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. (2003) International Worm Meeting "PLX-1 mediates a cell contact-dependent stop signal in elongating vulval precursor cells"

Hajime Fujisawa, Zhicen Liu, Rie Kurokawa, Shin Takagi

[International Worm Meeting, 2003]

The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos including the axonal guidance. We have generated and characterized putative null mutants of the plx-1 gene encoding C. elegans plexinA. PLX-1 interacts with transmembrane semaphorin Ce-Sema1a and 1b, and plx-1 mutants exhibited morphological defects in several organs of epidermal origin including the vulva. plx-1 adult hermaphrodites sometimes had a deformed vulva or extra vulva-like structures. A plx-1::gfp transgene was expressed in the vulval primordial cells throughout the vulval development. The epidermal cells of the vulval primordium visualized with ajm-1::gfp were aberrantly arranged in plx-1 mutants: the rotation-symmetric configuration of the primordum was often disrupted, and cells were sometimes not in contact with each other. Some cells failed to participate in the formation of the main vulva and formed an extra vulva-like structure. A mutation in smp-1, the gene encoding Ce-Sema1a, also caused the similar defects. At the early stage of the vulval development, the separate vulval precursor cells (VPCs) elongate aniterior-posteriorly and form a continuous single row of cells along the ventral midline in wild type animals. In plx-1 and smp-1 mutants, VPCs often made contact with each other along their lateral sides rather than at their tips, and were sometimes separated by hyp7. Continuous observation of single plx-1 animals revealed that some VPCs failed to stop elongation after making contact with each other, leading to the overlaps between cells. This observation suggests that PLX-1 and SMP-1 mediates a cell contact-dependent stop signal in elongating vulval precursor cells. It is known that cell fate specifications of VPCs are critically dependent on the positions. In plx-1 mutants, the arrangement of vulval precursor cells was disrupted sometimes so severely that the proxial-distal order of the cells was sometimes reversed. Analyses using cell-type specific markers revealed that cell fate specifications were sometime affected in plx-1 mutants. The frequency of defects in the cell fate specification, however, did not appear sufficiently high to fully account for the arrangements defects in later stages, suggesting that PLX-1 may be also involved in the regulation of arrangements and migrations of cells in the later phases of vulva morphogenesis.

Akira Nukazuka et al. (2003) International Worm Meeting "Analysis of plexin signaling events in C. elegans."

Akira Nukazuka, Shin Takagi, Hajime Fujisawa, Mie Chikuma

[International Worm Meeting, 2003]

Semaphorins are a family of proteins, which are known to function as axonal repulsive cues in vertebrates. Plexins, a family of transmembrane proteins, have been revealed to bind to semaphorins and transduce their signals into cytoskeletal rearrangement. In vertebrate cultured cells, it is shown that growth cone collapse induced by class 3 semaphorins requires an active form of small GTPase Rac protein and a phosphorylated form of actin depolymerizing factor, cofilin. In this study, we have adopted genetic analysis in C. elegans in order to elucidate the mechanism of plexin signaling. In C. elegans, plexin signals regulate the epidermal morphogenesis in the male tails; mutations in one of two plexin genes, plx-1, cause anterior displacement of ray 1, and this defect results from aberrant arrangement of the epidermal ray precursors. Here we found that loss-of-function (LF) mutations in Rac genes (mig-2, ced-10) cause ray 1 anterior displacement that is almost indistinguishable from that plx-1 mutants display, and that both mig-2 and ced-10 mutations enhance the phenotype of plx-1. We also found that LF mutations in a cofilin gene, unc-60, also cause ray 1 anterior displacement and this mutation enhances the phenotype of mig-2, but not that of plx-1 or ced-10. In addition to the analyses of known mutations, we have been searching for mutations that suppress the ray 1 phenotype of plx-1 mutants, in an attempt to identify downstream factors that genetically interact with plx-1 in a competitive or in an antagonistic fashion. For this purpose, we mutagenized plx-1 mutants with EMS and have obtained totally five suppressor mutations, which we tentatively named mutation a, b, c, e and f. Of these five mutations, mutation e and f suppress the ray 1 phenotype of mig-2 and ced-10 as well as plx-1, but not that of unc-60. Therefore, our results indicate that UNC-60 functions in the same pathway as PLX-1 and CED-10, and that the products of gene eand f may act as downstream factors of PLX-1, MIG-2 and CED-10, and as regulatory factors for UNC-60.

