indicate that the mutant huntingtin protein forms intracellular aggregates prior to symptom formation or cell death. In addition, perinuclear aggregates of the mutant protein have been associated with neurodegeneration in patient brains (reviewed in Martindale et al. 1998 Nature Genetics 18:150). However, the mechanism by which these aggregates contribute to (or are a byproduct of) neurodegeneration is unknown. We are using the osm-10 promoter to drive expression of an N-terminal fragment of the human huntingtin protein in the ASH, ASI, PHA and PHB sensory neurons of C. elegans (WBG 15(1):40). We have expressed both 2QHD and 150QHD constructs along with OSM-10::GFP to mark the cells which are affected in our transgenic animals. The results described below are exclusively from animals coexpressing the HD and GFP proteins. We find that up to 40% of the ASH neurons of 150QHD containing animals appear to die in 8 day old animals (WBG 15(1):42). We assessed cell death by the ability of the cells to fill with DiD, a fluorescent dye that fills 6 bilateral amphid neurons in the head including ASH. Defects in dye filling are usually caused by defects in the neuron's sensory cilia. ASH neurons that did not fill with dye and did not express OSM-10::GFP were considered dead in this assay. We have used immunohistochemistry to confirm that ASH cell death is age and polyglutamine dependent in this system. We stained 8 day old transgenic animals with antisera which recognize OSM-10 and GFP; failure of an individual ASH neuron to be visualized by both antibodies led us to conclude that the cell was dead. In this experiment, we confirmed that 2QHD does not cause ASH neurons to die, but that 150QHD causes cell death in old but not young animals. To determine whether our C. elegans model is consistent with human patient data and mouse models, we analysed the formation of intracellular protein aggregates in the ASH neurons of transgenic worms. We used the 3715(HP1) polyclonal huntingtin antibody, which recognizes the N-terminal fragment of the protein, to assess the expression level and subcellular localization of the various huntingtin protein fragments. While expression of all HD constructs was detected in a polyglutamine independent manner, we demonstrated a correlation between the level of 150HD accumulation and the percentage of ASH neurons affected in this system. In our transgenic animals, the N-terminal fragment of huntingtin forms cytoplasmic, axonal, dendritic and perinuclear aggregates in a time- and polyglutamine- dependent manner. Currently we are using other huntingtin antibodies, which have recognized intranuclear mutant huntingtin aggregates in human and mouse brains, to determine whether similar aggregates form in C. elegans ASH neurons expressing the mutant protein (Davies et al. 1997 Cell 90:537). The work presented here and other data (Faber et al. 1998 ECWM) suggest that the activity of mutant huntingtin is similar in our model and the human disease.

Faber PW et al. (1997) Worm Breeder's Gazette "A C. elegans Model of Huntington's Disease and Other CAG Repeat Diseases"

Alter JR, Hart AC, Faber PW, MacDonald ME

[Worm Breeder's Gazette, 1997]

There are striking similarities between vertebrate and C. elegans nervous systems, both at the cellular and molecular level lending this organism to the study of human diseases. We have developed a C. elegans model for Huntington!s disease (HD) and other disorders that are caused by the expansion of a CAG repeat, coding for a polyglutamine domain. The repeat is located in exon 1 of the HD gene, and is polymorphic with normal individuals having 10-34 repeats and affected individuals having greater that 36 repeats. This mutation places HD in a (still growing) group of diseases including spinal and bulbar muscular atrophy (SBMA) and a variety of spinal and cerebellar ataxia!s (SCA!s). Individuals with HD suffer progressive neurodegeneration which causes chorea, dementia and, eventually, death. HD is inherited in an autosomal dominant fashion and phenotypic data from three independent mouse knockouts suggest that the expanded CAG/polyglutamine domain results in a gain of function causing neuronal cell death. Huntingtin is a widely expressed 3144 a.a. novel protein of unknown function, with the N-terminal polyglutamine domain (the cloned allele has 23 repeats) followed by a proline rich region, HEAT repeats and apopain cleavage sites. Although huntingtin levels are highest in the brain, the affected neurons do not have the strongest expression levels. The biochemical process by which the expanded CAG/polyglutamine domain causes specific neuronal cell death in HD (and related diseases) is poorly understood. Transgenic mice expressing exon 1 of the human huntingtin gene containing over 100 glutamine codons display a progressive neurologic phenotype and nuclear inclusion bodies, but fail to show neuronal cell death. There is, in fact, no current animal or cellular model for HD that features cell death. Introduction of an N-terminal fragment of the human huntingtin gene containing an expanded CAG repeat causes neurodegeneration in C. elegans. In lieu of a C. elegans homolog, we used the osm-10 promoter to express the first 170 a.a. of normal and mutant (expanded) huntingtin. osm-10 is expressed in the ASH, ASI, PHA and PHB sensory neurons of C. elegans (Hart and Kaplan, in prep.). In our first set of experiments, ASH neuron survival was assayed by staining with DiO, a fluorescent dye that can be taken up by eight classes of neurons (staining is dependent on intact sensory processes). Based on DiO staining, wild-type and transgenic worms containing normal huntingtin had intact ASH neurons. However, in animals containing the mutant huntingtin, the ASH neurons failed to stain up to 40% of the time suggesting degeneration of the sensory processes or cell death. In a second set of experiments, we used osm-10::GFP to assay ASH survival and stained the cells with DiI (similar to DiO but on the rhodamine channel). After looking at five independent arrays, we found that animals transgenic for the mutant huntingtin had up to 39% ASH neurons that both failed to stain with DiI and were not detectable with the GFP fusion protein. These data suggest that the presence of an expanded CAG/polyglutamine domain within a fragment of the human huntingtin gene causes cell death in C. elegans. We are currently working to prove that the neurons are dying, to increase the rate of cell death by improving expression of the huntingtin constructs and toward integrating the arrays to eliminate mosiacism. In addition, we will determine if previously characterized C. elegans cell death pathways are required for the CAG/polyglutamine neurodegeneration/death. Currently, we plan to screen for suppressers of this effect and begin to define the process by which the expanded CAG/polyglutamine domain in HD and other CAG repeat diseases causes cell death.

