Inoue, Takao (2011) International Worm Meeting "Possible regulation of bed-3 by blmp-1."

Inoue, Takao

[International Worm Meeting, 2011]

blmp-1 encodes the C. elegans ortholog of Blimp1, a SET-domain containing transcription factor. The modENCODE project identified a binding site for BLMP-1 in the intron 3 of the bed-3 gene, within a region where a vulva and hypodermis specific enhancer element was previously identified. We found that RNAi against blmp-1 causes down-regulation of a bed-3::gfp reporter containing intron 3. Moreover RNAi against blmp-1 caused a molting defect similar to a strong bed-3 mutant. These results suggest that blmp-1 regulates bed-3.

Inoue, Takao et al. (2013) International Worm Meeting "Analysis of miro-1."

Ng, Li Fang, Shen, Yanqing, Gruber, Jan, Hagen, Thilo, Low, Natarie Pei Wen, Inoue, Takao

[International Worm Meeting, 2013]

miro-1 encodes the C. elegans homolog of mitochondrial rho (Miro), a mitochondrial outer membrane protein involved in mitochondrial movement. We found that a mutant carrying a deletion in miro-1 (tm1966) exhibits extended life span. In addition, miro-1(tm1966) mutants have altered distribution of mitochondria in intestinal cells and reduced amount of mitochondria in body wall muscles. Consistently, the mitochondrial DNA copy number is reduced in miro-1(tm1966) mutants. Interestingly, we found no difference in the oxygen consumption of miro-1(tm1966) mutant compared to the wild-type. We are carrying out further analysis to determine how the function of miro-1 relates to C. elegans life span.

Takao Inoue et al. (2005) International Worm Meeting "Reverse genetic analysis of C.elegans Sec14 family"

Hiroyuki Arai, Takahiro KANAMORI, Takao Inoue, Yumiko Ohi, Hiroyuki Kobuna, Yuka Suzuki

[International Worm Meeting, 2005]

Sec14 family is a novel cytosolic lipid-binding/transfer protein family that is defined by a conserved lipid-binding motif called Sec14 domain. The human Sec14 family consists of more than 20 members, including -tocopherol transfer protein (-TTP), cellular retinylaldehyde-binding protein (CRALBP), supernatant protein factor (SPF), and megakaryocyte protein-tyrosin-phosphatase (MEG2). It is hypothesized that members of the Sec14 family regulate cellular lipid transport and/or lipid metabolism, but their physiological functions and ligand lipids are largely unknown. In this study, we identified 15 members of the Sec14 family in C. elegans genome (ltp-1 ~ 15, lipid transfer protein-1 ~ 15). In these Sec14 family genes, ltp-1, 2, 3, 13, 14 and 15 show significant homologies with the corresponding human genes. To investigate the functions of these evolutionarily conserved Sec14 genes, we generated the deletion mutants of ltp-1, 2, 3, 13 and 15 by the UV/TMP method and analyzed their phenotypes. We also examined their expression pattern using the transgenic worms which express the promoter::GFP reporter genes. In this meeting, we will present phenotypic data on ltp-2 that is expressed predominantly in epithelial cells in C. elegans. At low temperatures, ltp-2(qa3102) deletion mutants showed variable abnormalities such as larval arrest, vulval bursting and egg-laying defects. We also observed that ltp-2(qa3102) exhibited abnormal cuticle structure. In a genetic screen for suppressors that rescue ltp-2 lethality at 12<sym05>C, we identified an obr-2(qa3117) mutation as a partial ltp-2 suppressor. obr-2 is an oxysterol-binding protein (OSBP)-related gene that encodes homologous sequences of mammalian ORP1, 2 and yeast Kes1p. Since Kes1p has been shown to act as a negative effector of the Golgi secretory function, ltp-2 may play roles as a positive regulator of the secretory pathway upstream of obr-2.

Takao Inoue et al. (2002) West Coast Worm Meeting "lin-17, lin-18 and patterning of the P7.p lineage"

Rashmi Deshpande, Paul W Sternberg, Russell Hill, Takao Inoue

[West Coast Worm Meeting, 2002]

We are interested in patterning of the 2 (secondary) vulval lineages P5.p and P7.p. In the wild-type, P5.p and P7.p produce stereotyped ABCD and DCBA patterns respectively. In lin-17 and lin-18 mutants, the polarity of the P7.p lineage becomes altered. We examined the P7.p lineage in lin-17 (encoding Frizzled Wnt receptor), lin-18 (encoding RYK receptor tyrosine kinase-related protein; W. Katz and P.W.S.), and double mutant using POP-1 (TCF/LEF) antibody staining and cell fate markers ceh-2::yfp and cdh-3::cfp.

