Takashi Fujii et al. (2001) International C. elegans Meeting "Regulation of the ray position by two Plexin/Semaphorin systems"

Fumi Nakao, Fumikazu Suto, Takashi Fujii, Shin Takagi, Zhicen Liu, Hajime Fujisawa

[International C. elegans Meeting, 2001]

Plexin is a family of transmembrane protein conserved across a wide range of animal species. Recent studies on Drosophilae and mice showed that plexins act as receptors for semaphorins, a group of proteins originally identified as a chemorepulsive factor for growing axons. C. elegans has two genes for plexins, plx-1 and plx-2 , (pka. cep-2 and cep-1 , respectively); two genes for transmembrane semaphorins, Ce-sema-1a , Ce-sema-1b ; and one gene for a secreted semaphorin, mab-20/Ce-sema-2a . We have generated deletion mutations for plx-1 and plx-2 in order to examine their function in vivo. Here we report the ray morphogenesis in these mutants.Putative null mutants of the plx-1 gene exhibited anterior displacement of Ray1 in the adult male tail. The similar ray phenotype was observed by suppression of Ce-sema-1a and/or Ce-sema-1b by RNAi. In plx-1 mutants, precursors for Ray1 were often mispositioned, suggesting that plx-1 regulates the arrangement of the ray precursors. Showing no defects in other rays, the phenotype of plx-1 mutants is distinct from that of mab-20 mutants reported previously. plx-2 (nc7) is presumed to cause a deletion within the extracellular region of PLX-2. While plx-2 (nc7) animals exhibited no apparent defects in the male tail by itself, nc7 enhanced the ray fusion traits of mab-20(bx-24) , an allele of medium strength, suggesting that these genes interact. By using cultured cells, we also showed that Ce-Sema-1a binds to PLX-1, but not to PLX-2, while Ce-Sema-2a binds to PLX-2 but not to PLX-1. The results indicate that PLX-1 interacts with the transmembrane semaphorins and PLX-2 interacts with the secreted semaphorin. Although both plexins are required for the proper positioning of rays, they appear to function rather independently.

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.

Zhicen Liu et al. (2002) Japanese Worm Meeting "PLX-1 plays an essential role in Vulva Precursor Cell positioning"

Shin Takagi, Hajime Fujisawa, Zhicen Liu, Takashi Fujii, Yuji KOHARA, Rie Kurokawa

[Japanese Worm Meeting, 2002]

Akira Nukazuka et al. (2002) Japanese Worm Meeting "Roles of C. elegans Rac proteins in the epidermal morphogenesis"

Mie Chikuma, Rie Kurokawa, Akira Nukazuka, Shin Takagi, Hajime Fujisawa, Takashi Fujii, Liu Zhicen

[Japanese Worm Meeting, 2002]

Plexins are the receptors for semaphorins, which are known to function as guidance cues during the development of vertebrate nervous system. We revealed that mutations in plx-1, a gene encoding one of two C. elegans plexins, caused anterior displacement of ray 1, and elucidated that this defect resulted from morphological defects in ray epidermal precursors. It was shown that growth cone collapse by semaphorins requires activities of small GTPase Rac in vertebrates. C. elegans genome contains three Rac genes, ced-10, mig-2 and rac-2. Though they are known to function in cell migration, axonal guidance and phagocytosis, their roles in the C. elegans epidermal morphogenesis are not clear. In this work, in an attempt to search for downstream factors of PLX-1, we examined whether Rac genes are involved in C. elegans epidermal morphogenesis in the male tail. In the adult stage, loss-of-function ced-10(n1993) and mig-2(mu28) alleles caused anterior displacement of ray 1, a defect similar to that of plx-1 mutants, and a constitutively active mig-2(gm38) allele exhibited anterior displacement of ray 2 and ray 3, as well as ray 1. Mutations in Rac-activators, CrkII (ced-2), DOCK180 (ced-5) and a Trio-like guanine exchange factor (unc-73), as well as a mutation in a downstream factor, Cofilin (unc-60), also affected ray 1 positions, suggesting that they may function in the same pathway as Racs in ray development. We also found that plx-1; mig-2(mu28) and plx-1 ced-10(n1993) double mutants exhibited stronger phenotypes as to ray 1 position than each of the single mutant did. In the L4 stage, the epidermal precursor clusters for rays were anteriorly displaced in all of these mutants, and ced-10::gfp::ced-10 and mig-2::gfp reporter transgenes were expressed in these epidermal precursor cells. Therefore, a balanced activity of Rac is necessary for the proper epidermal morphogenesis, and CED-10 and MIG-2, together with their multiple regulatory factors, appear to function both redundantly and differentially in this system.

Fujita, Mayu et al. (2017) International Worm Meeting "Isolation of genes required for cold acclimation."

