Guo X et al. (1989) International C. elegans Meeting "C elegans basement membrane collagens."

Kramer JM, Guo X

[International C. elegans Meeting, 1989]

Guo X et al. (2010) Biology of the C. elegans Male, Madison, WI "The Mechanism of Maintaining Mating Potency During Aging"

Guo X, Garcia R L

[Biology of the C. elegans Male, Madison, WI, 2010]

Every organism ages, and during aging, the function of the organism deteriorates including behaviors such as locomotion, mating and feeding. Many studies have focused on the molecular signaling, which can extend or shorten lifespan. But little is known about why a specific behavior deteriorates during aging. In our study, we are trying to figure out the mechanism by which C.elegans males mating behavior declines during aging. Through mating potency analyses, we find that wild-type C. elegans virgin males' mating behavior begins to decline at day 3 of adulthood. 80%, 70% and 40% males can mate at day 1, day 2 and day 3, respectively. Sir-2.1 null mutant males cannot mate well from day 2. 75% and 10% sir-2.1 null males can mate at day 1 and day 2, respectively. Since caloric restriction can extend the lifespan of C. elegans, we hypothesize that starvation can also prolong the mating potency of males. Indeed, when we starve L4 wild-type males for 20hrs and then re-fed them, those males mating potency can be prolonged. Then we ask if the caloric restriction works through SIR-2.1 to prolong mating potency. To our surprise, caloric restriction during L4 can actually rescue the sir-2.1 null males mating potency at day 2. Cycloheximide – a translational inhibitor can also extend the lifespan of C. elegans, so we check if cycloheximide can affect mating potency. It can prolong the mating potency of wild type, but not sir-2.1 null mutant males. Because the neuromuscular mating circuit of C. elegans males is mainly regulated by ACh signaling. We check the sensitivity of acetylcholine signaling during aging by exposing males to ACh agonist such as levamisole and arecoline, we find that males become more sensitive to Ach agonist when they are aged, which is a possible explanation for why aged males cannot mate well. Indeed, males that have decreased Ach signaling due to having one copy of functional ACh receptors genes unc-29, acr-16, acr-18 and gar-3, can mate better at day 3 than wild type. For sir-2.1 null mutant males, we find that they are more sensitive to ARE than age-matched him-5 males, which is consistent with their accelerated decline in mating behavior. Considering that all three factors that can affect mating potency can down-regulate gene expression: 1. SIR-2.1 is a histone deacetylase histone that can promote the chromosome condensation, thus attenuate gene transcription; 2. Starvation can reduce translation; And 3. Cycloheximide can inhibit elongation process during translation. We hypothesize that during aging more Ach receptors are expressed so that males become more sensitive to Ach agonist. And SIR-2.1, starvation, or cycloheximide can down-regulate the Ach receptors expression so that aged wild type males can mate more efficiently.

Guo S X et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Automated On-Chip Femtosecond Laser Axotomy Platform for Nerve Regeneration Studies"

Guo S X, Ben-Yakar A, Bourgeois F, Ellerton E L, Bovik A C, Everett N W

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

We report an automated on-chip approach for performing laser axotomies of fluorescently tagged mechanosensory neurons in living C. elegans. The optimized microfluidic design is capable of reducing the time required to perform axotomies in single worms by at least one order of magnitude as compared to manual axotomies. Specifically, the automated platform uses a T-shaped design that serially transports, positions, and immobilizes worms. Using image processing algorithms, the automated system can identify and ablate targeted axons at an average rate of ~30 seconds per axon. We tested the performance of the system on two mechanosensory neurons (ALM and PLM) of two different mutant strains (SK4005 and CX7123). Direct comparison of axotomies on-chip with those on agar pads using an anesthetic for immobilization, indicates no statistical difference in axon reconnection probabilities. This automated and high-throughput platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration.

Hodgkin, Jonathan et al. (2009) International Worm Meeting "Analysis of breakage and meiotic instability in attached-X-chromosome strains."

