H Sawa et al. (1999) International C. elegans Meeting "Mutations affecting asymmetric T cell division"

H Sawa, H Okano, H Kouike

[International C. elegans Meeting, 1999]

Asymmetric cell division is a fundamental mechanism for the production of cellular diversity during development. In C. elegans , the asymmetry of a number of cell divisions is affected by mutations in lin-17 or lin-44 . lin-17 encodes a receptor molecule that belongs to the Frizzled family. lin-44 encodes a signaling protein that belongs to the Wnt family. We have proposed that the polarities of cells undergoing asymmetric divisions are regulated by the LIN-44 signal which act through the LIN-17 receptor. How the signal is transduced from the LIN-17 receptor and how the consequent cell polarities are established are unknown. The polarities of the T cell division is regulated by lin-17 and lin-44 . In wild type, anterior daughter of the T cell produces hypodermal cells and posterior one makes neural cells including phasmid socket cells. In lin-17 mutants, both daughters produce hypodermal cells. To identify genes involved in asymmetric cell divisions, we are screening for mutants that lack phasmid socket cells (the phenotype is called Psa for p hasmid s ocket cell a bsent). We have so far identified more than 10 mutants with Psa phenotype which are not lin-17 , lin-44 nor egl-27 . Lineage analyses have shown that the T cell division can be symmetric in at least 5 of the mutants ( psa-1 through psa-5 ). psa-1 and psa-2 are temperature-sensitive maternal effect embryonic lethal. psa-2 specifically affects the elongation of embryo. Temperature shift experiments have shown that psa-1 is required for the T cell polarity before the T cell division. Molecular analyses of these mutants are underway. We have also found that a lit-1 mutation can disrupt the T cell polarity. lit-1 is involved in asymmetric cell divisions of the EMS blastmere that also requires mom-2/wnt and mom-5/frizzled . The result suggests that a signaling pathway similar to the one that controls the EMS polarity regulates the T cell polarity.

Sawa H et al. (1997) International C. elegans Meeting "IDENTIFICATION OF MUTATIONS AFFECTING THE ASYMMETRY OF B AND T CELL DIVISIONS"

Sawa H, Okano H, Horvitz HR

[International C. elegans Meeting, 1997]

Asymmetric cell division is a fundamental mechanism for the production of cellular diversity during development. In C. elegans, the asymmetry of a number of cell divisions is affected by mutations in lin-17 or lin-44. lin-17 encodes a receptor molecule that belongs to the Frizzled family (1). lin-44 encodes a signaling protein that belongs to the Wnt family (2). Frizzled protein appeares to function as a receptor of Wingless, a Drosophila Wnt protein (3). We have postulated that LIN-17 is likely to act as a receptor for LIN-44 to regulate the polarities of mother cells that undergo asymmetric divisions (1). How the signal is transduced from the lin-17 receptor and how the consequent cell polarities are established are unknown. To identify additional genes required for asymmetric cell divisions, we screened for mutants with a phenotype similar to that caused by lin-17 mutations. lin-17 mutant males have abnormal tails because of defects in many cell divisions, including those of the B and T cells. The phasmids of lin-17 hermaphrodites are defective, because the socket cells derived from the T cells are not produced. We performed an F1 clonal screen for F2 males with abnormal tails and for sibling F2 hermaphrodites with abnormal phasmids. We identified three new lin-17 mutants and 39 other mutants defective in both the male tail and the phasmids. We are analyzing the asymmetry of the B and T cell divisions in these mutants. In some of the mutants, we observed the absence of the phasmid socket cells. Genetic and molecular analyses of these mutants will help define components required for asymmetric cell divisions. (1) Sawa et al. Genes & Dev. 10, 2189-2197 (1996). (2) Herman et al. Cell 83, 101-110 (1995). (3) Bhanot et al. Nature 382, 225-230 (1996).

Sawa H et al. (1995) International C. elegans Meeting "lin-17 ENCODES A PROTEIN SIMILAR TO THE PRODUCT OF THE Drosophila TISSUE-POLARITY GENE Frizzled"

Horvitz HR, Sawa H

[International C. elegans Meeting, 1995]