Takao Inoue et al. (2001) International C. elegans Meeting "Analysis of genes involved in late patterning of the vulva"

Takao Inoue, Paul W Sternberg

[International C. elegans Meeting, 2001]

Based on EM reconstructions, two and five terminally differentiated cell types are generated by the primary and the secondary vulval lineages, respectively (Sharma-Kishore et al. 1999). We have assembled a panel of gfp , cfp , and yfp markers that label subsets of cell types, and together enable us to distinguish between six of these cell fates. We are using these marker lines to screen for new mutations that affect the patterning of these cell fates, and to analyze previously isolated mutations that affect the secondary VPC lineage execution. The process is complex and likely involve multiple cell signaling events and transcription factors. Previous analysis based on lineage have suggested a role of a Wnt signaling pathway in the P7.p secondary lineage patterning. Mutations in lin-17 (Frizzled homolog) and lin-18 (homolog of Ryk = r elated to receptor-t y rosine k inases; W. Katz and P. W. S.) cause a phenotype interpreted to be the anterior-posterior reversal of the P7.p (secondary) lineage. Preliminary examinations of marker expression patterns in these mutants suggest a more complex defect. We are analyzing the defects in these mutants using additional gfp markers. We are also investigating how lin-18 Ryk is involved in the Wnt signaling pathway. A number of transcription factors are also implicated in late patterning. egl-29, which affects expression of egl-17::gfp (M. Wang and P.W.S.) was found to be allelic with the heterochronic gene lin-29 , encoding a zinc-finger transcription factor. Re-interpretation of previous results and examination of additional gfp markers suggest that lin-29 mutations affect the timing of marker expression. Another gene under investigation is cog-1 , encoding a GTX homeodomain protein (R. Palmer and P.W.S.). A missense allele, cog-1(sy275) causes ectopic expression of egl-17::gfp (M. Wang and P.W.S.) and ceh-2::gfp in the outer cells (vulE) of the primary lineage. A partial deletion allele cog-1(sy607) (generated by D. Sherwood) causes loss of ceh-2::gfp and cdh-3::gfp expression from cells of the secondary lineage. By lineage analysis, sy275 delays or eliminates the terminal division of the inner primary cells (vulF), whereas sy607 eliminates the terminal division of secondary cells P5.ppa and P7.pap (vulC). cog-1 encodes two alternative transcripts, and the differences in the phenotypes of two alleles possibly reflect different functions of the two transcripts.

Takao Inoue et al. (2003) International Worm Meeting "LIN-17, LIN-18 and Wnt ligands control the orientation of the P7.p lineage"

Takao Inoue, Russell Hill, Paul W Sternberg, Rashmi Deshpande

[International Worm Meeting, 2003]

Many animal tissues are polarized with respect to the pattern of cell types and cell morphology, and mechanisms that control tissue orientation is an important issue in developmental biology. We are studying the mechanism by which Wnt, Frizzled and Ryk (receptor tyrosine kinase-related) signaling pathways control the orientation of the P7.p vulval lineage. The vulval precursors P5.p and P7.p execute the secondary lineage in opposite orientations. This difference in lineage orientation manifests itself at the molecular level in opposite patterns of POP-1 (TCF) localization and in the pattern of cell fates produced (assayed by ceh-2::yfp and cdh-3::cfp). In lin-17 and lin-18mutants, the orientation of the P7.p is reversed (P5.p-like), leading to a characteristic bivulva (Biv) phenotype as well as alteration of the expression pattern of molecular markers. We found that lin-17 and lin-18 function in parallel to establish the P7.p orientation. In lin-17 or lin-18 single mutants, we found frequent reversals in the VPC 2-cell stage (P7.p daughters), but less frequent reversals in the VPC 4-cell stage (P7.p granddaughters). In the lin-17; lin-18 double mutant, we found high frequency of reversals in both VPC 2 and 4-cell stages. As the mutual enhancement is observed with probable null alleles of lin-17 and lin-18, these genes appear to act independently of each other in parallel pathways. lin-17 encodes a Frizzled-type Wnt receptor. lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors which may function as Wnt receptors. To determine which Wnt proteins act as ligands, we tested various single, double and triple mutant combinations of the five C. elegans Wnt genes using genetic mutants and RNAi feeding. We found that the mom-2(-) lin-44(-) combination resulted in the Biv phenotype, suggesting that these two genes encode the principal ligands for establishing the orientation of P7.p. cwn-2 and egl-20 had minor effects in some genetic backgrounds. LIN-44 and MOM-2 may signal through LIN-17, LIN-18 or both. To test ligand/receptor specificities, we are testing wnt; receptor mutant combinations. Preliminary results suggest that some pathway specificity difference exist between LIN-44 and MOM-2. In vitro experiments are being carried out to test for receptor/ligand binding.

