Domeier ME et al. (1997) International C. elegans Meeting "EXPRESSION OF pha-1 DURING EMBRYOGENESIS"

Domeier ME, Mango SE

[International C. elegans Meeting, 1997]

At about the 200 cell stage of embryogenesis, 27 precursors are born that will ultimately produce the 80 cells of the pharynx. Initial studies using a pha-1::lacZ construct determined that pha-1 was expressed at the 200 cell stage, in pharynx and body wall muscle precursors1. To visualize the pharyngeal precursors in living embryos, we created two pha-1::GFP constructs. The first construct was a translational fusion that contained 3.7 kb of upstream sequences plus the entire pha-1 coding domain fused to GFP. The second was a transcriptional fusion carrying pha-1 5! sequences and a nuclear-localized GFP::lacZ cassette. GFP expression was assayed from stable lines in N2 embryos. Surprisingly, both GFP constructs were expressed in multiple cell types in addition to pharynx and body wall muscle precursors. For example, hypodermis, neurons and intestinal cells all expressed pha-1::GFP. Thus, pha-1 may be expressed more globally than first reported. Furthermore, the pha-1::GFP protein fusion was found predominantly in the cytoplasm. This construct was able to rescue pha-1 mutants, suggesting that endogenous PHA-1 may be a cytoplasmic protein as well. To determine the expression pattern of endogenous PHA-1, we are making anti-PHA-1 antibodies. 1.Granato M., Schnabel H. and Schnabel R. (1994) Development 120, 3005-3017. We thank Heinke and Ralf Schnabel for generously sending us alleles of pha-1 as well as pha-1 expression constructs.

Domeier ME et al. (2000) Science "A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans."

Morse DP, Knight SW, Portereiko MF, Mango SE, Bass BL, Domeier ME

[Science, 2000]

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

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

Quintin S et al. (1998) European Worm Meeting "The C. elegans HNF-3beta homolog pha-4 negatively regulates lin-26 expression and specifies pharynx and rectum fate."

Domeier ME, Horner MA, Labouesse M, Mango SE, Quintin S

[European Worm Meeting, 1998]

In a screen for genes acting upstream of lin-26, which is expressed in all non-neuronal ectodermal cells, we identified six genes required to activate or repress lin-26. We focused on one of them, pha-4, which is required to prevent lin-26 expression in the pharynx where it is normally not expressed. pha-4 was identified by Susan Mango who showed that it is necessary to generate the pharynx and the rectum (foregut and hindgut, respectively). Her lab recently cloned pha-4 and found that it is the same gene as the previously identified Cefkh-1, cloned on the basis of its homology with HNF-3!/Axial/forkhead by the group of Jim McGhee. The two groups showed that the PHA-4 protein is mainly expressed in the pharynx and in the rectum. We characterized the pha-4 mutant phenotype. pha-4 mutants, which lack both pharynx and rectum, have an excess of LIN-26 positive cells in their head. Using genetic methods we could show that these cells have a pharyngeal origin, suggesting that pharynx cells are tranformed into ectodermal-like cells. pha-4 mutants also ectopically express a lin-26::gfp transgene, raising the hypothesis that PHA-4 might directly bind to the lin-26 promoter to repress its expression. We reconstituted the embryonic lineage of four pha-4 mutant embryos using a time lapse video microscope and observed a defect starting 7 divisions after AB's birth: pharynx precursors fail to ingress within the embryo and instead divide on or close to the surface, thereby preventing formation of the pharynx primordium. Interestingly, ectopic expression of the LIN-26 protein at this moment can prevent pharynx formation. Thus pha-4 is necessary for the proper identity of the pharyngeal precursors and for their migration. An hypothesis is that pha-4 could maintain lin-26 expression off during pharynx development. These data show that pha-4 is necessary for pharynx development, but is it sufficient? We ectopically expressed the PHA-4 protein under a heat-shock promoter. We found that early ectopic expression of PHA-4 provokes a strong increase in the number of pharyngeal muscles and marginal cells as well as rectal valve cells. By contrast ectopic PHA-4 decreases the number of body wall muscle cells and epidermal cells. Thus PHA-4 is sufficient to implement both pharynx and rectum differentiation program. We suggest that pha-4 confers organ identity to the pharynx and the rectum and that HNF-3beta might act similarly.