Asymmetric cell divisions are involved in many aspects of development. lin-17 mutations disrupt the asymmetry of a number of cell divisions in C. elegans, suggesting that lin-17 is required to establish these asymmetries. We genetically localized lin-17 0.2 map units right of the RFLP nP58, which was identified by Leslie Lobel. Using cosmids and phage clones in this region, we searched for RFLPs in lin-17 mutant strains. A phage clone isolated by Kerry Kornfeld can detect RFLPs in rh41 and sy277. We identified cDNAs that detect both RFLPs. Because genomic clones containing the whole cDNA sequences are not available, we ligated a fragment of the genomic phage clone to one of these cDNAs and to the 3' region from the unc-54 gene. This construct rescued the Lin-17 phenotype. We determined the sequence of this cDNA. The predicted product of a 1.7 kb ORF is 28% identical to the product of the tissue-polarity gene frizzled of Drosophila. As in Lin-17, the polarities of a number of cells are disrupted in frizzled animals, resulting in the disordered orientation of hair, bristles, ommatidia and tarsi. Because lin-17 and frizzled encode proteins each with seven putative transmembrane domains, lin-17 is likely to be involved in cell-cell interactions required for the asymmetry of certain cell divisions. The similarity of lin-17 to frizzled raises the possibility that other genes related to tissue-polarity genes are involved in asymmetric cell divisions. The genome sequencing project identified an another frizzled-like cDNA that is different from lin-17 cDNAs. Also, a cDNA related to the Drosophila tissue-polarity dishevelled gene has been identified by the cDNA project led by Yuji Kohara. We are currently analyzing these cDNAs.

Mariko Sawa et al. (2004) East Asia Worm Meeting "Function of C. elegans WVE-1 and Abi in embryogenesis."

Shiro Suetsugu, Tadaomi Takenawa, Mariko Sawa

[East Asia Worm Meeting, 2004]

Cell migration is essential in morphogenesis in multi-cellar animals. In the cell culture assay and the reconstitution system with purified protein, mammalian WASP family protein, WASP, N-WASP and WAVE1-3, are revealed to be the important factors to generate cell motility, by activating Arp2/3 complex that functions as actin nucleation core. We previously reported that the sole C. elegans homologue WSP-1 for mammalian WASP and N-WASP and Arp2/3 complex function in ventral enclosure during embryogenesis. Although Arp2/3 complex is essential in cell migration, distribution of WSP-1 is partial (ref.1), indicating another pathway to Arp2/3 complex in ventral enclosure. Here, we report WVE-1, the sole homologue for mammalian WAVE1-3 in C. elegans , functions in cell migration during ventral enclosure. We performed feeding RNAi for wve-1 with rrf-3 at 15degC. It showed high frequency of embryonic lethality with the defect in hypodermal cell migration in ventral enclosure. Immunostaining with anti-WVE-1 antibody showed that WVE-1 localizes at the leading edge of hypodermal cells in ventral enclosure. However expression of AJM-1 and LIN-26 was not affected by wve-1(RNAi) , showing the normal differentiation of the hypodermal cells. In mammalian system, it is reported that WAVE2 forms a complex with Sra (specifically Rac1-associated protein), Nap (Nck-associated protein), and Abi (Abl-interacting protein) to prevent degradation (ref.2). Among those homologues in C. elegans , Sra( gex-2 ) and Nap( gex-3 ) are already shown to function in ventral enclosure(ref.3). Then, we performed RNAi for C. elegans Abi and found it also functions in ventral enclosure. Although we did not detect the decreased amount of WVE-1 in Abi(RNAi) , degradation of WVE-1 is promoted in the absence of GEX-2 or GEX-3. This indicates the physiological interaction among WVE-1, GEX-2 and GEX-3. With these results, we propose the regulatory pathway by WVE-1 in concert with GEX-2, GEX-3 and Abi to direct Arp2/3 complex in ventral enclosure. Reference 1. Sawa et al., 2003, Journal of Cell Science (116) 1505-1518. 2. Eden et al., 2002, Nature (418) 790-793. 3. Soto et al., 2002, Genes & Development (16) 620-632.

Downs WD et al. (1997) International C. elegans Meeting "THE GENE MOM-1 IMPLICATES A WNT/WINGLESS PATHWAY IN ESTABLISHING CELL POLARITY AND ASYMMETRIC LINEAGES IN C. ELEGANS"

Cha Y-H, Mello CC, Priess JR, Downs WD

[International C. elegans Meeting, 1997]

During the development of C. elegans, the majority of cell divisions are asymmetric, resulting in daughter cells that will assume different fates. Our work indicates that the gene mom-1 (MOre Mesoderm) is necessary to induce polarity in a cell that will lead to an asymmetry in its lineage. Two of these mom-1 dependent asymmetries rely upon cell signaling in C. elegans: 1) induction of the intestine in the 4-cell stage embryo (Goldstein, 1992) and 2) patterning of the vulva during postembryonic development (Katz & Sternberg, 1990). Cell signaling events involving glp-1/lin-12 (Notch-related), and lin-3 (EGF) mediated pathways remain operational in mom-1 mutants. However, the vulva defects observed in mom-1 mutants are also observed in loss of function alleles of lin-17, a gene previously implicated in wnt signaling in C. elegans and homologous to the putative Wingless receptor of Drosophila (Sawa & Horvitz, 1996). We have cloned the mom-1 gene and found that it is homologous to porcupine, a gene of the Drosophila wingless pathway. Based on these findings we now believe that mom-1 is acting through a wnt/wingless cell-signaling pathway.

