Chung Man Chan et al. (2008) Development & Evolution Meeting "Pheromone Perception - Molecularly Hardwired and Modulated by Paracrine Factors"

Chung Man Chan, King L. Chow, Kei Chu Fan

[Development & Evolution Meeting, 2008]

Comparison of C. remanei and C. elegans sexual behavior demonstrates that only females of the dioecious species can produce a sex attractant to lure males to its vicinity. However, males from both species respond to this remanei sex pheromone. In this study, we focus on the requirement of the cellular and molecular components for sex pheromone perception in Caenorhabditis males; and to examine whether the difference in pheromone response among males and females/hermaphrodites is due to the sexual identity of the cellular circuitry. Through genetic and physical characterization, two different sensory neurons (AWA, CEM) and an interneuron (AIZ) were shown to be required for this perception. A signaling pathway dependant on a G-protein regulated kinase activity is crucial for this chemosensory response. The process may act through the gating of channels in specific neurons. The evaluation of the function of these molecules in specific neurons through cell-specific expression and cell-specific knock-down will be presented. In addition, while males and hermaphrodites respond to the same olfactory attractants, they do have different preferences. Manipulating the gene activity of the sex determination components in specific cells of the peripheral and central nervous systems, we have investigated the effect of sexual identity in specific cell types on pheromone induced response. Our results will shed light on the differential roles of chemosensory neurons and the relay components. Moreover, the male-specific responsive behavior was found to be under the influence of an insulin signaling pathway. Mutants of genes acting in this pathway were non-responsive to the sex pheromone. Detailed experiments substantiating the involvement of paracrine signaling in behavioral control of perception paradigm will be discussed. (This study is funded by the Research Grants Council, Hong Kong.)

Chung Man Chan et al. (2006) East Asia C. elegans Meeting "Sex pheromone in dioecious nematode: its production and perception"

Wai Kar So, Chung Man Chan, Jeffrey Chasnov, King L. Chow

[East Asia C. elegans Meeting, 2006]

Two different mating systems are adopted by Caenorhabditis species, that is dioecy and androdioecy. For dioecious species, finding a mating partner is an obligatory process for survival. We have identified a sex-specific attractant produced by females of the dioecious Caenorhabditis remanei for attracting the mating partners. We have further characterized the nature and the constituents of this sex pheromone-like substance, the locale of its production, its perception requirement and related sex pheromones produced by other Caenorhabditis species. These attractant substances are produced by the somatic gonad as confirmed by laser ablation of gonadal progenitor cells in different combination. Their activity could be abolished by mating and can be recovered in the absence of further mating. While perception of these attractants could be observed in males of the androdioecious C. elegans, the perception pathway was delineated by repeated testing of various genetic mutants affecting either specific cell fate or molecular function. Our results show that two different sensory neurons and an interneuron are required. Moreover, a signaling pathway dependent of kinase activity and G-protein subunit are found to be crucial for the pheromone perception. A hypothesis of conservation of the pheromone perception pathway across different species will be discussed. (The research is funded by Research Grants Council, Hong Kong.)

Chung-Man Chan et al. (2007) International Worm Meeting "Perception of sex pheromone in Caenorhabditis: identification of cellular and molecular components in the signal relay."

Mei-Yee Wu, Chung-Man Chan, King L. Chow, Queenie W.K. So

[International Worm Meeting, 2007]

Finding a mating partner in a dioecious species is an obligatory process for species survival. We have used C. remanei and C. elegans as models to show that only females of the dioecious species can produce a volatile and stable sex pheromone to effectively attract males. On the other hand, males from both dioecious and androdioecious species respond to this same pheromone with the C. remanei males displaying a stronger attractive response. In this report, we focus our investigation on three specific issues; where is the sex pheromone produced? How is its production physiologically controlled, and how do males perceive the sex pheromone? Pheromone has been reported to be produced by a variety of tissues. To ascertain if gonadal tissues are required for its production, laser ablation was used to eliminate different combination of the gonadal precursor cells. Our results confirm that the somatic gonad is required for the production of this pheromone. The pheromone production peaks at the young adult stage, and mating with males would eliminate the attractiveness of a female. We showed that mating itself and the presence of sperms in the female body would not eliminate the pheromone attractiveness. The mechanism of mating on the attenuation of attractiveness will be discussed in light of a systematic analysis of the male gonad requirement. While the perception of the pheromone by androdioecious C. elegans males allows us to delineate molecularly and cellularly using genetic mutants with defective cell fate or molecular function. Two different sensory neurons and an interneuron are shown to be required for this perception. A signaling pathway dependant of G-protein coupled kinase activity and G-protein subunit are crucial for this chemosensory response. The process may act through the gating of channels in specific neurons. The evaluation of the function of these molecules in specific neurons will be presented. (This study is funded by the Research Grants Council, Hong Kong.).