Faber PW et al. (1998) East Coast Worm Meeting "A C. elegans Model for Huntington's Disease and other CAG Trinucleotide Repeat Diseases."

Faber PW, Hart AC, Alter JR, MacDonald ME

[East Coast Worm Meeting, 1998]

Huntington's Disease (HD) is a dominantly inherited human neurodegenerative disorder associated with progressively worsening chorea, psychiatric disturbances and cognitive impairment due to neuronal cell dysfunction and to cell loss in the basal ganglia and cerebral cortex. HD is caused by elongation of a polyglutamine (polyQ) tract in huntingtin, a novel 350 kDa protein of unknown function, over a critical length of 35 glutamine residues. This mutation places HD in a group of (momentarily) eight diseases including spinal and bulbar muscular atrophy, dentatorubropallidoluysian atrophy and spinocerebellar ataxia types 1, 2, 3, 6 and 7, also caused by expansion of a polyQ tract. Although the pathogenic mechanism underlying these diseases is still unclear, recent data suggests that protein fragments containing the expanded polyQ tract cause these disorders. The similarities between the vertebrate and C. elegans nervous system at the cellular and molecular level suggests that studies of these disease-causing proteins in C. elegans might provide relevant clues regarding the pathogenic mechanism in patients. In lieu of a C. elegans homolog of huntingtin, the osm-10 promoter (WBG 15(1):40) which drives expression in the ASH, ASI, PHA and PHB sensory neurons, was used to express N-terminal human huntingtin fragments (amino acids 1-171) containing the polyQ tract. 4 to 5 independent transgenic lines were analyzed for constructs expressing the huntingtin fragment with either 2, 23, 95 or 150+ glutamine residues (For details of huntingtin expression, see Alter et al., ECWM98). From our first set of experiments we conclude: 1) Based on PCR analysis, all CAG-repeats are stably propagated over large periods of time (> 90 generations). 2) Only 8 (not 3) day old animals expressing the 150+ polyQ tract have dye filling defects. Up to 40% of the ASH neurons fail to stain with DiD [8 classes of neurons, including ASH, backfill with DiD, for which an intact sensory cilium is required]. No defects were observed for polyQ tracts of other sizes. As predicted, introduction of a translational stop codon before the 150+ CAG-repeat prevented this phenotype. 3) Immunohistochemical analysis with an osm-10 antisera demonstrated that the ASH neurons are still present, indicating a polyQ dependent dye filling defect (Dyf) but not ASH cell death. In a second, related set of experiments, huntingtin fragments with polyQ tracts of either 2 or 150+ residues were co-expressed with an osm-10::GFP fusion protein. Expression of this fusion protein in the ASH neuron is detrimental, resulting in a sensitized background. In these lines failure of the ASH to stain with DiD in combination with absence of a detectable GFP signal was interpreted as ASH cell death. No cell death or Dyf defects were caused by 2 glutamines but the 150+ polyQ tract resulted in up to 19% ASH cell death in 8 day old animals, accompanied by an additional 32% of Dyf ASH neurons. Importantly, ASH cell death in these lines was confirmed using the osm-10 antisera. Our results indicate that expressing an N-terminal human huntingtin protein fragment in C. elegans neurons results in a polyQ-dependent, progressive degeneration which can continue to polyQ-dependent cell death in a sensitized background. Currently, we are addressing the role of previously characterized C. elegans genes in polyQ dependent cell death and degeneration. For example, we are testing if apoptosis is involved in these processes by assessing the effects of the 150+ polyQ tract in ced-3 animals. Also, we are analyzing animals that express the huntingtin fragments in additional cell types (panneuronal/ubiquitous). We plan genetic screens for genes involved polyQ dependent degeneration and death to address the underlying neuropathology of HD and related disorders.