Kenji Sugioka et al. (2006) East Asia C. elegans Meeting "Roles of microtubules and their regulators in asymmetric cell divisions regulated by Wnt signaling."

Kenji Sugioka, Hitoshi Sawa

[East Asia C. elegans Meeting, 2006]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many blast cells are regulated by Wnt signaling. Previously, we showed that nuclear localization of WRM-1/β-catenin::GFP was asymmetric after the T cell division; stronger in the posterior nucleus (T.p) than the anterior one (T.a) (Takeshita and Sawa 2005). The nuclear asymmetry is regulated by APR-1/APC that localized asymmetrically on the cortex (abstract by Mizumoto and Sawa). However, it is not known how cortical APR-1 regulates WRM-1 localization. Because APC binds microtubules (Munemitsu et al. 1994), one possibility is that APR-1 regulates WRM-1 through microtubules. This hypothesis was also supported by the observation that the T cell divisions are defective in mutants of rmd-1 that regulates microtubule dynamics in embryos (Oishi et. al. IWM 2005). To investigate involvements of microtubules, we analyzed mutants of 15 genes that regulate microtubules. Among them, 4 mutants zen-4(or153), cyk-4(t1689), air-2(ok298) and lin-5(ev571) have abnormality either in cell divisions themselves or asymmetry of the T cell lineage. One of these, zen-4(or153) showed symmetric nuclear localization of WRM-1::GFP at telophase. In embryos, ZEN-4/kinesin-like protein and CYK-4/RhoGAP form a complex named central spindlin which is required for central spindle assembly and cytokinesis (Mishima et al. 2002). In contrast to zen-4 mutants, cyk-4(t1689) mutants were not defective in WRM-1::GFP localization, even though they showed stronger cytokinesis defects in the T cell than zen-4 mutants. These results suggest that ZEN-4 may regulate the asymmetric nuclear WRM-1 localization in a CYK-4-independent manner. To further analyze involvements of microtubules in the asymmetric division, we will examine effects of air-2, lin-5 and rmd-1 mutations and microtubule destabilizing drugs on the nuclear WRM-1 localization.

Kenji Sugioka et al. (2007) International Worm Meeting "Roles of microtubules and their regulators in asymmetric cell divisions regulated by Wnt signaling."

Hitoshi Sawa, Kenji Sugioka

[International Worm Meeting, 2007]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many blast cells are regulated by Wnt signaling. Previously, we showed that nuclear localization of WRM-1/<font face=symbol>b</font>-catenin::GFP was asymmetric after the T cell division; stronger in the posterior nucleus (T.p) than the anterior one (T.a) (Takeshita and Sawa 2005). The nuclear asymmetry is regulated by APR-1/APC that localized asymmetrically on the cortex (Mizumoto and Sawa 2007). However, it is not known how cortical APR-1 regulates WRM-1 localization. Because APC binds microtubules (Munemitsu et al. 1994), one possibility is that APR-1 regulates WRM-1 through microtubules. This hypothesis was also supported by the observation that the T cell divisions are defective in mutants of rmd-1 that regulates microtubule dynamics in embryos (Oishi et. al. IWM 2005). To investigate involvements of microtubules, we analyzed mutants of 15 genes that regulate microtubules. Among them, 4 mutants zen-4(or153), cyk-4(t1689), air-2(ok298) and lin-5(ev571) have abnormality either in cell divisions themselves or asymmetry of the T cell lineage. And only zen-4(or153) showed symmetric nuclear localization of WRM-1::GFP at telophase. In embryos, ZEN-4/kinesin-like protein and CYK-4/RhoGAP form a complex named central spindlin which is required for central spindle assembly and cytokinesis (Mishima et al. 2002). In contrast to zen-4 mutants, cyk-4(t1689) mutants were not defective in WRM-1::GFP localization, even though they showed stronger cytokinesis defects in the T cell than zen-4 mutants. These results suggest that ZEN-4 may regulate the asymmetric nuclear WRM-1 localization in a CYK-4-independent manner. To further analyze involvements of microtubules in the asymmetric division, we will examine effects of rmd-1 mutations and microtubule destabilizing drugs on the nuclear WRM-1 localization.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.