Takao Inoue et al. (2010) East Asia Worm Meeting "Multivesicular body formation requires OSBP-related proteins and cholesterol"

Hiroyuki Kobuna, Keiko Gengyo-Ando, Takao Inoue, Shohei MItani, Hiroyuki Arai

[East Asia Worm Meeting, 2010]

In eukaryotes, different subcellular organelles have distinct cholesterol concentrations, which is thought to be critical for biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesterol trafficking in cells; however, their in vivo functions and therefore the biological significance of cholesterol in each organelle are not fully understood. Here, by generating deletion mutants of ORPs in C. elegans, we show that ORPs are required for the formation and function of multivesicular bodies (MVBs). In an RNAi enhancer screen using obr quadruple mutants (obr-1; -2; -3; -4), we found that MVB-related genes show strong genetic interactions with the obr genes. In obr quadruple mutants, late endosomes/lysosomes are enlarged and membrane protein degradation is retarded, although endocytosed soluble proteins are normally delivered to lysosomes and degraded. We also found that cholesterol content of late endosomes/lysosomes is significantly reduced in the mutants. In wild-type worms, cholesterol restriction induces the formation of enlarged late endosomes/lysosomes as observed in obr quadruple mutants, and increases embryonic lethality upon knockdown of MVB-related genes. Finally, we show that knockdown of ORP1L, one of the mammalian ORP family members, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings indicate that proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB-mediated membrane protein degradation.

Takao Inoue et al. (2003) International Worm Meeting "Type II PAF-AH, paf-2, is essential for hypodermal organization in C. elegans"

Yuka Suzuki, Hiroyuki Arai, Takao Inoue, Junken Aoki, Asako Sugimoto, Masayuki Yamamoto

[International Worm Meeting, 2003]

PAF-acetylhydrolase (PAF-AH) is an enzyme that inactivates PAF (Platelet-activating factor) by hydrolyzing the acetyl group attached at the sn-2 position of the glycerol backbone. Type II PAF-AH (PAF-AH (II)) is a 40-kDa monomeric enzyme and myristoylated at the N-terminus. PAF is not detected in C. elegans, however, orthologous genes of PAF-AH (II) are conserved in many species, including C. elegans. The physiological function and substrate(s) of this enzyme have not been fully understood so far. In this study, we cloned two PAF-AH (II) genes in C. elegans (paf-1, paf-2) and disrupted these genes to elucidate the biological function. Amino acid sequences of PAF-1 and PAF-2 exhibited 70% identity with each other and 35% identity with mammalian PAF-AH (II)s. Both PAF-1 and PAF-2 conserved a Gly-X-Ser-X-Gly catalytic center and the N-myristoylation sequence, and exhibited similar substrate specificity as mammalian PAF-AH (II)s in vitro. Next we generated deletion mutants of these two enzymes and examined their phenotypes. paf-2(-/-) was lethal during embryogenesis, whereas paf-1(-/-) grew normally. All paf-2(-/-) embryos failed to elongate and the development was arrested at the lima bean stage. Analysis using SEM (scanning electron microscope) and immuno-staining revealed that the organization of the hypodermis in the paf-2(-/-) mutant embryo is severely obstructed. To further analyze the function of paf-2 during elongation process and following postembryonic development, we generated transgenic animals in which heat-shock induces paf-2 RNAi, producing both senses of paf-2 RNAs in vivo. Heat-shocked hsp::paf-2 RNAi animals showed negligible PAF acetylhydrolase activity, indicating that PAF-2 is a major isoform for enzyme activity in C. elegans. These worms exhibited variable abnormal morphology (Vab) at the late embryonal and early larval stages including ectopic protrusions due to epithelial organization defects. These observations demonstrate that PAF-AH (II) is required for hypodermal formation and suggest that particular lipids, the substrates or products of PAF-2, are critically involved in epithelial morphogenesis.

