Shin KH et al. (2008) Mol Cells "Analysis of C. elegans VIG-1 expression."

Cho NJ, Choi B, Park YS, Shin KH

[Mol Cells, 2008]

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta -908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Shin Murakami (2001) International C. elegans Meeting "Longevity and gerontogenes in model organisms"

Shin Murakami

[International C. elegans Meeting, 2001]

Mutations leading to life-extension have revealed molecular mechanisms that regulate longevity and aging in model organisms. Approximately sixty-five gerontogenes have been identified in model organisms, ranging from nematodes to mammals. The vast majority have been identified in the nematode, Caenorhabditis elegans. Thus, C. elegans represent a paradigm for understanding the specification of lifespan in other organisms. Most C. elegans gerontogenes act as negative regulators of longevity and also affect resistance to environmental stressors. Such gerontogenes may regulate a group of genes needed for increased longevity and other biological processes including environmental perception, metabolism, reproduction, stress resistance and others. This poster describes gerontogenes in organisms ranging from C. elegans to the mouse and discusses molecular mechanisms specifying longevity.

Shin Y et al. (2017) Science "Liquid phase condensation in cell physiology and disease."

Brangwynne CP, Shin Y

[Science, 2017]

Phase transitions are ubiquitous in nonliving matter, and recent discoveries have shown that they also play a key role within living cells. Intracellular liquid-liquid phase separation is thought to drive the formation of condensed liquid-like droplets of protein, RNA, and other biomolecules, which form in the absence of a delimiting membrane. Recent studies have elucidated many aspects of the molecular interactions underlying the formation of these remarkable and ubiquitous droplets and the way in which such interactions dictate their material properties, composition, and phase behavior. Here, we review these exciting developments and highlight key remaining challenges, particularly the ability of liquid condensates to both facilitate and respond to biological function and how their metastability may underlie devastating protein aggregation diseases.

Shin Takagi et al. (2001) International C. elegans Meeting "Involvment of PlexinA in vulval morphogenesis"

Shin Takagi, Hajime Fujisawa, Akira Nukazuka, Go Shioi, Yukimasa Shibata, Takashi Fujii

[International C. elegans Meeting, 2001]

Involvment of PlexinA in vulval morphogenesis Shin Takagi, Takashi Fujii, Akira Nukazuka, Yukimasa Shibata, Go Shioi, Hajime Fujisawa Laboratory of Developmental Neurobiology, Division of Biological Science, Graduate School of Science, Nagoya University, Japan, CREST, JST The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos including axonal guidance. We have generated and characterized putative null mutants of the plx-1 ( pka. cep-2 ) gene encoding C. elegans plexinA. plx-1 mutants exhibited morphological defects in several organs of epidermal origin, such as Ray1 in the male tail (see Fujii et al . in this Meeting) and alae. Here we report on the defects in vulvae of plx-1 mutants. plx-1 adult hermaphrodites sometimes had a deformed vulva or extra vulva-like structures. plx-1::gfp transgene was expressed in the vulval primordial cells. Since the epidermal cells composing the vulval primordium are known to undergo dynamic changes in shape and position during the vulval morphogenesis, we analyzed the vulval primordium with MH27::gfp . The epidermal cells of the vulval primordium were aberrantly arranged in plx-1 mutants: the rotation-symmetric configuration of the primordum was often disrupted, and some cells were not in contact with each other. Some cells failed to participate in the formation of the main vulva and formed an extra vulva-like depression. The results indicate that plx-1 regulates the arrangement and/or migration of epidermal cells in this system.

Shin T-H et al. (1997) International C. elegans Meeting "A HIGH THRESHOLD FOR PIE-1 ACTIVITY?"

