Liu, Wan-Ju et al. (2011) International Worm Meeting "Identification of transcription factors regulating lin-39 expression."

Liu, Wan-Ju, Eisenmann, David

[International Worm Meeting, 2011]

Our lab studies the development of the vulva in C. elegans. The C. elegans Hox gene, lin-39, which patterns the midbody region of the worm, is required for vulval formation during development. During vulval development, lin-39 expression is regulated by two extracellular signaling pathways, the RTK/Ras signaling pathway and the Wnt signaling pathway. To further understand how LIN-39 regulates cell fate specification and pattern formation we wish to identify the transcription factors that regulate lin-39 expression in the embryo and larva. Previous work by our lab and others has identified several transcription factors and chromatin regulatory proteins that regulate lin-39 expression. To identify other transcription factors necessary for lin-39 expression, the Yeast-1-Hybrid system was used with a C. elegans transcription factor library. The lin-39 promoter regions from C. elegans, C. briggsae and C. remanei were aligned and 31 conserved regions were found (referred to as triple conserved regions (TCRs)). We grouped the 31 TCRs into 12 fragments (RF1-RF12) that were used as 'baits' in yeast One Hybrid (Y1H) screens. From three different library screens (in collaboration with the Walhout lab), we found several transcription factors that might bind to lin-39 promoter regions, and are validating the results with further experiments. First, to determine if the protein regulates lin-39 in worms, we performed RNAi against the transcription factor genes in worms carrying lin-39::GFP constructs to look for changes in GFP expression. Second, to validate the interaction between the transcription factors and the lin-39 promoter regions, we performed gel shift assays. Third, we performed RNAi against the transcription factor genes on wild type worms to observe whether there are any phenotypes in lin-39-dependent processes. The transcription factors we are currently working on are nhr-43, alr-1, ztf-17, lin-26, tbx-9, and bed-3. The results and progress for each gene will be described in more detail. Finally we are also trying to identify factors that bind to a 338 bp fragment of the lin-39 promoter that may be required for initiation of lin-39 expression in the embryo. We identified a GATA factor, ELT-6, which binds to a GATA site in this fragment of lin-39 and positively regulates lin-39 expression in the embryo. An update on these results will also be described.

Kim, Dong-Wan et al. (2011) International Worm Meeting "Physiological roles of ESCRT complexes in Caenorhabditis elegans."

Sung, Hyun, Lee, Sun-Kyung, Ahnn, Joohong, Lee, Sangho, Kim, Dong-Wan, Shin, Donghyuk, Shen, Haihong

[International Worm Meeting, 2011]

ESCRT (Endosomal Sorting Complex Required for Transport) complexes are required for protein degradation, cytokinesis and viral budding. Extensive genetic, biochemical and structural studies on the ESCRT system have been carried out using yeast and mammalian models. However, the functions of ESCRT system at whole organism-level are less well studied. In C. elegans, we performed RNAi experiments to knock-down the gene expression of ESCRT components and profiled their effects on protein degradation and endocytosis of YP170, a yolk protein. Knockdown of ESCRT-I (vps-23 and vps-28) and ESCRT-III (vps-24 and vps-32.1) components interfered with protein degradation. knockdown of ESCRT-II (vps-25 and vps-36) and ESCRT-III (vps-20 and vps-24) components hampered endocytosis. In contrast, the knock-down of vps-37, another ESCRT-I component, showed no defect both in YP170 uptake and degradation. Depletion of at least one component from each complex - ESCRT-0 (vps-27), ESCRT-I (vps-23, vps-28, and vps-37), ESCRT-II (vps-36), ESCRT-III (vps-24) and Vps4 (vps-4) - resulted in abnormal distribution of embryos in uterus of worms, possibly due to abnormal ovulation, fertilization and egg-laying. These results suggest differential physiological roles of ESCRT-0, I, II and III complexes in organism context of C. elegans.

Wan, Xuan et al. (2017) International Worm Meeting "Molecular basis of the nematode volatile sex pheromone perception."

WAN, Xuan, Chow, King L.

[International Worm Meeting, 2017]