Hodgkin, Jonathan, O'Rourke, Delia, Jethwa, Nirmal

[International Worm Meeting, 2009]

The isolation of an attached X chromosome (X^X), consisting of two apparently intact X chromosomes joined at their left telomeres, was previously reported (Hodgkin and Albertson 1995, Genetics 141: 527). In contrast to attached X chromosome constructs in other organisms, and also to X-autosome fusions in C. elegans, this X^X configuration is unstable, frequently breaking down to give an apparently normal X chromosome, or else an X chromosome with a deletion of the left end, or an X chromosome carrying a duplication of the left end. The extent of these deletions and duplications is variable, and they are generated at high frequency (in at least 5% of all X^X oogenic meioses). X^X strains therefore provide a useful source of aberrations in this region of the genome, which contains both a meiotic pairing site and numerator sites for sex determination. It seemed likely that X^X breakage was occurring at meiosis, and might be dependent on the meiotic recombination machinery. To test this hypothesis, X^X was crossed onto a series of meiosis-defective backgrounds (him-1, him-3, him-5, him-6, him-8, him-14, xnd-1, etc.). X^X breakage, as measured by the production of self-progeny males, was significantly or greatly reduced in most of these backgrounds, indicating that the breakage events are partly or wholly dependent on meiotic recombination. Previous FISH studies using YAC probes indicated that the end junction between the two X chromosomes was symmetrical and involved no deletion. We attempted to clone the junction fragment by single primer PCR, in order to determine its exact structure. However, PCR amplification was not successful, perhaps because the junction may include a substantial stretch of telomere repeats or local rearrangements. Further investigation of the structure and properties of the end junction is in progress.

Chun Yi Huang et al. (2005) International Worm Meeting "A new player in the C. elegans programmed cell death pathway"

Yi-Chun Wu, Tsung-Yuan Hsu, Ruey-Hwa Chen, Chun Yi Huang, Pei-ju Lu, Jia-Yun Chen

[International Worm Meeting, 2005]

Human CED-X was identified as a strong interacting protein with the death domain of DAPK (death associated protein kinase) in a yeast two-hybrid screen. To investigate the potential involvement of CED-X in apoptosis, we study the function of its C. elegans homolog ced-x. Inactivation of ced-x by either RNAi or a deletion mutation resulted in decreased numbers of cell corpses during embryogenesis. Ectopic killing caused by transcriptional overexpression of egl-1 or ced-4 but not ced-3 in touch cells was suppressed by the ced-x(0) mutation. Therefore, ced-x genetically acts downstream of or in parallel to egl-1 and ced-4 and upstream of or in parallel to ced-3 in the programmed cell death pathway. In addition, the observation that the ced-x(0) mutation partially suppressed ectopic cell deaths caused by the icd-1(RNAi) mutation indicates that ced-x genetically acts downstream of or in parallel to icd-1. To further explore CED-X function, we generated antibodies against CED-X. The immunostaining signal revealed a filamentous CED-X localization pattern in the cytosol during embryogenesis. We are currently investigating if CED-X is associated with organelles or cytoskeleton. In summary, our results show that ced-x is important for promoting or proper execution of programmed cell death in C. elegans. As human ced-x can functionally substitute C. elegans ced-x in a ced-x mutant, the function of ced-x in apoptosis is likely conserved in evolution.

Lau, Alyssa et al. (2015) International Worm Meeting "Deciphering the mechanism of X-upregulation in C. elegans dosage compensation."

Lau, Alyssa, Csankovszki, Gyorgyi, Zhu, Kevin

[International Worm Meeting, 2015]

In C. elegans dosage compensation is required to balance X-linked gene expression to autosomes. It is hypothesized that an unknown mechanism causes X upregulation in both sexes. This mechanism balances the X to autosomal expression in males, but creates X overexpression in hermaphrodites. Therefore, to restore the balance, hermaphrodites downregulate gene expression two-fold on both X chromosomes. While many studies have focused on X chromosome downregulation, the mechanism of X upregulation is not known.Using 3D FISH microscopy to measure the volume of chromosome territories we found that the X chromosome, but not chromosome 1, territories in males are unexpectedly decondensed. We found that this X chromosome decondensation requires the activity of the histone acetyltransferase, MYS-1 (homologous to human TIP60). Interestingly, MYS-1 acetylates H4K16, the key factor responsible for male-specific X-upregulation in Drosophila melanogaster dosage compensation. This suggests that H4K16ac may be responsible for higher gene expression levels on the male X in both species. Depleting other members of a putative C. elegans TIP60-like complex led to similar X phenotypes as MYS-1 depletion. We hypothesize that a TIP60-like complex decondenses the X chromosome in C. elegans males, and this decondensation contributes to upregulation of gene expression on the chromosome. By analyzing X chromosome volumes in young male embryos we determined that the developmental time window for this male X chromosome decondensation occurs around the 30-to-50-cell stage, surprisingly the same stage as the downregulation process in hermaphrodites. Overall, our data indicates that histone acetylation by the MYST family HAT MYS-1 may play an important role in the highly debated X upregulation mechanism in C. elegans. .

