Shin, Hanna et al. (2015) International Worm Meeting "The small GTPase Ral signals via an Exo84-GCK-2/MAP4K-p38-MAPKAPK cascade to induce 2deg vulval fate.."

Reiner, David J., Der, Channing J., Kaplan, Rebecca E.W., Shin, Hanna

[International Worm Meeting, 2015]

Vulval precursor cells are patterned through graded action of EGF from the anchor cell in conjunction with LIN-12/Notch-mediated lateral signaling. Together these signals form the highly reproducible 3deg-3deg-2deg-1deg-2deg-3deg pattern that develops to form the vulval epithelial duct. A key protein in this process is LET-60/Ras, which in mammals is the most mutated oncoprotein in cancer. In presumptive 1deg cells EGFR promotes LET-60/Ras activation to induce 1deg fate via the canonical Raf-MEK-ERK MAP kinase cascade. We previously demonstrated that, in presumptive 2deg cells, LET-60/Ras switches effectors to activate the lesser known Ras effector RalGEF-Ral to induce 2deg fate in support of Notch. In general 1deg- and 2deg-promoting signals are strongly antagonistic. Thus, in equipotent cells Ras switches between Raf and RalGEF-Ral effectors to promote mutually antagonistic 1deg and 2deg cell fates, respectively. However, the Ral signaling cascade is poorly understood. We therefore screened candidate Ral effectors from the literature. Two subunits of the Exocyst complex have been previously shown to bind Ral proteins in other systems; we found that loss of Exo84 but not Sec5 caused a phenotype similar to loss of Ral and blocked the 2deg-promoting activity of transgenic mutationally activated Ral, indicating that Ral partners with Exo84 to promote 2deg fate. To move further downstream we analyzed a little known family of MAP4 kinases. In Drosophila Sec5 and Msn MAP4K, the ortholog of C. elegans MIG-15, physically interact and promote JNK signaling, but function in opposition to Ral signaling. Since Sec5-Msn did not act like a downstream Ral partner, we hypothesized that the paralogous CNH domain MAP4K subfamily, C. elegans GCK-2 and Drosophila Hppy, constitutes the real Ral effector. We found that loss of GCK-2 function conferred a defect similar to loss of Exo84, and Ral-Exo84 signals through a conserved GCK-2-p38 MAP kinase cascade. This cascade may further activate MAK-2/MAPKAP kinase. We propose that antagonistic ERK and p38 MAP kinase cascades promote opposing 1deg and 2deg fates, respectively. We further investigated MIG-15, and our data suggest that MIG-15 antagonizes LIN-12/Notch 2deg induction in the absence of EGF signal, an activity opposing the Ral-Exo84-GCK-2 EGF-dependent 2deg-promoting signal supporting LIN-12/Notch. We have discovered two functionally opposed CNH domain MAP4 kinases that function in vulval patterning. Perhaps Ral-GCK-2 and MIG-15 work in opposition to clearly demarcate 1deg and 2deg signals compartments through LIN-12/Notch regulation, thus increasing developmental fidelity.

Shin, Hanna et al. (2017) International Worm Meeting "LET-60/Ras recruits its 2 deg -promoting effector, RGL-1/RalGEF, specifically to the apical plasma membrane of presumptive 2 deg cells."

Shin, Hanna, Reiner, David

[International Worm Meeting, 2017]

EGF from the anchor cell (AC) induces the vulval precursor cells (VPCs) to assume the highly reproducible 3 deg -3 deg -2 deg -1 deg -2 deg -3 deg pattern of cell fates. Two potentially competing models describe this event. In the morphogen gradient model, position of VPCs in the EGF gradient dictates cell fates. In the sequential induction model, LET-23/EGFR in the VPC closest to the AC triggers the canonical Ras-Raf-MEK-ERK MAP kinase cascade to promote 1 deg fate, which in turn synthesizes DSL ligands to activate LIN-12/Notch in the two neighboring VPCs to induce 2 deg fate. We reconciled these models by identifying a signaling mechanism by which the EGF gradient promotes 2 deg fate. We found that in vulval patterning, LET-60/Ras dynamically switches effectors, from the canonical Raf to the poorly understood RalGEF-Ral, which promotes 2 deg fate in support of the main LIN-12/Notch signal. Ras-RalGEF-Ral is required for 2 deg cells to respond fully to the 2 deg -promoting EGF-EGFR signal, and ectopic constitutively active RAL-1/Ral promotes 2 deg fate (see Zand et al., 2011). We are also investigating the signal downstream of Ral (see poster by Shin/Kaplan). LET-23/EGFR::GFP shows general plasma membrane localization in VPCs. Mammalian Ras is uniformly expressed at the plasma membrane, where upon activation it is thought to recruit effectors. Traditional heterologous means of expressing proteins run the risk of missing regulatory sequences or, through over-expression, swamping delicate mechanisms of subcellular trafficking. We therefore used CRISPR to tag the N-terminus of endogenous RGL-1/RalGEF with mNeonGreen (mNG). Prior to induction, mNG::RGL-1 was diffuse and cytosolic in VPCs. After induction, mNG::RGL-1 was concentrated at the apical plasma membrane in presumptive 2 deg - but not presumptive 1 deg - VPCs, and not to basolateral membranes. Our observations raise the intriguing possibility of spatial compartmentalization of potentially conflicting signals, 1 deg -promoting LIN-45/Raf vs. 2 deg -promoting RGL-1/RalGEF, as a novel mechanism of interpreting the EGF gradient. Consequently, to complement our data with mNG::RGL-1, we are tagging endogenous LET-23/EGFR, LET-60/Ras and LIN-45/Raf to observe spatial regulation of signaling activity over time. We intend to have these new tools ready to present preliminary data.