Akira Nukazuka et al. (2004) East Asia Worm Meeting "A mutation in gcn-1, a gene coding for a regulator of translation initiation, suppresses the ray defect of plx-1 mutants"

Akira Nukazuka, Shin Takagi, Mie Chikuma, Hajime Fujisawa

[East Asia Worm Meeting, 2004]

Semaphorins, commonly known as repulsive axonal guidance cues in vertebrates and insects, regulate epidermal morphogenesis in Caenorhabditis elegans . Plexins, constituting a family of transmembrane proteins, function as receptors for semaphorins and transduce their signals downstream, though little has been understood about the semaphorin-plexin signaling events. In C. elegans , loss-of-function semaphorin ( smp-1 , smp-2 ) and plexin ( plx-1 ) mutants exhibit anterior displacement of ray 1 in the male tail, which is presumably the consequence of an aberrant arrangement of the epidermal ray precursors. To gain insight into the semaphorin-plexin signaling, we performed a genetic screen for mutations that suppress the ray 1 phenotype of plx-1 mutants. A mutation induced by EMS, nc40 , strikingly suppressed the phenotype of plx-1 , as well as that of smp-1 and smp-2 . SNP mapping, a rescuing experiment, RNAi and a sequence analysis confirmed that nc40 was a loss-of-function allele of a GCN1 homolog gene ( gcn-1 ). GCN1, which is highly conserved among eukaryotes, functions as a negative regulator for eukaryotic translation initiation factor 2 alpha (eIF2alpha) by participating in its phosphorylation. A loss-of-function mutation in PEK/PERK ( pek-1 ), which would normally phosphorylate and inactivate eIF2alpha independently of GCN1 cascade, also suppressed the phenotype of plx-1 , smp-1 and smp-2 mutants. Western blot analysis revealed that the level of eIF2alpha phosphorylation was reduced in gcn-1 and pek-1 mutants, indicating that gcn-1 and pek-1 mutations increase the activity of eIF2alpha and thus the translation initiation is up-regulated. In addition, inactivation of eIF2alpha by RNAi caused ray 1 anterior displacement, the same defect as can be seen in plx-1 , smp-1 and smp-2 mutants. These results are consistent with the model that the semaphorin-plexin signaling stimulates the translation initiation. Possible mechanism of how translation initiation would regulate epidermal morphogenesis will also be discussed.

Shin Takagi et al. (2004) East Asia Worm Meeting "C. elegans PlexinA PLX-1 mediates a cell contact-dependent stop signal in vulval precursor cells"

Zhicen Liu, Akira Nukazuka, Hajime Fujisawa, Shin Takagi

[East Asia Worm Meeting, 2004]

PLX-1 is a PlexinA transmembrane protein in C. elegans, and the transmembrane-type semaphorin, SMP-1, is a ligand for PLX-1. The SMP-1/PLX-1 system has been shown to be necessary for proper epidermal morphogenesis in the male tail and seam cells. Here we show that PLX-1 also regulates vulval morphogenesis. In plx-1 and smp-1 mutants, hermaphrodites sometimes exhibit a protruding vulva or multiple vulva-like protrusions. Throughout the vulval development of plx-1 mutants, the arrangement of vulval primordial cells is often disrupted: the configuration of the vulval primordium is not rotation-symmetrical and primordial cells form separate cell clusters. In the initial step of vulval morphogenesis, the vulval precursor cells (VPCs) are generated normally but are subsequently arranged abnormally in plx-1 mutants. Continuous observation revealed that plx-1 VPC fails to terminate longitudinal extension after making contact with neighbor VPCs. plx-1::egfp and smp-1::egfp transgenes are both expressed in all vulval primordial cells, including VPCs, throughout vulval development. We propose that the SMP-1/PLX-1 system is responsible for a cell contact-mediated stop signal for VPCs extension. Analyses using cell-fate-specific markers showed that the arrangement defects of VPCs sometimes lead to aberrant cell-fate specification and abnormal cell lineages in plx-1 mutants. The frequency of cell specification defects, however, is relatively low, suggesting that plx-1 may also directly regulate vulval morphogenesis in later stages.

Akira Nukazuka et al. (2007) International Worm Meeting "Semaphorin - Plexin signal controls epidermal ray morphogenesis by stimulating mRNA translation via eIF2a in C. elegans."