Takao Inoue et al. (2005) International Worm Meeting "Ryk/Wnt and Frizzled/Wnt pathways control patterning of the P7.p vulval lineage"

Paul W Sternberg, Takao Inoue

[International Worm Meeting, 2005]

The Wnt family of secreted signaling proteins controls many aspects of animal development. In many systems, Wnt signals are transduced by Frizzled seven-transmembrane receptors and downstream components. More recently, the Ryk family of receptor tyrosine kinases has emerged as alternative receptors for Wnts. Although the Ryk family is conserved in vertebrates, insects and nematodes, little is known regarding its mode of action, including whether it functions as a coreceptor of Frizzled or in an independent pathway.We are analyzing Wnt signaling in late vulval patterning. During this process, three vulval precursors (P5.p, P6.p and P7.p) divide and differentiate to produce seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, vulF) in a specific spatial pattern. Availability of multiple (&gt;15) gene expression markers makes this phase of vulval development an ideal system in which to analyze molecular mechanisms of organogenesis.We found that mutations in lin-17/Frizzled and lin-18/Ryk control the anterior/posterior order of cell types produced by the P7.p precursor cell. Analysis of mutants and RNAi also indicated that lin-44/Wnt, mom-2/Wnt and cwn-2/Wnt function redundantly in this process. Mutations in lin-17 and lin-18 mutually enhance each other in the double mutant. Also, the pattern of enhancement in receptor; ligand double mutants indicated that lin-17 and lin-18 exhibit distinct ligand preferences. Thus, our results indicate that lin-17/Frizzled and lin-18/Ryk function in parallel Wnt pathways, arguing against the model in which LIN-18/Ryk functions as a coreceptor for LIN-17/Frizzled.We found that a source of the signal that orients the P7.p lineage is the somatic gonad. Laser ablation experiments by Wendy Katz and P. W. S. suggested that the gonad is required for proper patterning of the P7.p lineage. We found that mom-2::gfp is expressed in gonadal cells, including the anchor cell. These cells are positioned anterior to the P7.p lineage, and thus can provide the directional cue that determines the anterior/posterior orientation. We are continuing to investigate two aspects of this system. One is the mechanism of signaling by Ryk and its relationship to other Wnt signaling pathways. Second, we are interested in the mechanism by which Wnt signaling establishes the cell fate pattern. To this end, we are searching for additional components of this pathway using genetic screens.

Takao Inoue et al. (2002) Japanese Worm Meeting "paf-2 RNAi induces abnormal hypodermal organization during elongation process in C. elegans"

Hiroyuki Arai, Junken Aoki, Masafumi Tsujimoto, Masayuki Yamamoto, Asako Sugimoto, Yuka Suzuki, Takao Inoue

[Japanese Worm Meeting, 2002]

We previously discovered a unique N-myristoylated phospholipase [called type II platelet-activating factor-acetylhydrolase: PAF-AH (II)], and showed that paf-2, an orthologue of PAF-AH (II), is essential for hypodermal organization during embryogenesis. To further analyze the function of paf-2 during the elongation process and following postembryonic development, we generated transgenic animals in which heat-shock induces paf-2 RNAi, producing both senses of paf-2 RNAs in vivo. Heat-shocked hsp::paf-2 RNAi animals showed negligible PAF acetylhydrolase activity, indicating that PAF-2 is a major isoform for enzyme activity in C. elegans. In about 11% of the heat-shocked hsp::paf-2 RNAi transgenic animals, development was arrested during embryogenesis and in about 5% of these animals it was arrested at the early larval stages. The arrested hsp::paf-2 RNAi animals showed variable abnormal morphology (Vab) due to defects of hypodermal integration during the elongation process. These observations confirm PAF-AH (II) is required for hypodermal formation and suggest that particular lipids, the substrates or products of paf-2, are critically involved in epithelial morphogenesis. Identification of such lipid molecule(s) is in progress using hsp::paf-2 RNAi transgenic animals in our laboratory.