Fakieh, Razan et al. (2021) International Worm Meeting "The RAP-2 Small GTPase and MIG-15 MAP4 kinase promote tertiary fate in C. elegans VPC Patterning"

Fakieh, Razan, Reiner, David, Shin, Hanna

[International Worm Meeting, 2021]

During C. elegans development, graded EGF signal from the anchor cell (AC) induces the six equipotent vulval precursor cells (VPCs) to assume a pattern of 3 -3 -2 -1 -2 -3 cell fates. The VPC closest to the AC is induced via the Ras-Raf-MEK-ERK MAP kinase cascade to assume 1 fate. Presumptive 1 cells generate DSL ligands to induce the two neighboring cells via the Notch receptor to assume 2 fate. 1 and 2 developmental programs have been shown to be mutually antagonistic. Our lab showed that lower EGF dose causes Ras to switch effectors, from Raf to RalGEF-Ral, which functions to promote 2 fate in support of Notch. We further showed that Ral signals through GCK-2, a member of the Ste20 family of mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), to trigger a p38 MAP kinase cascade to promote 2 fate (Shin et al., 2018). 1 and 2 cells execute distinct and stereotyped division patterns to form the vulva. In contrast, 3 fate is typically referred to as the "ground" or "uninduced" cell fate; 3 cells divide once and fuse with the surrounding epithelium. We have found that a paralog of GCK-2, MIG-15, also plays a role in VPC patterning. Upon mutation or RNAi depletion of MIG-15 or RAP-2, we observed an increase in ectopic 1 as well as ectopic 2 cells. MIG-15 is also required for expression of a putative cell fate reporter in 3 cells. Both RAP-2 and mig-15 are necessary for full expression of the 3° biomarker. Thus, we hypothesize that, like 1 - and 2 -promoting signals, 3 -promoting signals antagonize other vulval cell fates. Using CRISPR-Cas9, we engineered an insert of fluorescent protein and epitope tag into the 5' end of the endogenous mig-15 gene, revealing ubiquitous expression in the animal, localized to the cytosol and cell-cell junctions. We also inserted auxin inducible degron (AID), which mediates conditional degradation of tagged proteins. We will use complementary degradation experiments and tissue-specific transgenic rescue to test whether MIG-15 functions in the VPCs to repress 1 - and 2 - signals. We will also use CRISPR to mutationally activate MIG-15, as we did previously with the paralogous GCK-2. Preliminary data suggest that RAP-2 functions similarly to MIG-15, and RAP-2 has been shown to activate MIG-15 in other systems. We hypothesize that RAP-2-MIG-15 promotes 3° fate, counter to the notion of 3 fate as "uninduced." Our work presents positions us to explore signals that promote "ground" developmental state and perhaps informs the relationship cancers and surrounding stromal cells.

Ashraf S et al. (1997) International C. elegans Meeting "DOMINANT MATERNAL EFFECT STERILE MUTANTS"

Shin T-H, Ashraf S, Mello CC

[International C. elegans Meeting, 1997]