Kei-Chu Fan et al. (2008) "Sex pheromone perception in Caenorhabditis is modulated by the insulin-like signaling pathway."

King L. Chow, Kei-Chu Fan, Chung-Man Chan, Queenie W.K. So

[2008]

"Sex pheromone is a volatile chemical released by C. remanei females, used to attract males for mating. The response to sex pheromone is sex- and stage-specific. Only adult males are attracted. This response, however, is not species-specific; males of the other Caenorhabditis species are also attracted by this pheromone. In our study, we use C. elegans, which can be attracted by the sex pheromone and has a rich collection of mutants with defective sensory circuit, as a model organism to investigate what are required for the males to execute their pheromone response. Males of its mutants will be tested in the chemoattraction assay for the Chemoattraction Index. C.I. value ranges from -1 to 1 where a higher value indicates a stronger response. As a result, males of a C. elegans mutants having C.I. value lower than that of wildtype C. elegans males implicate genes required for sex pheromone perception.In C. elegans, the insulin-like signaling pathway coordinates multiple biological functions such as mediating the choice of dauer formation, affecting the rate of aging and searching of food. These activities are elicited by various chemical inputs like the dauer pheromone, signals from the gonad and food. Recent studies also suggested that this pathway affects the mate searching ability in C. elegans males (Lipton et al., 2004). It is possible that cross talks among cells involved in sex pheromone perception exist. Base on this speculation, mutants of gene involved in the daf-2/daf-16 insulin-like signaling pathway were tested in the chemoattraction assay for their response to sex pheromone. Among them, several mutants have their C.I. values dramatically reduced. These genes encode various receptor components and signaling molecules like phosphoinositide 3-kinase (PI3K) and serine/threonine kinase (Akt/PKB) in the daf-2/daf-16 insulin-like signaling pathway. In this report, more detailed experiments substantiating the involvement of insulin-like signaling pathway in sex pheromone perception will be discussed. (This study is funded by the Research Grants Council, Hong Kong.)"

Jeffrey R Chasnov et al. (2005) International Worm Meeting "Evidence of a sex attractant in dioecious Caenorhabditis"

Wai Kar So, Chung Man Chan, Jeffrey R Chasnov, King L Chow

[International Worm Meeting, 2005]

Four species in the Elegans group of the subgenus Caenorhabditis are tested for evidence of a sex attractant. We show that single females from the dioecious Caenorhabditis species C. remanei and C. sp. strain CB5161 placed overnight on an agar plate attract conspecific males from a distance, while single hermaphrodites from the androdioecious species C. elegans and C. briggsae do not. These worm attraction experiments are simplified further by soaking females in buffer overnight to collect a biologically active pheromone solution capable of attracting males from a distance. A serial dilution of this pheromone solution demonstrates that male pheromone detection is concentration dependent, with the pheromone solution becoming less attractive with increasing dilution. Using the fully active pheromone solution, we investigate the species-, sex-, and stage-specificity of pheromone attraction. The female sex pheromone is determined not to be completely species-specific, with males of all four species attracted to both the C. remanei and C. sp. female sex pheromones; however, males of the dioecious species are significantly more attracted than males of the androdioecious species. The pheromone is, however, sex-specific, with only females secreting the pheromone and only males responding. Furthermore, testing of various C. elegans male mutants implicates specific molecular and cellular components in the males required for pheromone detection. Female pheromone production and male response are both shown to be stage-specific, with female secretion and male detection of the pheromone beginning at sexual maturity, as would be expected for a sex attractant used by females to attract males for immediate mating. Virgin adult females continue to secrete pheromone as they age; however, solution obtained from mated females immediately after mating no longer attracts males. Mated females after males have been removed do recover their ability to attract males, with full pheromone potency returning over a couple of days, well before the females are sperm depleted. The androdioecious species C. elegans and C. briggsae and the dioecious species C. remanei and C. sp. strain CB5161 are thought to be descendant from a common dioecious ancestor. Our results suggest natural selection favored the loss of a sex attractant from the androdioecious species, presumably because hermaphrodites that self-fertilize have higher fitness than those that outcross with males.

Dwyer, Donard et al. (2017) International Worm Meeting "Protophenotypes in C. elegans relevant for schizophrenia and major depression."