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Albritton, Sarah, Ercan, Sevinc

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

Satoru Uzawa et al. (2007) International Worm Meeting "Visualization of the Spreading of the Dosage Compensation Complex on X by Microscopy."

Satoru Uzawa, Barbara Meyer

[International Worm Meeting, 2007]

For proper development of XX and XO animals, the dosage of X-chromosome-linked genes have to be compensated. In free-living soil nematode C. elegans, the dosage compensation of the X-linked genes is accomplished by reducing the gene expression from the X chromosomes in XX hermaphrodites by half. This process is done by the molecular machinery known as the dosage compensation complex (DCC), which resembles condensin complex. DCC loads specifically onto the X chromosome in early embryos (~20 cell stage) and remains on the X chromosome throughout the life of the hermaphrodite. The DCC is recruited onto the X chromosome through specific DNA sequences on the X chromosomes, rex (recruitment element on X). The X chromosome regions that lack a rex site receive DCC through spreading from other rex sites in cis. The nature and molecular mechanism of the spreading of the DCC on the X chromosomes is unknown. We are trying to visualize this spreading process through various microscopic methods using FISH and IF double staining for high resolution images and live cell imaging for time domain analysis.

Briglin A et al. (1998) East Coast Worm Meeting "The role of PAR-1 in establishing polarity in the early C. elegans embryo."

Briglin A, Kemphues KJ

[East Coast Worm Meeting, 1998]

PAR-1 protein is required for establishing polarity during early embryogenesis in the nematode C.elegans. Mutants in par-1 have an abnormal first cell division and patterning defects. PAR-1 protein is localized asymmetrically, to the posterior periphery of the one cell embryo. The conserved C-terminal domain of PAR-1 was found to interact specifically, both in vivo and in vitro, with NMY-2, a non-muscle myosin type II heavy chain (Guo and Kemphues 1996). RNA interference experiments suggest that removing NMY-2 from the early embryo results in a mislocalization of the PAR-1 protein. At present, the role of the interaction between PAR-1 and NMY-2 in establishing polarity in the early embryo is still unclear. To understand better the role of this interaction in vivo, I will generate mutant PAR-1 proteins that disrupt binding to NMY-2 in vitro and test these proteins for rescue with germline transformation. PAR-1 is predicted to be a serine/threonine protein kinase (Guo and Kemphues 1995). Two par-1 alleles contain mutations in conserved residues of the kinase domain. These mutants suggest that PAR-1 kinase activity is also required to correctly establish polarity. In order to understand the role that PAR-1 plays in the early cell divisions, it will be necessary to identify the substrates for this kinase activity in vivo. To do this, I am conducting a screen for PAR-1 substrates. Guo, S. and K.J. Kemphues. 1995. Cell 81:611-620. Guo, S. and K.J. Kemphues. 1996. Nature 382:455-258.

LaMunyon CW et al. (1995) International C. elegans Meeting "SPERM COMPETITION IN HERMAPHRODITIC CAENORHABDITIS SPECIES"

Ward S, LaMunyon CW

[International C. elegans Meeting, 1995]

As in C. elegans, male sperm take precedence over hermaphrodite sperm after C. briggsae worms mate. However, in C. briggsae the initial outcross progeny are principally hermaphrodites and become male-biased with time. Thus, X-bearing male sperm have a striking fertilization advantage over non-X-bearing male sperm. Results from controlled matings show that the X-bearing sperm neither outnumber nor activate faster than their non-X-bearing counterparts. The advantage (meiotic drive) of the X-bearing sperm is therefore due to a competitive edge over non-X-bearing sperm. Thus, in C. briggsae, male X-bearing sperm outcompete male non-X-bearing sperm which themselves outcompete hermaphrodite sperm. While we do not know the mechanism underlying the competitive effect of the X chromosome, we do have evidence from artificial insemination that the precedence of male sperm over hermaphrodite sperm in C. elegans is independent of both seminal fluid and the mode of sperm activation (male or hermaphrodite). Male sperm precedence in hermaphroditic Caenorhabditis maximizes outcrossing after mating, and the X-bearing sperm advantage in C. briggsae delays the associated cost of making males which are by themselves incapable of reproduction.