Mello CC, Miliaras NB, Shin T-H, Ashraf S

[International C. elegans Meeting, 1997]

In early C. elegans embryos, the PIE-1 protein functions to protect germline blastomeres from somatic differentiation. We are using reverse and forward genetic approaches to identify the factors that regulate PIE-1 localization and function. Through a yeast two hybrid screen, we identified a protein ALP-1 (asymmetric localization of PIE-1) that is required for the germline localization of PIE-1 and of the P-granules. ALP-1 is related to Drosophila Bicaudal-C (Bic-C) and is predicted to contain multiple KH domains, motifs implicated in RNA-binding. Bic-C is necessary for posterior localization of the Polar granules and of the germline determinant Oskar, in Drosophila, suggesting that the localization machinery controlling germline development may be evolutionarily conserved. Additional proteins involved in PIE-1 localization include a worm homologue of the human UBC9 (a ubiquitin-conjugating enzyme) and ALP-2, a protein related to Saccharomyces cerevisiae Nfi1, which in the budding yeast appears to function at the site of cytokinesis. Antisense RNA injections for alp-1 and alp-2 induce sterile phenotypes. Using this phenotype as a guide, we initiated genetic screens to look for mutations in these and other genes required for PIE-1 localization (see Ashraf et al. ). We have identified one maternal effect sterile mutation (ne157) that maps to the alp-1 locus and contains a missense mutation in the alp-1 coding region. Subsequently, we isolated a suppressor of ne157 which carries a reversion of this point mutation. We have also identified new sterile alleles of previously identified maternal genes, mex-1 and par-2, and we now know that both of these as well as the previously identified mes-1 gene are all required for PIE-1 localization. Our sterile mutants exhibit mislocalization of PIE-1 into somatic cells and yet somatic development is apparently normal in these strains. These findings suggest that PIE-1-dependent suppression of somatic development is controlled by a high threshold. In wildtype embryos somatic sisters of the germline blastomeres often inherit small amounts of PIE-1 protein, perhaps a high threshold for PIE-1 activity minimizes the consequences of this imperfect segregation. A high threshold for germline function could also explain why some genes needed for PIE-1 localization such as alp-1 and par-2 appear to be haploinsufficient sterile loci. Bic-C in Drosophila also appears to be a haploinsufficient locus. Could the same logic be at work in the germline of flies?

Tadasu Shin-i et al. (2001) International C. elegans Meeting "NEXTDB: The nematode expression pattern database."

Tadasu SHIN-I, Yuji KOHARA

[International C. elegans Meeting, 2001]

Jiyeon Shin et al. (1999) International C. elegans Meeting "Characterization of Flectin -like protein in C.elegans"

Joohong Ahnn, Jungsu Lee, Sunja Kim, Jiyeon Shin

[International C. elegans Meeting, 1999]

Flectin is a new extracellular matrix protein which was identified from eye extract of chick embryo. Homologous proteins were found in many other vertebrates suggesting that flectin may be an evolutinary conserved protein. A flectin-like gene (ZK783.4) was found in C. elegans genome data base, showing approximately 40% similarity (20% identity) to chick flectin. In order to examine the localization of this gene, reporter gene fused with this gene were expressed in germ-line transformed transgenic animals. Green Fluorescent protein was expressed in various neurons, hypodermis and distal tip cells from early embryonic stage throughout larval and adult stages. Interestingly, strong expression patterns were observed in neuronal cells and hypodermal cells which compose the ectodermal tissue during early embryogenesis. This observation suggest that flectin-like protein may be important for the development of hypodermis and neurons. In addition, majority of cells expressing reporter gene are migrating cells during development. In order to study its function , we are preparing C.elegans flectin antibodies. We have screened for deletion mutant by treating worm with EMS and isolated a deletion mutant candidate. We are currently following this deletion mutant candidate in order to characterize its function in C.elegans.

