β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions
2008; Springer Nature; Volume: 27; Issue: 12 Linguagem: Inglês
10.1038/emboj.2008.102
ISSN1460-2075
AutoresTakahiro Kanamori, Takao Inoue, Taro Sakamoto, Keiko Gengyo‐Ando, Masafumi Tsujimoto, Shohei Mitani, Hitoshi Sawa, Junken Aoki, Hiroyuki Arai,
Tópico(s)Plant Molecular Biology Research
ResumoArticle22 May 2008Open Access β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions Takahiro Kanamori Takahiro Kanamori Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan Search for more papers by this author Takao Inoue Takao Inoue Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Taro Sakamoto Taro Sakamoto School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Search for more papers by this author Keiko Gengyo-Ando Keiko Gengyo-Ando CREST, Japan Science and Technology Agency, Saitama, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Masafumi Tsujimoto Masafumi Tsujimoto Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan Search for more papers by this author Shohei Mitani Shohei Mitani CREST, Japan Science and Technology Agency, Saitama, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Hitoshi Sawa Hitoshi Sawa Laboratory for Cell Fate Decision, RIKEN Center for Developmental Biology, Kobe, Japan Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan Search for more papers by this author Junken Aoki Junken Aoki Department of Molecular & Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan PRESTO, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Hiroyuki Arai Corresponding Author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Takahiro Kanamori Takahiro Kanamori Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan Search for more papers by this author Takao Inoue Takao Inoue Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Taro Sakamoto Taro Sakamoto School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Search for more papers by this author Keiko Gengyo-Ando Keiko Gengyo-Ando CREST, Japan Science and Technology Agency, Saitama, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Masafumi Tsujimoto Masafumi Tsujimoto Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan Search for more papers by this author Shohei Mitani Shohei Mitani CREST, Japan Science and Technology Agency, Saitama, Japan Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Hitoshi Sawa Hitoshi Sawa Laboratory for Cell Fate Decision, RIKEN Center for Developmental Biology, Kobe, Japan Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan Search for more papers by this author Junken Aoki Junken Aoki Department of Molecular & Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan PRESTO, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Hiroyuki Arai Corresponding Author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Author Information Takahiro Kanamori1,2, Takao Inoue1,3, Taro Sakamoto4, Keiko Gengyo-Ando3,5, Masafumi Tsujimoto2, Shohei Mitani3,5, Hitoshi Sawa6,7, Junken Aoki8,9 and Hiroyuki Arai 1,3 1Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan 2Laboratory of Cellular Biochemistry, RIKEN, Saitama, Japan 3CREST, Japan Science and Technology Agency, Saitama, Japan 4School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan 5Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan 6Laboratory for Cell Fate Decision, RIKEN Center for Developmental Biology, Kobe, Japan 7Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan 8Department of Molecular & Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan 9PRESTO, Japan Science and Technology Agency, Saitama, Japan *Corresponding author. Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: +81 3 5841 4720; Fax: +81 3 3818 3173; E-mail: [email protected] The EMBO Journal (2008)27:1647-1657https://doi.org/10.1038/emboj.2008.102 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/β-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as β-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A1 (PLA1), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of β-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA1 gene, cortical β-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of β-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of β-catenin. Introduction Asymmetric division is an attractive means for stem cells to balance self-renewal and differentiation (Morrison and Kimble, 2006). To divide asymmetrically, stem cells become polarized prior to asymmetric divisions; Drosophila neural stem cells, for example, establish an axis of polarity to localize cell-fate determinants to one side of the cells and to orient the mitotic spindle correctly (Betschinger and Knoblich, 2004; Yu et al, 2006). In Caenorhabditis elegans, the polarity of the most asymmetric divisions is regulated by the Wnt signalling pathway, dubbed as the ‘Wnt/β-catenin asymmetry pathway’ (Thorpe et al, 2000; Herman, 2002; Mizumoto and Sawa, 2007b). In the canonical Wnt pathway, binding of Wnt to its receptor, Frizzled (Fz), results in activation of Dishevelled (Dsh), which in turn inactivates the β-catenin destruction complex, leading to β-catenin stabilization. The stabilized β-catenin enters the nucleus, where it interacts with TCF transcription factors and converts them from repressors to activators (Logan and Nusse, 2004; Clevers, 2006). In the Wnt/β-catenin asymmetry pathway, Wnt signalling components become polarized in the mother cells by Wnt proteins, and then polarized Wnt signalling produces two daughter cells with distinct transcriptional activities of TCF pop-1. Model asymmetric divisions for the Wnt/β-catenin asymmetry pathway include that of the T blast cell. Before T cell division, the Wnt protein lin-44, which is expressed posterior to the T cell, induces asymmetric cortical localizations of the canonical Wnt pathway components; Fz lin-17, Dsh dsh-2 and Dsh mig-5 localize to the posterior side of the T cell, and β-catenin wrm-1 and some