The PIE-1 protein is localized to cells of the germ lineage in the early C. elegans embryo where it functions to suppress somatic cell fates. We have found that mutant strains in which PIE-1 protein is mislocalized to somatic cells exhibit a sterile phenotype, but in most cases undergo fairly normal somatic differentiation. These observations suggest that while the threshold for PIE-1 function in suppressing somatic development is set high, slight reductions in PIE-1 levels may be sufficient to prevent germline protection leading to sterility. This may explain why certain PIE-1 genetic backgrounds as well as strains defective in functions required for PIE-1 localization appear to exhibit haplo-insufficient dominant sterile phenotypes (See Shin, et al). These observations led us to initiate screens for Dominant Maternal Effect Sterile mutations (DMES). Steriles are a very common phenotypic class. In standard genetic screens dominant maternal effect steriles will arise in the F2 among a huge background of recessive zygotic steriles. To avoid this difficulty we decided to ask if the diploid oocytes within the proximal arms of a mutagenized animal might be considered as independent mothers. If so then this would permit us to screen for dominant maternal effects among the F1 progeny of a mutagenized animal. We know from our previous dealings with pie-1 associated sterility that affected animals can often produce a few fertile viable offspring so we screened in an egg-laying defective background for F1 animals with only a few embryos developing inside. We first did a pilot screen of 30,000 F1 mutagenized animals. From this screen we obtained two mutations that behave like bona fide DMES loci, and a few that appear to be zygotic effect Dominants. One of the DMES mutants (ne146) , exhibits mislocalization of both the PIE-1 protein and of the P-granules. This mutation fails to complement previously identified maternal effect lethal alleles of the par-2 locus. The second DMES mutation mapped genetically to the location of a gene, alp-1 that encodes a PIE-1 binding protein, and indeed our mutant allele appears to contain a point mutation in this gene (See Shin et al.). This initial success has encouraged more screens which are presently underway.

Awani, Awani et al. (2011) International Worm Meeting "Genome wide RNAi screen to study uv1 cell necrosis."

Awani, Awani, Hanna-Rose, Wendy, Crook, Matt

[International Worm Meeting, 2011]

Necrosis is more than a phenomenon of uncontrolled cell death. There is increasing evidence to suggest that necrosis is genetically programmed. We are exploiting a new model to investigate necrotic cell death. In worms that lack pnc-1 nicotinamidase, the four uv1 cells die by necrosis (1). We want to investigate why uv1 cells die in pnc-1 mutants and to identify genes that promote or inhibit uv1 cell necrosis. In pnc-1 mutant animals with a uv1 cell specific GFP transgene, we can screen for rescue of uv1 cell necrosis by screening for animals that retain the uv1 GFP signal. We aim to screen using the genome-wide RNAi library and expect to gain insight into how and why uv1 cells die in pnc-1 mutants. We are using a split GFP system to engineer animals with uv1 cell specific GFP expression for this screen (2), and we will discuss progress in engineering the strain and preparing for the screen. Reference: 1. EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus. Li Huang , Hanna-Rose Wendy 2. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Indraneel Ghosh , Andrew D. Hamilton , Lynne Regan.

Hyun-Ok Song et al. (2007) International Worm Meeting "Calcineurin, a Ca2+/calmodulin dependent Ser/Thr protein phosphatase, is important for endocytosis in C. elegans."

Joohong Ahnn, Hyun-Ok Song, Meenakshi Dwivedi

[International Worm Meeting, 2007]

Endocytosis is an essential cellular mechanism required for the internalization of fluid from the extracellular medium, nutrient uptake, and the recycling of membrane components. The significance of endocytosis has been shown in the down-regulation of signaling, synaptic vesicle recycling, and antigen presentation in immune cells. One dramatic example of receptor-mediated endocytosis is yolk uptake observed in C. elegans oocytes. Yolk proteins are synthesized in intestine and secreted into pseudocoelomic cavity. Finally oocytes take yolk protein up by receptor-mediated encodytosis. Many rme (receptor mediated endcytosis defective) mutants have been identified using vitellogenin::GFP (YP170::GFP) fusion system (Barth Grant et al., 1999). In addition, C. elegans use coelomocytes, scavenger cells, to endocytose fluid from pseudocoelom. The endocytosis using coelomocytes has been suggested to be mediated by clathrin. Coelomocyte uptake defective (cup) mutants have been identified based on the uptake of fluid-phase GFP markers from the pseudocoelom by coelomocytes (Hanna Fares and Iva Greenwald, 2001). Here, we report a possible function of calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, in C. elegans endocytosis using above analyses. We show that mutants of calcienurin A and B [tax-6(jh107gf), tax-6(p675lf), cnb-1(jh103null)] have partial defects in endocytosis.

Reboul J et al. (2001) Nat Genet "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans."

Doucette-Stamm L, Lamesch PE, Reboul J, Temple GF, Hartley JL, Brasch MA, Hill DE, Vaglio P, Thierry-Mieg N, Shin-i T, Lee H, Moore T, Vandenhaute J, Kohara Y, Vidal M, Jackson C, Thierry-Mieg J, Tzellas N, Thierry-Mieg D, Hitti J

[Nat Genet, 2001]

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM

Shin, Heaji J. et al. (2013) International Worm Meeting "Characterization of SYGL-1, a novel regulator of germline stem cells."