Shin JY et al. (1997) International C. elegans Meeting "A FLECTIN-LIKE PROTEIN IN C. ELEGANS"

Shin JY, Ahnn JH

[International C. elegans Meeting, 1997]

Flectin is a new extracellular matrix protein which has been identified in chick using a monoclonal antibody F22. Discrete spatiotemporal pattern of flectin expression during heart looping suggested that it could play a significant role in heart development. (Developmental Biology 173, 39-50, 1996) When we stained C. elegans with the monoclonal antibody of chick flectin, positive stainings were observed in egg shell, pharynx and basement membrane of gonad. These staining patterns indicate that it recognizes extracellular matrix in C. elegans. As a result of the homology searching in C. elgans genome data base, A ZK783.4 clone was found to be in high similarity with flectin. In order to prove that the antigen is encoded by ZK783.4 clone, we are currently constructing gfp tagging fusion protein with promoter region of ZK783.4. The gfp expression pattern in transgenic animal will be observed so as to test whether its expression pattern is consistent with F22 staining pattern. For the further characterization of this gene, Northern and Southern analysis will be performed with cDNA fragment. In addition, we are trying to elucidate the functional role of this putative flectin homologue of C. elegans. For this purpose, we are planning to screen the Tc1 induced library to isolate the mutant.

Shin-i T et al. (1997) International C. elegans Meeting "EXPRESSION PATTERN MAP ON THE INTERNET"

Kohara Y, Shin-i T

[International C. elegans Meeting, 1997]

We are constructing a database to integrate all the information of our expression pattern map of the C.elegans genome (see the abstract by Kohara et al.). The database consists of three parts; (1) "Clone tag DB" treats the cDNA tag sequencing data automatically to map the sequences onto the genomic sequence and to cluster them into unique cDNA groups. The processed data will be released quickly to the public domain, such as our WWW server, public DB and ACEDB. (2) "In situ hybridization DB" functions as an annotation workbench which supports us to add various information, particularly about the positive cell(s) and cell lineages in the hybridization, to the individual images of in situ analysis. Once the annotation is made by us, the in situ images are arranged appropriately and are made available at the WWW server. (3) "Feedback info DB" accommodates a wide range of information provided by the users of the cDNA clones. We plan to have a demonstration of the database at the meeting.

Shin-Yi Lin et al. (2001) International C. elegans Meeting "lin-64, A NEW GENE INVOLVED IN DEVELOPMENTAL TIMING"

Mary Abraham, Amy Pasquinelli, Gary Ruvkun, Shin-Yi Lin, Frank Slack, Mona Carlson

[International C. elegans Meeting, 2001]

Genes of the heterochronic pathway regulate developmental timing in C. elegans . let-7 , a late-acting heterochronic gene, regulates the switch from proliferation to terminal differentiation in seam cells during the L4 to adult transition. let-7 , a 21-nucleotide RNA molecule negatively regulates lin-41 , and possibly other heterochronic genes as well through interaction with let-7 complementary sites in the 3’UTR of these genes. However, the molecular mechanism of let-7 -mediated regulation remains unclear. To clarify the mechanism, we sought to identify other genes involved in let-7 regulation. We performed a screen for mutants showing a Let-7-like phenotype (e.g. retarded development, egg-holding, protruding-vulva). One gene isolated from this screen is lin-64 , for which we have at least one mutant allele, mg285 . Genetic mapping has placed lin-64 near lon-2 on chromosome X. Further mapping utilizing polymorphisms and deficiencies is being done. Complementation analysis is being carried out between mg285 and other mutant alleles isolated in this screen to determine whether we have additional alleles of lin-64 . lin-64 appears to complement daf-12 and lin-64 mutants are not dauer defective. We have taken advantage of gfp constructs that mark either seam cell nuclei ( ssm::gfp ) or cell junctions (jam-1::gfp ) to further characterize lin-64 ’s mutant phenotype. Preliminary phenotypic analysis indicates that mg285 animals are unable to appropriately make the seam cell differentiation vs. proliferation decision during the transition to adulthood. These mutants have extra seam cells and abnormal alae. We are also investigating possible genetic interactions between lin-64 and other heterochronic genes. We are studying the ability of lin-64 mutants to suppress the precocious heterochronic phenotypes of lin-41 mutants and lin-28 mutants. We are also examining lin-41 expression in the lin-64 mutant using a col-10-lacZ-lin-41 3’UTR reporter construct to determine whether lin-64 participates in the regulation of lin-41 by let-7 .