Wong A et al. (1993) International C. elegans Meeting "clk-1 is involved in the regulation of developmental and behavioral rhythms."

Wong A, Hekimi S

[International C. elegans Meeting, 1993]

Wong HT et al. (1997) International C. elegans Meeting "ANALYSIS OF A NCAM-LIKE GENE IN CAENORHABDITIS ELEGANS."

Chow K-L, Wong YM, Wong HT, Siddiqui SS, Ali MY

[International C. elegans Meeting, 1997]

The differentiation of a tissue is a dynamic process with cell migration, change of cell shape and remodeling of cell-cell contact. The involvement of cell adhesion molecules and extra-cellular matrix proteins in this process has been documented in a number of model systems. We are interested in understanding the differentiation of male tail sensory organs and their morphogenesis. While most of the genes identified to take part in this morphogenetic process are playing more a regulatory role than guiding the final structural cellular modification, structural and adhesion components would likely be the targets of these regulatory genes. We therefore try to identify these components in order to understand better the regulatory mechanism of this differentiation process. It was reported that antibody against NCAM molecule recognized a number of neurons in the male tail. We try to characterize these NCAM-like molecules in worm to examine their expression pattern and function in male tail development. We noticed that there was a cDNA from the Kohara collection, yk9g7, which encodes a sequence with similarity to neural cell adhesion molecules. Although early mapping data of this partial cDNA was not informative, we have confirmed recently that this NCAM-like gene is on chromosome X on the left arm close to mab-7. We have obtained cosmids encompassing this NCAM-like gene. We are conducting rescue experiments with them to test whether there is any corresponding mutations of this gene. We are also characterizing the promoter of this gene as well as its expression pattern. The progress of this analysis together with the potential implication will be discussed.

Wong YM et al. (1997) International C. elegans Meeting "REGULATION OF THE SUBCELLULAR LOCALIZATION OF MAB-21 PROTEIN IN MAMMALIAN CELLS."

Chow K-L, Wong YM

[International C. elegans Meeting, 1997]

The development of sensory ray in male C. elegans requires the function of HOM-C genes, which act in a dosage dependent fashion. These genes interact with a battery of modifier genes in defining the sensory ray identity. One of these genes, mab-21, has been shown to play a crucial role in determining the identity of thick ray 6. Mutation of this gene results in transformation of ray 6 to ray 4. Experiments by expressing tagged MAB-21 protein in both worm and yeast showed that MAB-21 protein could partition between the cytoplasm and the nucleus. While genetic data suggest that mab-21 is acting downstream of a cell surface receptor, daf-4, we are interested to examine the control of the subcellular localization of this protein in response to external signals. We have succeeded in constructing a mammalian vector to express a GFP-tagged and a b-galactosidase-tagged MAB-21 protein. In transient and stable transfection assay in cos-7 and R6 fibroblasts. Both constructs are actively expressing the tagged protein in the cytoplasm with occasional nuclear expression. The localization appears to be related with cell morphology and cell density. Such observation prompts us that the partition of MAB-21 in the cell may be biologically regulated. We are currently utilizing the stable lines generated from the above experiments to examine what the external input signal is. Through direct application of growth factors suspected to play key role in the mab-21 signalling pathway or using conditioned medium from cell lines expressing relevant signalling molecules, we would like to define what molecules are important in regulating the MAB-21 localization. The transduction mechanism and the implication on mab-21 function will be discussed.

Wong WL et al. (1997) International C. elegans Meeting "PATTERN OF MAB-21 GENE EXPRESSION IN CAENORHABDITIS ELEGANS IS REGULATED DEVELOPMENTALLY."

Lee EF, Wong WL, Chow K-L

[International C. elegans Meeting, 1997]

The differentiation of the male specific peripheral sensory organs in C. elegans is a complex process. A gene mab-21, which encodes a protein of 386 amino acids, is required for the choice of alternate cell fates of several cells in the male tail through its interaction with homeobox containing genes. Tagged MAB-21 protein has been shown to be localized in the nucleus with residual expression in the cytoplasm in nematode by both galactosidase assay and anti-b-galactosidase antibody staining against the fusion protein in transgenic worms. Such observation has also been confirmed by immnostaining in yeast and subcellular fractionation analysis. Using antibody against b-galactosidase protein, we have analyzed the expression pattern at different stages in transgenic lines. mab-21 promoter is active in distinct cells in the body wall muscle around the pharynx, lateral hypodermis, and male tail. The observation, which is at a higher resolution, is consistent with our previously reported galactosidase staining result. To confirm the expression pattern at the protein level, a maltose binding protein (MBP)-MAB-21 fusion protein was expressed and purified from bacteria for raising antisera from mice, rats, and rabbits. ELISA and western blotting analysis showed the antisera could detect both the fusion protein expressed in bacteria and affinity purified fusion protein. However, only the rabbit serum could recognize the MAB-21 protein in worm. This antiserum is currently used to determine the expression pattern of MAB-21 protein in wildtype and mutant nematode by immunostaining. We intend to perform co-immunoprecipitation to identify additional cellular components that interact with the mab-21 products.

Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

YM Wong et al. (1999) International C. elegans Meeting "Effect of overexpressing mab-21, isolation and identification of mab-21 interacting cellular components in C. elegans by epitope tagging"

KL Chow, YM Wong

[International C. elegans Meeting, 1999]

In C. elegans male tail, the peripheral sensory structures develop during the late larval stages. They comprise of nine pairs of sensory rays embedded in a acuticular fan. These rays play a crucial role in the mating behavior of male nematodes. Each ray has its own specific morphological and functional identities. A set of genes has been defined to regulate ray differentiation. Among them mab-21 regulates ray 6 formation. Since its isolation, mab-21 's cellular role is by large unknown. To establish the role of mab-21 in normal development, we have overexpressed mab-21 cDNA with a variety of promoters. The results showed that mab-21 expression could perturb the identity determination of a number of rays. Most dramatically, tail spike could be induced in male animals. The relationship of the tail spike phenotype and mab-21 expression in the tail hyp cells will be examined. These dominant phenotypes once established in stable transgenic lines will be useful for screening of suppresser mutations to identify the regulatory targets of mab-21 . To further establish mab-21 's role in the signaling process, attempts were made to study the signaling molecules interacting with MAB-21. While multiple attempts to generate antibody against MAB-21 did not yield good reagent with high specificity, we have switched to protein tagging. Expression vector with fusion proteins gene expressing MAB-21 tagged with glutathione S-transferase (GST) or consecutive histidine residues (His tag) were constructed. Bacterial expressed protein was purified by affinity chromatography specific for the tags. Using these protein reagents, isolation of other signaling molecules involved in the ray formation process by immunoprecipitation or affinity chromatography is underway. Similar tagged constructs in worm expression vector have been made and will also be introduced into animals for parallel in vivo interaction study. These transgenic animals will be useful for subcellular localization of the mab-21 gene product, and to identify interacting components in worms.

Yan Fung Wong et al. (2006) East Asia C. elegans Meeting "The zinc finger protein MAB-30 controls patterning of sensory rays"

Yan Fung Wong, King L. Chow

[East Asia C. elegans Meeting, 2006]

The reduction of mab-21 gene activity results fusion of sensory ray 6 and 4 in the male tail. We previously reported the identification of mab-30, which encodes a zinc finger transcription factor in an RNAi knock-down experiment. Mutant animals showed a remarkable Mab-21 phenotype. Since the expression pattern of mab-30 overlaps temporally and spatially with that of mab-21 in ray 6 structural cell, we speculate that mab-30 is a key component in the mab-21 pathway to control ray identity. We have recently obtained a mab-30 deletion mutant from National BioResource Project (NBRP) and started both molecular and phenotypic characterization. RT-PCR analysis revealed that the mab-30 locus may encode two alternative MAB-30 proteins both with three intact zinc- finger DNA binding domains. Hence, the deletion allele may not have eliminated the mab-30 function completely. Indeed, consistent with this notion, the brood size of mab-30 mutant was reduced to only one-third of the wild type. Mab-21 phenotype was also displayed in mutant males similar to that revealed by RNAi knock-down experiments. In order to incorporate mab-30 into the mab-21 signaling pathway, genetic interaction of mab-30 with genes in the mab-21 pathway will be investigated. Meanwhile, a rabbit polyclonal antibody for MAB-30 is generated with a polypeptide corresponding to the N-terminal 205-amino acids of the protein. This reagent would allow us to identify the direct downstream target genes of MAB-30 by applying an in vitro genome-wide selection or an in vivo chromatin immunoprecipitation (ChIP). The preliminary data will be discussed in the presentation. (This study is funded by Research Grants Council, Hong Kong.)

Wong, Ming-Ching et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Identification of genes required for distal tip cell migration."