Haupt, Kimberly, Kershner, Aaron M., Shin, Heaji J., Kimble, Judith

[International Worm Meeting, 2013]

C. elegans germline stem cells (GSCs) are maintained by GLP-1/Notch signaling from the stem cell niche. Two GLP-1/Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic germline proliferation defective), act redundantly to maintain GSCs throughout development and in both sexes (Kershner, Shin, and Kimble, manuscript in preparation). Here we focus on our characterization of the sygl-1 gene, which had not been analyzed previously. The sygl-1 locus encodes a single transcript (T27F6.4) that is predicted to generate a novel protein with no folded domains or motifs. To begin to investigate sygl-1 function, we have characterized a strong loss of function sygl-1(tm5040) mutant and generated a rescuing epitope-tagged SYGL-1 transgene. The sygl-1(tm5040) homozygote is viable, but it possesses a smaller than normal mitotic zone. The sygl-1 mRNA is restricted to the distal mitotic zone and is dependent on GLP-1/Notch signaling. The SYGL-1 protein is similarly restricted to the distal mitotic zone and is cytoplasmic. Our characterization so far is therefore consistent with a role for SYGL-1 in GSC maintenance, but we still know very little about this enigmatic locus. Experiments to explore sygl-1 regulation and function are in progress, and results will be reported.

I Maeda et al. (1999) International C. elegans Meeting "RNAi Screening with a non-redundant cDNA set"

I Maeda, Y Kohara, A Sugimoto, M Yamamoto

[International C. elegans Meeting, 1999]

RNA interference (RNAi) is a powerful tool for the transient inactivation of specific gene expression. We aim to establish a new screening method in which RNAi is performed systematically, to identify genes involved in specific developmental processes. To maximize the efficiency, we use a tag-sequenced, non-redundant cDNA set (made by Y. K.) as templates for preparing dsRNA. To deliver dsRNA into worms, we use the "soaking method" (in which worms are soaked in dsRNA solution) instead of microinjection, so that multiple RNAi can be performed concurrently. Although the soaking method has been known to be less potent and to give variable results, we optimized the condition to achieve a satisfactory efficacy to be used for the screen. In the control experiments, all nine genes tested ( unc-22 , glp-1 , mei-1 , mes-3 , fem-1 , glh-1 , glh-2 , pgl-1 and emo-1 ) with this method exhibited expected phenotypes with high penetrance. We are currently screening for the RNA species that cause sterility or embryonic lethality, however, we will record any other noticeable phenotypes. We plan to make the RNAi data available to the public at the NEXTDB WWW server at NIG (See the abstract by Shin-i et al. ). In the pilot screen, 10/64 gave embryonic lethality of the F1s, and 6/64 resulted in sterility of the P0s. Progress on the screen will be presented.

Kohara Y et al. (1997) International C. elegans Meeting "EXPRESSION PATTERN MAP OF THE C.ELEGANS GENOME"

Shin-i T, Sano M, Motohashi T, Tabara H, Mitani Y, Ohba T, Hirono K, Miyata A, Uesugi H, Obara M, Watanabe H, Iida K, Kohara Y

[International C. elegans Meeting, 1997]

Aiming at understanding of the gene expression networks in development of C. elegans, we are constructing an expression pattern map of the 100Mb genome through identifying and characterizing cDNA clones of all the genes whose total number is estimated to be 15,000. Thus far, 37,000 clones have been subjected to tag-sequencing from both ends. The analysis gave 25,000 clean 3'-tag sequences, which were classified into >6,500 unique cDNA species (nearly half of the total genes) by comparing the 3'-tags. This set covers 70% of the C.elegans entries in GenBank (excluding those for tRNA and non-coding sequences). Most of them were mapped on the genome either by in silico mapping or hybridization to the YAC filters. We are systematically analyzing the expression patterns of the classified cDNA species using in situ hybridization on whole mount specimen of embryos, larvae and adults (see the abstract by Motohashi et al.). Rough estimation is that 39% of the cDNA species show specific pattern of expression during embryogenesis, out of which 1/3 shows zygotic expression and 1/3 maternal expression. mRNA of 1/4 of the maternally expressed genes (about 4% of all) disappears in very early stages before gastrulation. Classifying of the expression patterns leads to identification of sets of genes which show the very similar expression patterns. Determination of the regulatory regions of these genes is also in progress. We are preparing to make the information available on the internet (see the abstract by Shin-i et al.).