Aamodt, Eric, Dagenhardt, Julie, Dwyer, Donard

[International Worm Meeting, 2017]

Efforts to deconvolute the complexity of genetic contributions to psychiatric illnesses such as schizophrenia and major depressive disorder (MDD) have led to the concept of endophenotypes - heritable traits that reflect intermediary pathological processes of a complex disorder. We have coined the term 'protophenotypes' to describe endophenotypes that have been conserved across species during evolution. The goal of our studies is to characterize the genetics and pharmacology of protophenotypes in C. elegans related to pre-pulse inhibition and startle defects in schizophrenia, social withdrawal (a prominent feature of both schizophrenia and MDD) and avolition/anhedonia, which is characteristic of various affective disorders in man. To model pre-pulse inhibition we evaluated touch suppression of pharyngeal pumping in various mutant strains and with pharmacological agents. To gain insight into social withdrawal, we studied aggregation in social feeding strains in the absence and presence of drugs. Lastly, we developed a model of avolition/anhedonia or diminished motivation based on defects in insulin/IGF-1 signaling, exposure to DMSO and food deprivation. In this model, we observed "suicidal" worms that failed to seek food or escape, but remained in place until they died, despite preservation of responsiveness to external stimuli. Genetic and pharmacological studies revealed that dopamine affects touch suppression via a primitive counter-circuit that presages the arrangement of dopaminergic pathways in man controlling emotions and logical thought. We found that serotonin regulated social feeding, which was inhibited by 5-HT2 receptor antagonists. This finding was consistent with serotonergic defects in MDD and schizophrenia. Studies of the third protophenotype related to diminished motivation revealed that defects in insulin receptor signaling (DAF-2) and proteins that regulate insulin secretion (UNC-64, but not UNC-31) produced rapid immobility in animals subjected to food deprivation in the presence of 1% DMSO. The immobility state in daf-2(lf) mutants was maintained by elevated serotonergic and muscarinic cholinergic signaling. Immobility was reversed with antidepressants (e.g., amitriptyline, imipramine and amoxapine) and antipsychotics (e.g., clozapine and olanzapine, but not haloperidol) that decrease immobility in the forced swimming test - an established model of depression in man. These findings may help to explain the 2-3-fold increase in MDD in patients with diabetes. Finally, this work may establish mechanisms for how fundamental behaviors conserved through evolution contribute to the emergence of psychiatric illness in man.

Ahmed, Rina et al. (2011) International Worm Meeting "Multi-platform sequencing identifies novel mirtron with an embryonic lethal phenotype in Caenorhabditis elegans."

Gunsalus, Kristin, Ahmed, Rina, Sar, Funda, Dieterich, Christoph, Chang, Zisong, Miska, Erik

[International Worm Meeting, 2011]

MicroRNA genes (miRNAs) are post-transcriptional regulators of mRNA stability and/or translation and are regulated themselves on the level of gene transcription, processing and decay. MiRNAs regulate many biological processes and aberrant miRNA expression has been implicated in several disease states. Mirtrons are a special class of miRNAs since they originate from properly sized introns (~ 70 nt) of protein-coding genes. Precursor miRNAs (pre-miRNAs) of mirtrons are excised by the splicing machinery from the host gene, debranched, and directly processed by Dicer. The expression pattern of a mirtron is consequently similar if not identical to its host gene's expression pattern. Only a few mirtrons have been identified in vertebrates and invertebrates since their discovery in 2007. None of them has been linked to any phenotype so far. We revisited the repertoire of miRNAs in Caenorhabditis elegans with a multi-platform sequencing approach (ABI SOLiD and Illumina GA II) to screen for novel miRNA gene candidates. Both platforms differ in sequencing bias, which is usually expressed in divergent normalized cross-platform read counts for any given miRNA. Consequently, both platforms complement one another in the gene discovery process. With this approach, we were able to extend the known set from four to six mirtrons. The modENCODE consortium (Chung et al., 2011) has independently confirmed our discovery. However, one novel mirtron caught our attention and we started a functional characterization of this candidate gene. At the time of writing, we are certain that a knockout of the host gene has an embryonic lethal phenotype and shows greatly reduced levels of hatching worms. The knockout phenotype can be, at least partially, rescued by a mirtron-expressing transgene. Most surprisingly, this mirtron has been acquired recently and is not present in any of the other available Caenorhabditis genomes. We will give an update of our experimental findings at the International Worm Meeting. References: Chung, W.J. et al. Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans. Genome Res. 21, 286-300 (2011).

Cirillo, L. et al. (2015) International Worm Meeting "Coordinating Cdk1 and Plk1 activation for mitotic entry in early C. elegans embryos and human cells."

Cirillo, L., Van Hove, L., Gotta, M., Martino, L., Schwager, F., Panbianco, C., Santamaria, A., Thomas, Y., Pindard, L.