Both sexually matured C. remanei virgin females and self-sperm exhausted C. elegans hermaphrodites release a long-range volatile sex pheromone to attract adult males from afar. Previously, we reported that this chemotaxis behavior requires CEM, AWA and AIZ neurons. In AWA, a GPCR SRD-1 has been demonstrated as the receptor of this pheromone. Ectopic expression of srd-1 in AWB in srd-1 male elicited distinct repulsive behaviour towards pheromone; this result indicated the sufficiency of the SRD-1 receptor acting in AWA. From calcium imaging, we further confirmed the receptor was necessary for the excitation of AWA upon pheromone induction. Male C. elegans is the least attracted to pheromone in comparison to C. briggase, C. remanei, and C. brenneri. Endogenous srd-1 expression level is lower than that of its orthologue, cre-srd-1, in male C. remanei. Transformation rescue of the male srd-1 mutant in C. elegans by cre-srd-1 cDNA was shown to be more responsive to pheromone than those rescued by srd-1 cDNA. Based on protein structure analysis and protein sequence alignment, SRD-1 receptors across four nematode species were highly conserved in terms of their sequences and structure, except their C-terminal region (CT). Six amino acid sequences are polymorphic in C. elegans SRD-1 extracellular loop regions (ECL). The ECL polymorphisms substituted versions of SRD-1 cDNA cannot elicit a higher responsiveness to this pheromone. However, CT truncated cre-srd-1 and srd-1 cDNA are unable to rescue srd-1 chemotaxis mutant phenotype, suggesting the CT region to be critical for SRD-1 function. Specifically, after the CT domain was swapped between Cre-SRD-1 and SRD-1, the transgenic line carrying cre-srd-1-CT-swapped-in-srd-1 cDNA is more attracted to this pheromone than the one carrying srd-1-CT-swapped-in-cre-srd-1 cDNA. Since the CT region in GPCR is usually related to the intracellular signal transduction, we envision that both the expression level of gene product and intracellular signal transduction relay component may account for the differential response of different species in sex pheromone perception. (The study is supported by Research Grants Council, Hong Kong.)

Liu, Wan-Ju et al. (2009) International Worm Meeting "Identification of transcription factors regulating lin-39 expression."

Liu, Wan-Ju, Eisenmann, David

[International Worm Meeting, 2009]

The C. elegans Hox gene, lin-39, which patterns the midbody region of the worm, is required for vulval formation during development. During vulval development, lin-39 expression is regulated by two extracellular signaling pathways, the RTK/Ras signaling pathway and the Wnt signaling pathway. To further understand how LIN-39 regulates cell fate specification and pattern formation, especially in vulva formation, we wish to identify the transcription factors that regulate lin-39 expression. Previous work by our lab and others has identified several transcription factors and chromatin regulatory proteins that regulate lin-39 expression. To broadly identify other transcription factors necessary for lin-39 expression, the Yeast-1-Hybrid system was used with a C. elegans transcription factor library. The lin-39 promoter region from C. elegans, C. briggsae and C. remanei was aligned, and 33 conserved regions were found and referred to as triple conserved regions (TCRs). 12 fragments (frf-1~frf-12) containing the TCRs were cloned into LacZ and HIS3 reporter constructs that were integrated into yeast, which were transformed with the transcription factor library to identify transcription factors binding to particular lin-39 promoter regions. We have found three transcription factors that interact with different fragments of the lin-39 genomic region: nhr-43, a nuclear hormone receptor, ztf-17, a zinc finger protein, alr-1, a transcription factor containing Hox domain. We are doing further experiments to validate these interactions in C. elegans and determine a role for the factors. To determine if the protein regulates lin-39 in worms, we are performing RNAi in worms carrying lin-39::GFP constructs. To identify the interaction between transcription factors and the lin-39 promoter regions, we will perform gel shift assays. We will also perform RNAi on wild type worms to observe whether there is any phenotype in lin-39 dependent processes. Currently we found lin-39 expression seems to go down in P5,p, P6,p and P7,p when treated with ztf-17 RNAi.

Wan-Suk Oh et al. (2008) ""HWY-4213, a novel nematocide and fungicide for prevention and eradication of the pinewood nematode, Bursaphelenchus xylophilus""

Yil-Seong Moon, Wan-Suk Oh, Pan-Young Jeong, Young-Ki Paik, Jung-Ho Kim, Jeong-Eui Lee, Hyoe-Jin Joo

[2008]

"The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of pine wilt disease, which has become a major threat to pine forests and a socioeconomic burden in Eastern Asia, North America, and some parts of Europe. No therapeutic drug for eradication of PWN currently exists. In addition, although several preventive chemical agents are currently available (e.g., morantel tartarate, avermectin, emamectin benzoate), each is associated with drawbacks (e.g., poor water solubility, efficacy, and specificity) that limit their use as of trunk-injection agents against PWN. Ideal trunk-injection drugs against PWN would be highly water-soluble and possess both nematocidal and antifungal activity (against blue stain fungi, the food source of PWN). To search for such a multifunctional nematocidal agent, we first established a high-throughput screening method, which yields potential hits within 6 hours. Using this high-throughput method, we screened of a large set of antifungal chemical libraries for agents with antinematodal activity. Among the compounds identified was HWY-4213 (1-n-undecyl-2-(2-fluorphenyl)methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a water-soluble agent that exhibited potent antifungal and nematocidal activity. The potent nematocidal activity of this compound was confirmed with cotton ball assays. Further development of HWY-4213 as a therapeutic and preventive trunk injection agent against PWN is warranted. (Supported by a grant from a Forest Science & Technology Project [No. S110707L0501501 to YKP] through the Korea Forest Service)"

Wan Ju Liu et al. (2007) International Worm Meeting "Wnt pathway function in C. elegans VCN fate specification."