Toyoda, Atsushi, Sakai, Shiori, Okahata, Misaki, Ohta, Akane, Fujita, Mayu, Kuhara, Atsushi, Minakuchi, Yohei

[International Worm Meeting, 2017]

Animals can sense and memorize environmental temperature to survive and proliferate in variety of temperature condition. In this study, we are studying about molecular mechanisms for temperature acclimation and its memory by using cold acclimation. Wild-type animals can not survive at 2 degree after cultivation at 25 degree, whereas they can survive at 2 degree after cultivation at 15 degree. After 25 degree-cultivated animals were transferred to and stayed at 15 degree for 3 hours, these animals can survive at 2 degree, as previously reported (Ohta, Ujisawa et al., Nature commun, 2014). This phenomenon is regulated by CREB that is a transcription factor in mammalian memory, suggesting that gene expression is involved in the regulation of cold acclimation or temperature memory (Fujii et al. this meeting). (1) We quantified gene expression-changes depending on temperature shift, by using RNA sequencing analysis with next generation sequencer. We collected mRNA of the animals cultivated at various temperature conditions. By the RNA sequencing analysis, expression levels of about 950 genes were strongly changed, in which mutants of about 520 genes were available in the stock centers. So far, abnormal cold acclimation was observed in hacd-1 mutant defective in HADH, 3-hydroxyacyl-CoenzymeA dehydrogenase. HADH is involved in beta-oxidation regulating fatty acid metabolism in a mitochondrial matrix. GFP reporter analysis indicated that hacd-1 was expressed in neurons and intestine. We found that abnormal cold acclimation of hacd-1 mutant was rescued by the expression of hacd-1cDNA in neurons and intestine. We are planning to determine which neuron is required for hacd-1-dependent cold acclimation. (2) We also used DNA microarray analysis on temperature shift to identify more genes involved in cold acclimation. We found that RB2549 sms-3(ok3540) showed abnormal cold acclimation. When 25 degree-cultivated animals transferred to 15 degree and stayed for 3 hours, RB2549 mutant can survive at 2 degree, while wild-type can not survive at 2 degree. However, another null mutant sms-3(tm4022) did not show abnormal phenotype. Therefore, we hypothesized that background mutation(s) excepting sms-3 mutation in RB2575 causes abnormal cold acclimation. To identify the responsible gene, we decoded whole genome DNA sequences by using next generation sequencer (NGS). So far, the candidate responsible genes were narrowed down to 28 genes. We are isolating recombinant strains, and are introducing SNP analysis to determine the responsible gene for abnormal cold acclimation of RB2575 strain.

Yoshiya Nakano et al. (2001) International C. elegans Meeting "Mutants with larger body size and elongated life-span in C. elegans"

Yoshiya Nakano, Takashi Misumi, Yasumi Ohshima

[International C. elegans Meeting, 2001]

In order to analyze the mechanisms for body size determination, at least ten larger body size mutants were isolated after EMS mutagenesis. Four of them had up to twice as much volume as that of the wild type in adults.These four mutants have similar phenotypes which are larger body size, weak egg laying defect, dark intestine, reduced brood size, abnormal male tail structure, and elongated life span. Complementation tests showed that the four mutations ( ks16 , ks 60 , ks61 and ks62 ) are allelic with one another, and the corresponding gene encodes a cyclic GMP-dependent protein kinase (see abstract by Takashi Hirose and Yasumi Ohshima). To reveal biological roles of this gene in the body size determination and life span, we have analysed mutant phenotypes mainly on body size and life span. As a basis to study the body size, we developed a system to measure body length and diameters, and to estimate volume, of a worm by an automatic image analysis. Using this, we obtained growth curves of the four mutants. They showed that large mutants continue to grow in adults to be approximately twice as big four days after becoming adults. Measurements of the life span of four large body size mutants and wild type N2 showed that, all the four large size mutants have elongated lifespan. Especially, in a condition without FUDR, N2 had 14.28 +/- 0.56 days mean life span and 29 days maximam life span (n=71). On the other hand, ks61 mutant had 20.4 +/- 0.97 days mean life span and 43 days maximam life span (n=75). However, in a condition with FUDR which inhibits DNA synthesis, mean lifespan and maximam lifespan of this mutant were shorter than those without FUDR. Also, body length and volume were smaller than the animals grown without FUDR. These results suggest that long life span and large body size of the mutants requrire DNA synthesis. How much body size do the aging mutants have? We measured the body size of daf-16 mutant and daf-2 mutants. daf-16 lives shorter than wild-type and daf-2 lives longer. Although both of them had the same body length as wild-type N2, daf-2 was thinner and less in volume than wild type, and daf-16 was wider and more in body volume. This relation between body size and life span is oposite to the case of our large mutants. Furthermore, a double mutant of daf-16 and ks61 was much larger than each of the single mutants. These results suggest that body size penotypes of large mutants are independent of daf-16 dependent size determination. Also, role of life span regulation of this gene may be independent of IGF signal dependent life span regulation.