Chow, King L. et al. (2013) International Worm Meeting "The role of the claudin-like gene nsy-4 in C. elegans sensory ray development."

Chow, King L., Fan, Kei C.

[International Worm Meeting, 2013]

During the ray development, homeobox protein CEH-43 and T-box transcription factor TBX-2 are key transcription factors regulating the cellular assembly of the organ structure. Yet, downstream structural genes that execute the process are largely unknown. Here we present our recent findings on a potential structural gene, nsy-4. Based on our genome-wide RNAi screen, nsy-4 is required for the ray differentiation. We showed with structural cell (Rnst)-specific marker that Rnsts of most missing ray in nsy-4RNAi males are born, indicating that nsy-4 is essential for the assembly but not the lineage of ray cells. Reporter gene analyses revealed that nsy-4 is expressed in both the hypodermis and Rnst. In Rnst, nsy-4 expression is positively regulated by ceh-43 and tbx-2. The nsy-4 reporter signal in both ceh-43(RNAi) and tbx-2 mutants was reduced significantly, suggesting that the expression of nsy-4 is dependent of CEH-43 and TBX-2. These results collectively imply nsy-4 to be a ray structural gene downstream of this transcription factor network. As for its function, though nsy-4 is a worm-specific protein with no obvious homolog outside nematodes, it structurally resembles claudin in the vertebrates. Studies done on claudin-like molecules in C. elegans had shown that they have similar functions as their mammalian counterparts in establishing distinct apical junctions. This type of cell junctions are found in rays and is important for the adhesion between ray cells. Using two translational reporters, NSY-4 was confirmed to be subcellularly localized at the apical junctions in all rays throughout the assembly process. Phenotypic analysis reveals morphological defects in the developing ray, including malformation of papillae and lack of Rnst in the extending rays, both of which are associated with improper apical junction establishment. We will continue to confirm the function of nsy-4 as a claudin in establishing cellular adhesion between ray cells during the ray development. Ultimately the study would shed light on detailed mechanism guiding the assembly of this simple sensory organ. (The study is supported by Research Grants Council, Hong Kong).

FU, Lan et al. (2011) International Worm Meeting "The functional connectivity of male-specific CEM neurons in C. elegans."

Fu, Lan, Chow, King L.

[International Worm Meeting, 2011]

CEMs are the only four male-specific cephalic neurons located in the head region of Caenorhabditis elegans. We have reported that CEMs along with a pair of amphid neurons AWA and interneurons AIZ are required for female sex pheromone perception (Chasnov et al. 2007; Chan, 2007). While EM sections of hermaphrodites revealed the synaptic formation between AWA and AIZ (White et al. 1986), the connectivity of CEM is still unknown. In order to define the functional connectivity of CEMs in the sex pheromone perception pathway, we recorded the physiological behavior of CEMs and AWAs upon different stimulations using GCaMP2. The activation of either AWA or CEM by excitation of an optogenetic reagent, ChR2, could induce the excitation of the other. It implies a bi-directional communication between CEM and AWA, even when the physical connectivity remains unclear. Although CEM could respond to sex pheromone with or without the input from AWA, it was not activated by sex pheromone to its full potential in the wild-type males. When additional activation of CEM by ChR2 was presented together with the sex pheromone, AWA exhibited a stronger influx of Ca2+. Based on these observations and comparison with various controls, we hypothesize three different modes of CEM connectivity in the pheromone perception circuit for integrating input. (1) CEM and AWA are connected by gap junction while two different thresholds exist for AWAs' activation modulated by CEMs' input; (2) Same as (1) together with chemical synapses where two different thresholds for CEM to communicate with AWA electrically or chemically; (3) CEM, AWA and AIZ formed a classic tri-angular circuit for sex pheromone perception. The evaluation of these possibilities will be discussed. The future study of the CEMs' gene expression of components making up the chemical and electrical connections would provide more clues to the pheromone response circuitry in males. (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, 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. (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.)

XIE, Dongying et al. (2015) International Worm Meeting "The role of a claudin superfamily gene nsy-4 in male sensory organ development of C. elegans."

Chow, King L., Xie, Dongying

[International Worm Meeting, 2015]