Wong, Ming-Ching, Slone, R Daniel, Schwarzbauer, Jean E

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The C. elegans hermaphroditic gonad consists of two U shaped arms. The morphology of this organ relies on the migration of the distal tip cells (DTC). DTCs must first migrate longitudinally away from the mid-body along the ventral basement membrane. The cells then turn to migrate dorsally and turn again to migrate longitudinally back toward the mid-body along the dorsal basement membrane. Previously, a whole genome RNAi screen was conducted to reveal that 99 genes are required for DTC migration (Cram et al., 2006, J. Cell Sci, 119:4811). To determine whether these genes are required cell autonomously in the DTC, we conducted a DTC-specific RNAi screen. This screen revealed that 29 of the 99 genes are required specifically in the DTCs. Here, we focus on one gene, cbp-1 , which encodes for a transcriptional coactivator that possesses histone acetyltransferase activity. DTC-specific RNAi knockdown of cbp-1 revealed that cbp-1 is necessary for DTCs to properly turn during migration. 70% of cbp-1 RNAi treated DTCs either failed to achieve the first ventral-to-dorsal turn or turned away from the mid body during the second turn, which resulted in gonad arms that extended to the pharynx or the tail. Because cbp-1 has a role in transcriptional control, we took a candidate gene approach to identify CBP-1 target genes that potentially have a role in DTC turning. Using this approach, we have identified genes that encode integrin adhesion receptors and integrin signaling proteins whose expression is dependent on CBP-1. We are currently exploring the mechanisms in which these genes influence DTC-basement membrane interactions and cell turning to further our understanding of precise cell migrations.

Tzeng T et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The Role of Reishi Polysaccharide in Promoting Lifespan of C. elegans"

Hsu T, Tzeng T, Wong C, Yang W

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Using Caenorhabditis elegans as a model, we showed that culturing with acetic acid or Reishi polysaccharide fraction 3 (RF3) could induce longevity of C. elegans. In addition, acetic acid and RF3 showed synergistic effects on inducing longer lifespan in C. elegans. Our results showed the up-regulation of daf-16, tir-1 and rab-1, and the down-regulation of daf-2 were involved in this phenomenon. Knock-down experiments confirmed that tir-1 and daf-16 were required for RF3- promoted longevity. We are currently examining the downstream signal/event of daf-16 activation mediated by RF3.

Lianna Wong et al. (2003) International Worm Meeting "FMI-1, the C. elegans homolog of Flamingo, is important for axon guidance and synapse formation"

Yishi Jin, Jim Rader, Julie Ahringer, Alexander Goncharov, Nikki Alvarez, Mei ZHEN, Lianna Wong, Gian Garriga

[International Worm Meeting, 2003]

To understand the processes of axon guidance and synapse formation, we performed two independent genetic screens. One was for mutants defective in axon guidance, focusing on the HSN axons. The HSNs are a bilaterally symmetric pair of serotonergic motor neurons that innervate the vulval muscles and stimulate egg laying. The mature HSN axon morphology can be visualized using gmIs1, a GFP reporter that illuminates the HSN cell bodies and axons. In wild-type animals, each HSN axon extends ventrally to the ventral nerve cord (VNC), turns anteriorly and extends along the VNC to the nerve ring. At the vulva, the HSN axons branch and elaborate varicosities. Two of the mutants isolated, gm188 and gm204, have abnormal HSN axons that circle around the vulva, elaborate unusually long branches, and cross between the left and right VNC tracts multiple times.The second screen was for mutants defective in synapse formation, using the synaptic vesicle marker Punc-25-SNB-1::GFP. In wild-type animals, this marker labels the presynaptic terminals of VD and DD motor neurons, and GFP appears as discrete puncta spaced evenly along the ventral and dorsal nerve cords. Two mutants isolated, ju43 and ju58, frequently lose SNB-1::GFP puncta in the presynaptic regions of VD1 and VD2 neurons, while the puncta from the remaining VDs exhibit subtle abnormalities in morphology and spacing. Some mutant animals also lack SNB-1::GFP puncta in presynaptic regions of DD neurons. Some of these neurons show axon outgrowth defects which could account for the loss of synapses. Specific synaptic defects are also observed in preliminary ultrastructural analysis.These mutations map to the same region of chromosome V and fail to complement. We mapped gm188 to a region containing 30 ORFs. Unable to achieve rescue of the mutant phenotype by cosmid injection, we phenocopied the HSN axon phenotype by RNAi to F15B9.7. We have identified molecular lesions in F15B9.7 for three of the four alleles.F15B9.7 encodes the C. elegans homolog of Flamingo, hereby designated FMI-1, a large, seven-pass transmembrane cadherin involved in the planar polarity pathway and dendritic morphogenesis in Drosophila. We are currently making GFP fusions and antibodies to study expression and localization of FMI-1 in C. elegans.