Wan Ju Liu, David. M. Eisenmann

[International Worm Meeting, 2007]

Wnt signal transduction pathways have been highly conserved during evolution and are used during the development of all animals. Wnt signaling functions in a number of developmental processes in C. elegans , controlling cell fate specification, cell migration and cell polarity. C. elegans utilizes two types of Wnt signaling pathways, BAR-1-dependent Wnt signaling pathway and WRM-1/SYS-1-dependent Wnt signaling pathway. My project focuses on the role of the Wnt signaling pathway in ventral cord neuron fate specification. The descendants of the P cells give rise to ventral cord neurons (VCNs) and vulval precursor cells (VPCs). There are five different classes of VCNs that are post-embryonically derived from Pn.a neuroblasts, which control the muscles used for locomotion (VA, VB, VC, VD, and AS). Previous data from our lab suggests that Wnt signaling may regulate ventral cord motor neuron function; a) bar-1 and other Wnt pathway mutant have an Unc phenotype, b) bar-1 and several Wnt genes are expressed in VCNs, and c) expression of in bar-1 the VCNs rescues the Unc phenotype. To address the hypothesis, different reporter genes that are markers for distinct VCN fates will be used to determine the cell fates adopted in Wnt signaling pathway mutants. Our current results suggest that a WRM-1-dependent Wnt signaling pathway may regulate the VA fate. The number of unc-4::gfp (VA marker) expressing cells goes down in cwn-1, cwn-1; cwn-2, cwn-1; egl-20 mutants and wrm-1, pop-1 RNAi animals; while the number goes up in hs::<font face=symbol>D</font>NTbar-1 animals. The number of neurons is the same in these strains by DAPI staining. Other cell fates are going to be tested in the same way to further examine the role of Wnt signaling in VCN cell fate specification.

Wan, Y. et al. (2019) International Worm Meeting "Glucose metabolism among different tissues regulates mating performance and durability."

Garcia, L., Wan, Y.

[International Worm Meeting, 2019]

Glucose metabolism is essential for both neurons and muscles' normal function. However, glucose homeostasis is also tightly maintained in mammals to support all cells' function. We are interested in how glucose metabolism among different tissues affects neuromuscular function. In aging C. elegans males, we found a correlation between the decline of mating potency and the upregulation of mRNA levels of metabolic genes involved in both catabolism and anabolism of glucose, including glycolytic enzyme hexokinase (hxk-1) and cataplerotic enzyme phosphoenolpyruvate carboxykinase (pck-1 and pck-2). hxk-1 has overlapped expression with pck-1 in neurons and muscles, and overlapped expression with pck-2 in intestines and muscles. Using CRISPR-Cas9 and CRE-Lox technology, we generated homozygous and tissue specific knockouts of hxk-1. While homozygous or neuronal+muscular knockout of hxk-1 had no effect on mating potency or performance in virgin males, pharyngeal muscular+intestinal knockout significantly enhanced their mating performance in day 3 aged virgin males. These results suggest intestinal glucose catabolism might contribute to the decline of mating performance in aging C. elegans virgin males. Consistent with this, overexpression of intestinal hxk-1 can accelerate the performance decline to day 1. pck-2 knockout was previously shown to reduce glycogen and lipid content, and also can accelerate the performance decline. We found that knocking out of hxk-1 in pck-2 mutants rescues mating performance at high-, but not low-competitive setting. These results suggest that glucose homeostasis between intestine and other tissues influences mating performance. We are also interested in how the durability of the mating circuit is affected by glucose metabolism. To test this, we assayed how many females one male can impregnate in its lifetime. While hxk-1 or pck-1 single knockout showed no obvious effect on mating durability, hxk-1; pck-1 double knockout lowered the durability by 50%. This suggests the redundant function of hxk-1 and pck-1 in maintaining the durability and highlights the possible function of neuronal and muscular glucose metabolism in maintaining the durability of the mating circuit.

Wan, Xuan et al. (2017) International Worm Meeting "Identification of nematodes volatile sex pheromone and the molecular basis of its perception."

WAN, Xuan, Chow, King L.