[International Worm Meeting, 2015]

Entry into mitosis is regulated by several kinases including Cdk1, Aurora A and Plk1. However, how the activity of these kinases is coordinated in space and time is not fully understood. Our laboratories have recently shown that SPAT-1, an activator of Plk1, is phosphorylated by Cdk-1 at several sites in C. elegans. Our in vivo and in vitro data revealed that phosphorylation of the N-terminal sites is crucial to promote the interaction with PLK-1 and its activation. Together, our results suggest the existence of a positive feedback loop that connects CDK-1 and PLK-1 via SPAT-1 and ensures a robust control of cell division timing during embryonic development.The human ortholog of SPAT-1, Bora, regulates several aspects of mitosis (Bruinsma et al. 2014, Chan et al 2008, Macurek et al. 2008, Seki et al. 2008). Although phosphorylation of Bora is not strictly required to activate Plk1 (Seki et al. 2008, our unpublished data) we find that Cdk1 phosphorylation of Bora greatly increases Plk1 activation in vitro. We have mapped the Cdk1 phosphorylation sites of Bora. Based on the results obtained in C. elegans, we have mutated a number of N-terminal sites and found that Bora ability to activate Plk1 is greatly reduced in vitro. We are currently testing the function of these sites in human cells by monitoring these mutants in vivo. Our data will help to shed light on the activation mechanisms of Plk1 by Bora. Since Plk1 is overexpressed in many cancers, understanding the precise mechanisms of activation of this kinase will help the design of anti-cancer drugs.References:Bruinsma W. et al. J Cell Sci. 2014; Chan EH. et al. Chromosoma. 2008; Macurek L. et al. Nature. 2008; Seki A. et al. Science. 2008.

Saleel Raut et al. (2008) C.elegans Neuronal Development Meeting "HLH-3, a C. elegans Achaete/Scute protein, has a role in the axonal pathfinding of hermaphrodite-specific motor neuron (HSN) that is independent of UNC-40."

Saleel Raut, Aixa Alfonso

[C.elegans Neuronal Development Meeting, 2008]

The basic helix loop helix protein, HLH-3, is a member of the achaete/scute family in C. elegans hlh-3 mutants have an egg-laying defective (Egl) phenotype. We have shown (Doonan et al., 2008 submitted) that the mutant phenotype results from abnormal axonal pathfinding in hermaphrodite-specific motor neurons (HSNs) that fail to innervate the vulval muscles. The NETRIN/UNC-6 receptor UNC-40/DCC is necessary for the guidance of the HSN axons (Garriga et al.,1993; Chan et al., 1996; Gitai et al., 2003; Adler et al., 2006). We hypothesized that mutant animals are not uncoordinated (Unc). Currently we are looking at SYG-1, a receptor on HSN''s that binds with the guidepost signal SYG-2 for proper synapse formation (Shen et al., 2004), as a possible target of HLH-3.

KA Hagstrom et al. (1999) International C. elegans Meeting "Characterization of an SMC-like protein in C. elegans"

J Lieb, BJ Meyer, M Albrecht, D Pasqualone, KA Hagstrom

[International C. elegans Meeting, 1999]

Members of the highly conserved SMC (structural maintenance of chromosomes) protein family are involved in several aspects of chromosome dynamics. From yeast to man, SMC proteins are essential for processes such as mitotic chromosome condensation, sister chromosome cohesion, and recombinational DNA repair. We are investigating the C. elegans homologs of this chromosomal protein family, and have found that they play critical roles in dosage compensation, mitosis, and meiosis. The SMC proteins DPY-27 and MIX-1 are part of the dosage compensation protein complex that binds to hermaphrodite X chromosomes and downregulates their transcription. In addition, MIX-1 associates with all mitotic chromosomes in both sexes and is essential for their proper segregation. A third SMC family member, HIM-1, appears to function in meiosis, perhaps for sister chromatid cohesion (see abstract from A. Chan, D. Pasqualone, T. Wu, and B.J. Meyer.) Characterization of the only other C. elegans SMC homolog, SLP-2 (SMC-like protein 2) is presented here. SLP-2 RNA interference produces dead embryos with abnormal DNA bodies, most likely resulting from defective mitotic chromosome segregation. Transgenic worms carrying extra wild type copies of the SLP-2 gene exhibit lethality, with a high incidence of males among the survivors. This Him phenotype implies that misexpression of SLP-2 also disrupts chromosome segregation during meiosis. Preliminary immunolocalization studies indicate that the SLP-2 protein is not specifically localized to the X, but rather is associated with all chromosomes in both the germline and in embryos. To investigate potential associations between SLP-2 and other SMC proteins, co-immunoprecipitation studies were performed. In embryonic extracts, SLP-2 co-immunoprecipitates with MIX-1, but not with DPY-27. Based on these results, we speculate that while DPY-27 is a partner for MIX-1 within the dosage compensation protein complex, SLP-2 is the partner for MIX-1 in a complex that executes mitotic chromosome segregation. We hope that continued study of the composition and function of SMC protein complexes will provide a greater understanding of their influence on chromosome mechanics.