Epithelial cell junctions are crucial for cell adhesion, cell-cell communication and morphogenesis. In C. elegans, how the components of adherens junctions are assembled in organogenesisis is largely unknown. We have previously reported that a claudin-like protein NSY-4, located at the apical junctions, is required for sensory ray assembly. nsy-4 knockdown in either ray hypodermis or structural cells alone is sufficient to generate ray-missing males. Like its claudin homologs in vertebrates, NSY-4 contains a functional PDZ binding motif and intracellular C-terminus, which are dispensable for correct localization, while mutation of the GLW motif leads to intracellular retention of the protein. These results suggest that NSY-4 functions similarly as its vertebrate counterparts do and may mediate adhesion by direct homophilic binding. NSY-4 also requires HMR-1, the classical cadherin, for correct localization and the two co-localize at the ray tips. hmr-1 and nys-4 double knockdown enhances the ray missing phenotype, implying functional interaction between the two proteins. In addition, homeobox transcription factor ceh-43 knockdown reduces nsy-4 expression level via a transcriptional reporter assay, pointing nsy-4 as a downstream target of ceh-43. Given these results, we propose NSY-4 is an additional building block in C. elegans male sensory ray. Further studies on NSY-4, such as homophilic interaction, mapping of membrane localization domain and interaction with other adherens junctional proteins, should reveal more information on the roles of claudin-like proteins in nematode organ formation.(This study is supported by Research Grants Council, Hong Kong).

Li, Rachel C.K. et al. (2011) International Worm Meeting "Genetic and molecular investigation of PKD-2 and LOV-1 involved in male C. elegans perception of sex pheromone."

Li, Rachel C.K., Chow, King L.

[International Worm Meeting, 2011]

Transient receptor potential polycystin (TRPP) complex PKD1/PKD2 in human are important in kidney's function, and their mutations are associated with polycystic kidney disease. The C. elegans counterparts, PKD-2/LOV-1 localized to the ciliated endings of the male specific neuron CEMs, however, are indispensable for the male response to sex pheromone. In this study, we aim to identify additional PKD-2/LOV-1 polycystin signaling molecules required for sex pheromone perception. atp-2 and cwp genes are potential pkd-2 and lov-1 interacting partners based on their roles in male sensory behavior and their co-expression in CEMs. In our pheromone assays, all cwp mutant males respond normally while males with atp-2 knocked-down are defective, which suggest the distinct requirement of atp-2 in this sex-specific behavior. The interdependence between pkd-2, lov-1 and atp-2 will be ascertained by testing atp-2 RNAi males or its cell specific over-expression in pkd-2 and/or lov-1 mutants by pheromone assay. In addition, a pkd-2 promoter deletion analysis has been performed to identify upstream regulatory components of PKD-2/LOV-1 TRPP channel synthesis. As genes co-expressed in a specific cell type may share a common cis-regulatory motif, identifying the signature motif for CEM-specific expression may help uncover potential pkd-2 interacting partners by a genome-wide search for genes with a CEM-signature sequence. For the cellular function of PKD-2/LOV-1 TRPP complex in CEMs, we hypothesize that this complex is directly activated by sex pheromone and the signal is relayed intracellularly. This hypothesis will be evaluated by an in vitro system using Drosophila Schneider cells to investigate if physiological response can be triggered via PKD-2/LOV-1 TRPP complex by sex pheromone stimulation. Results from this experiment would be informative in defining the functional activity of these TRPP channels in the sex pheromone perception, shedding light on the biology and signaling of polycystin complex. (This study is supported by Research Grants Council, Hong Kong.).

Hui, Agnes K. Y. et al. (2009) International Worm Meeting "The requirement of stress response and autophagy to cope with abnormal sensory ray morphogenesis."

Hui, Agnes K. Y., Chow, King L.

[International Worm Meeting, 2009]

ER stress is generated by accumulation of misfolded proteins, deprivation of calcium ion or change in redox environment. Unfolded protein response (UPR) pathways are selectively activated by the ER stress to maintain the ER homeostasis. Autophagy/lysosomal degradation pathway and cell death may be triggered in severely stressed cells. We have been using ram-2(bx32) mutant as a model to study the impact of misfolded proteins in ray development. Previous studies have documented ER expansion, autophagy activation and signs of cell death in male tail of ram-2(bx32) mutant. In this report, we addressed the following issues: (1) which UPR pathway is required to handle the stress? (2) what is the role of autophagy? And (3), is cell death pathway activated in ram-2(bx32) mutant? Transcriptional activation of hsp-4 and enhancement of Ram phenotype in ram-2(bx32);xbp-1(zc12) and ram-2(bx32);crt-1(bz50) double mutants suggest that ire-1/xbp-1 signaling pathway is required for handling the stress generated by ram-2(bx32) products. A potential downstream target of pek-1, atf-5, was shown to be activated in ram-2(bx32) mutant. Attenuation of autophagy activity can enhance the severity of Ram phenotype of ram-2(bx32) mutant, while elevation of autophagy by resveratrol treatment showed partial suppression of its Ram phenotype. Hence, autophagy is likely required for removal of aberrant organelle. As for the punctate nuclear membrane and positive acridine orange staining in the ram-2(bx32) mutant male tail, both TUNEL and annexin V staining showed negative results and ruled out the occurrence of cell death in ram-2(bx32), a notion supported by the absence of specific effect of egl-1(n487) and ced-4(n1162) on ram-2(bx32) in double mutants. Absence of genetic interaction in ram-2(bx32); unc-68(e540) and ram-2(bx32); itr-1(sa73) double mutants also eliminate the possibility of having necrosis occurring in ram-2(bx32). Interestingly, altered nuclear architecture in ram-2(bx32) mutant resembling that of aging nuclei can be revealed by confocal microscopy. The finding suggests the leakiness of nuclei may account for the acridine orange staining and implicates a potential link between aberrant collagen production with cellular senescence. (The research is funded by Research Grants Council, Hong Kong.).