[International Worm Meeting, 2017]

Nematodes rely on their sensory modalities to locate mating partner. Volatile sex pheromone attracts potential mating partners from afar. Intriguingly, sexually matured females C. remanei and self-sperm exhausted hermaphrodites C. elegans produce a volatile attractant that is detectable by both con-specific and its relative adult males, qualifying this attractant as a long-range sex pheromone. We previously reported a class D GPCR serpentine receptor (SRD-1), a receptor in AWA neuron, is responsible for detecting this pheromone and several components in the olfactory pathway are involved in this pheromone signal transduction. The GC-MS results of chemo-attractants from female C. remanei verified several volatile chemicals. Two of them exhibited sex-, stage-, and species-specific attractiveness on wild-type males. Noticeably, these compounds could neither attract receptor-defective mutant nor pheromone-perception-defective mutant males. We also visualized AWA neuron excitation by employed a calcium indicator, GCaMP5. The excitation of the AWA neurons was observed only in wild-type males, but not in srd-1 mutants, upon induction by both candidates separately. We concluded that these two chemicals are the active components of this pheromone through the GC-MS analysis, signal transduction pathway mutant strain studies and also the demonstration of ligand-receptor relationships in calcium imaging and behavioural assays. (The study is supported by Research Grants Council, Hong Kong.)

WAN, Xuan et al. (2015) International Worm Meeting "Identification of nematodes volatile sex pheromone and the molecular basis of sex pheromone perception."

Chow, King L., WAN, Xuan

[International Worm Meeting, 2015]

Our lab studies the molecular basis of chemosensory behaviors. Sexually mature C. remanei females produce an attractant detectable by conspecific adult males and its relative C. elegans. The volatile attractant acts in a sex-, stage- and species-specific manner and thus qualifies as a long-range sex pheromone. Analysis on GC-MS data suggested several chemicals and two among them were functionally verified by chemo-attraction assays. A mixture of the two exhibited stronger attractiveness than either one alone, suggesting a synergistic effect behind. On another focus, we search for the corresponding receptors. Previously we reported cell ablation experiments that demonstrated the necessity of CEM, AWA and AIZ neurons in this process. In AWA, SRD-1 was identified as a GPCR responding for sex pheromone perception, corroborated by the localization, single cell microarray data, mutation strain function examine and cell specific cDNA rescue experiments. Ectopically srd-1 expression from AWA to AWB elicits a distinct repulsive behavior towards pheromone in males, demonstrating its sufficiency as a pheromone receptor. Noticeably, the two pheromone candidates above could not attract srd-1 mutant worms, further strengthening the ties between the suggested ligands and receptor. To screen for potential receptors in the CEM, we conduct a cell-specific RNA pull down on transgenic hermaphrodites. Hermaphrodites without CEM neurons to carrying CEM neuron with increasing male-fated mutations show gradually increased attraction towards sex pheromones. We thus hypothesize CEM cells of the right fate must be expressing sets of molecules that confer masculinity. Comparison between transcriptome profiles should reveal key differences between non-functional and functional CEM neurons, and screen out relevant receptors. (The study is supported by Research Grants Council, Hong Kong.).

Wan-Ju Liu et al. (2008) Development & Evolution Meeting "Identification of Transcription Factors Regulating lin-39 Expression and LIN-39 Downstream Targets"

Wan-Ju Liu, David Eisenmann

[Development & Evolution Meeting, 2008]

Our lab studies the development of the vulva in C. elegans. It has been shown that lin-39, a Hox gene patterning the midbody region of the worm, is required for vulval formation during development. During vulval development, lin-39 expression is regulated by two extracellular signaling pathways, the RTK/RAS signaling pathway and the Wnt signaling pathway. To further understand how LIN-39 regulates cell fate specification and pattern formation, especially in vulva formation, we wish to identify the transcription factors that regulate lin-39 expression, as well as LIN-39 downstream targets. Previous work by our lab and others has identified several transcription factors and chromatin regulatory protein that regulate lin-39 expression. To broadly identify other transcription factors necessary for lin-39 expression, the Yeast-1-Hybrid system will be used with a C. elegans transcription factor library. The lin-39 promoter region from C. elegans, C. briggsae and C. remanei was aligned, and 33 conserved regions were found and referred to as triple conserved regions (TCRs). 12 fragments containing the TCRs were cloned into LazZ and HIS3 reporter constructs that will be integrated into yeast, which will be transformed with the transcription factor library to identify transcription factors binding to particular lin-39 promoter regions. Further experiments will be done to validate these interactions in C.elegans and determine a role for the factors in vulva development. To identify LIN-39 targets, Affymetrix microarray analysis was performed using RNA from a lin-39 over-expressing strain and a lin-39/ceh-20 over-expressing strain, and compared with RNA from a wild-type strain. The potential LIN-39 target genes were further validated by qRT-PCR. To determine whether validated genes are really targets of LIN-39, we created transcriptional YFP reporter constructs of several candidate genes by using SOEing (Splicing by Overlap Extension) PCR. Using these reporters we will determine whether the expression pattern of the potential target gene overlaps with the lin-39 expression pattern, and whether expression of the target gene is dependent on LIN-39.