Hajime Fujisawa, Mie Chikuma, Shin Takagi, Yoichi Oda, Toshifumi Inada, Akira Nukazuka

[International Worm Meeting, 2007]

Semaphorins comprising a protein family in animals regulate diverse developmental events, and plexins serve as semaphorin receptors to transduce the signals into cytoskeletal reorganization. Genetic analysis has revealed that the semaphorin/plexin system regulates epidermal morphogenesis in C. elegans: in semaphorin (smp) and plexin (plx-1) mutants, epidermal cells in the larval male tails are aberrantly arranged, which results in an ectopic positioning of sensory ray 1 in the adult stage. We here report on in vivo evidence that mRNA translation is stimulated by SMP - PLX-1 signal via reduced phosphorylation of a translational regulator, eukaryotic initiation factor 2<font face=symbol>a</font> (eIF2<font face=symbol>a</font>). Higher phosphorylation of eIF2<font face=symbol>a</font>, which was observed in smp / plx-1 mutants, reduced to the normal level by restoring the SMP - PLX-1 signal, implying that this signal is necessary to lower the eIF2<font face=symbol>a</font> phosphorylation. Overexpression of a phosphomimetic eIF2<font face=symbol>a</font>, as well as inactivation of translation machineries (eIF2<font face=symbol>a</font>, eIF2<font face=symbol>b</font>, eIF4G), produced the ray phenotype comparable to smp / plx-1 mutants, indicating that downregulated protein synthesis is attributed to the ray defect in mutants. On the other hand, lowered eIF2<font face=symbol>a</font> phosphorylation caused by the loss-of-function mutations in GCN1 and PEK/PERK, both of which normally inhibit mRNA translation by participating in the eIF2<font face=symbol>a</font> phosphorylation, suppressed the ray phenotype in smp / plx-1 mutants, suggesting that enhanced protein synthesis is sufficient for SMP - PLX-1 signaling. Thus, these data demonstrate an essential role for translation stimulation mainly via reduced eIF2<font face=symbol>a</font> phosphorylation in SMP-induced proper epidermal ray morphogenesis. In our current research, we aim to identify target protein(s) that is synthesized in response to the SMP - PLX-1 signal. In this meeting, we will also make a progress report on this issue and further discuss the mechanism of cytoskeletal reorganization during the semaphorin/plexin-mediated cellular morphogenesis.

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.

Akira Nukazuka et al. (2006) East Asia C. elegans Meeting "Semaphorin - Plexin signal controls epidermal ray morphogenesis by stimulating mRNA translation via eIF2α in C. elegans"

Toshifumi Inada, Shin Takagi, Yoichi Oda, Mie Chikuma, Akira Nukazuka, Hajime Fujisawa

[East Asia C. elegans Meeting, 2006]

Semaphorins, which comprise a protein family in animal kingdom, regulate diverse developmental events including axonal guidance. Transmembrane protein plexins serve as receptors for semaphorins and transduce their signals into cytoskeletal reorganization. Genetic analysis has revealed that the semaphorin/plexin system regulates epidermal morphogenesis in C. elegans: in semaphorin (SMPs) and plexin (PLX-1) mutants, epidermal cells in the larval male tails are aberrantly arranged, which results in an ectopic positioning of sensory ray 1 in the adult stage. We here report on necessity and sufficiency of translation stimulation for SMPs - PLX-1 signaling unexpectedly by reduced phosphorylation of a master translational regulator, eukaryotic initiation factor 2&alpha; (eIF2&alpha;). In SMPs / PLX-1 mutants, higher phosphorylation of eIF2&alpha; was observed, implying downregulated global protein synthesis. Overexpression of a phosphomimetic form of eIF2&alpha; phenocopied SMPs / PLX-1 mutants, indicating that the elevated eIF2&alpha; phosphorylation is attributed to the ray defect in mutants. On the other hand, lowered eIF2&alpha; phosphorylation caused by the loss-of-function mutations in GCN1 and PEK/PERK, both of which normally inhibit mRNA translation by participating in the eIF2&alpha; phosphorylation, suppressed the ray phenotype in SMPs / PLX-1 mutants, raising a possibility that enhanced protein synthesis is sufficient for SMPs - PLX-1 signaling. Thus, these data indicate an essential role for translation stimulation via reduced eIF2&alpha; phosphorylation in SMPs-induced proper epidermal ray morphogenesis. In our current research, we aim to identify target protein(s) that is synthesized in response to the SMPs - PLX-1 signal. In this meeting, we will also make a progress report on this issue and further discuss the mechanism of cytoskeletal reorganization during the semaphorin/plexin-mediated cellular morphogenesis.

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.