Leung, David C K et al. (2009) International Worm Meeting "Autoregulation of mab-22 expression takes place during Caenorhabditis elegans male sensory ray."

Leung, David C K, Chow, King L.

[International Worm Meeting, 2009]

We have reported that mab-22 encoding a T-box containing transcription factor, TBX-2, is indispensable for ray cells assembly. mab-22(bx59) mutant exhibits extensive ray-missing phenotype in a temperature-sensitive fashion, which also infers its action in all rays. However, the mab-22::gfp reporter containing key regulatory elements to rescue the mutant ray phenotype displays activity only in the structural cells of rays 1, 5 and 7 in wild-type animals. We hypothesize that mab-22 expression is under its own negative regulation in other rays. To test this notion, mab-22::gfp reporter line was crossed into mab-22 mutant. The incidence of expression and intensity of GFP signals in the structural cells of rays 1, 5 and 7 surged. In addition, substantial level of reporter expression was detected in all rays. We probe into this hypothesis of feedback regulation and test if it is mediated by direct binding of the mab-22 product onto the mab-22 locus. By adjusting the mab-22 dosage with the bx59 allele cultured at different temperatures or by changing the copy of wild-type and bx59 alleles, the mab-22 reporter expression level was examined. The results consistently indicated that reduction of mab-22 activity boosted the mab-22 reporter expression level in the structural cells. We have identified two putative MAB-22 binding sites on its own intron 1. We show that when these sites were deleted altogether, the mab-22 reporter expression in wild-type animals could be detected in all structural cells with a higher level in rays 1, 5 and 7. Besides, site-directed mutagenesis of the two binding sites individually was performed to confirm the bona fide MAB-22 effective sequence. Both the mutant reporters with only one of the two binding sites mutated lead to full expression of the reporter in all the structural cells. The observation substantiates that mab-22 exerts a repressive effect on its own expression via this intron 1 with two MAB-22 recognition sequences. To the best of our knowledge, it is the first report that a T-box gene expression is modulated by a negative autoregulatory mechanism. Biochemical analyses are in progress to decipher the binding specificity of MAB-22 to yield additional insights into the mechanism of the sequence-specific DNA binding of T-box protein. (This study is supported by the Research Grants Council, Hong Kong).

Hui, Agnes K. Y. et al. (2009) International Worm Meeting "Cellular basis of Ram (RAy Morphology abnormal) phenotype in C. elegans."

Chow, King L., Hui, Agnes K. Y.

[International Worm Meeting, 2009]

Organ morphogenesis is an important process during development. The male tail sensory rays of nematode C. elegans serve as a good model for the study of morphogenesis, particularly when many ram (RAy Morphology abnormal) mutants with lumpy rays are available. Based on their molecular natures, these ram genes can be classified into distinct groups according to the product features, e.g., ram-1, ram-2, ram-4 encoding male tail-specific collagens; ram-5 coding for ZP protein; and mab-7 and mab-29 encoding EGF-like domain containing proteins. However, the cellular basis of the sensory ray abnormality is not known. To define the cellular basis of the specific ray morphology, cellular, molecular biology and genetic techniques were employed to reveal biological events occurring inside these swollen rays in different ram mutants, ram-2(bx76), ram-2(bx32), ram-5(bx81) and mab-7(e1599), each representing a subclass of the ram genes. Fluorescent subcellular organelle markers revealed expanded ER, mislocalized nuclei, altered nuclear structure, autophagosomes and lysosomes accumulation in the swollen rays in ram-2(bx76), ram-2(bx32) and mab-7(e1599) mutant animals, while only expanded ER, mislocalized nuclei were observed in ram-5(bx81) mutants. The causal-relationship between the observed subcellular defects and the phenotype was tested. Modulation of autophagy level in ram-2(bx76), ram-2(bx32) and mab-7(e1599) mutants showed differential effects on the ray swelling, suggesting different mechanisms are responsible for the manifestation of Ram phenotype and distinct roles of autophagy in shaping the tissue morphology. In addition, two of the three stress response pathways were found to contribute to the subcellular defects in ram-2(bx32), but not other ram mutants. In summary, this study helps classify ram genes not by their molecular properties but by their differential biological impacts on subcellular events in the abnormal rays. (The research is funded by Research Grants Council, Hong Kong.).