of the destruction complex proteins localize to the anterior cortex (Takeshita and Sawa, 2005; Goldstein et al, 2006; Mizumoto and Sawa, 2007a). In telophase, WRM-1 accumulates into the posterior nucleus, where it appears to promote the nuclear export of POP-1 (Lo et al, 2004), and the nuclear level of POP-1 becomes asymmetric between two daughter cells (POP-1 asymmetry). Recently, it was shown that another β-catenin, SYS-1 acts as a coactivator for POP-1 (Kidd et al, 2005), and that in many A–P divisions, the nuclear localization of SYS-1 is also asymmetric in a manner reciprocal to POP-1 asymmetry (Huang et al, 2007; Phillips et al, 2007). This reciprocal asymmetry of POP-1 and SYS-1 provides a robust change in POP-1 transcriptional activity and seems to be a universal mechanism for cell-fate specification during animal development (Huang et al, 2007; Phillips et al, 2007). Among the cells that undergo asymmetric divisions during C. elegans development, the lateral epithelial seam cells are unique in that they have characteristics of stem cells. First, seam cells repeatedly divide to generate one daughter with a seam cell fate (self-renewal) and one daughter that fuses with the hypodermal syncytium (differentiation). Second, like many stem cells in other organisms, seam cells can also divide symmetrically to expand in number during development. Therefore, seam cells can provide a good model for the study of stem cells (Mizumoto and Sawa, 2007b). Phospholipids, in addition to acting as structural components of cell membrane, also regulate many biological processes by acting as lipid mediators, second messengers and subcellular microenvironment. In many cases, these functions are mediated by a diverse group of phospholipases (PLs) that are classified into four groups (PLA, PLB, PLC and PLD) according to the bond hydrolysed on phospholipid substrates. PLA is represented by the two isoenzymes PLA1 and PLA2 that differ in the fatty acid they remove from a glyceride; PLA1 hydrolyses sn-1 fatty acids attached to phospholipids to produce 2-acyl-lysophospholipids, whereas PLA2 hydrolyses sn-2 fatty acids. PLA2 enzymes, the largest group of PLs, have essential functions in the generation of lipid mediators such as prostaglandins and leukotrienes. In contrast, the molecular identity of PLA1 enzymes was not clarified until recently (Higgs et al, 1998). In mammals, at least three members of the intracellular PLA1 family exist, namely PA-PLA1, KIAA0725 and p125, none of which show sequence homology with other known PLs (Inoue and Aoki, 2006). Intracellular PLA1 is highly conserved in a wide range of eukaryotic organisms from yeast, plants to mammals. However, most of their physiological functions remain to be elucidated. Our laboratory is currently interested in studying the intracellular PLA1 family. Using a reverse genetic approach, we show here an intracellular PLA1, ipla-1, as a regulator of asymmetric divisions in C. elegans. Disruption of the ipla-1 gene causes defects in spindle orientation, asymmetric cell-fate determination and asymmetric cortical localization of WRM-1 in the terminal asymmetric divisions of seam cells. We have also used a forward genetic approach to identify genes that functionally interact with ipla-1 in the asymmetric divisions, and provide evidence to suggest that ipla-1 regulates membrane trafficking to control cortical asymmetry of WRM-1 during the terminal asymmetric divisions of seam cells. Results Isolation and initial characterization of ipla-1 mutants Our database searches identified one intracellular PLA1 family member, which we named ipla-1, in C. elegans. ipla-1 is predicted to encode a protein of 840 amino acids, including a serine esterase consensus sequence motif (GxSxG) and the DDHD domain, and shows 31% identity to human PA-PLA1 (Supplementary Figure 1B and C). To investigate the functional roles of ipla-1, we isolated two ipla-1 deletion alleles, designated ipla-1(xh13) and ipla-1(tm471), by PCR-based screening of UV-TMP-mutagenized libraries. The xh13 allele deleted 1138 bp encoding exons 6 and 7, whereas tm471 harboured a 1172 bp deletion at exon 8 (Supplementary Figure 1A). None of the two alleles of ipla-1 showed detectable IPLA-1 protein by western blot, suggesting that they are strong loss-of-function or null alleles (Supplementary Figure 1D). These two ipla-1 mutants were viable and fertile; however, some of ipla-1 mutants exhibited vulval defects, including a protruding vulva and occasional vulval bursting (Supplementary Figure 2A and B). Because xh13 and tm471 were phenotypically indistinguishable in the initial characterization, we used ipla-1(xh13) mainly in subsequent analyses. ipla-1 expression in seam cells is sufficient for proper morphology of the vulva The vulva is formed from the descendants of three vulval precursor cells (VPCs). The fates of the VPCs are specified by an inductive signal from the gonadal anchor cell, which appears to be controlled by an inhibitory signal from the major hypodermal syncytium (hyp7) (Sternberg, 2005). In addition, recent studies have suggested that maintenance of seam cells is important for structural integrity of the vulva (Pellis-van Berkel et al, 2005; Smith et al, 2005). To identify the cells in which ipla-1 is required for proper morphology of the vulva, we expressed an ipla-1 minigene in subsets of these cells using specific promoters. Proper vulval morphology was restored to ipla-1 mutants by expression of ipla-1 with the dpy-7 promoter, which drives expression in hyp7 and weakly in seam cells (Gilleard et al, 1997), and with the seam cell-specific scm promoter (Koh and Rothman, 2001). In contrast, expression of ipla-1, under the control of either the hyp7-specific egl-15 enhancer elements (Huang and Stern, 2004) or the VPC-specific lin-31 promoter (Tan et al, 1998) failed to rescue the vulval defects (Supplementary Figure 2C). These results suggest that defects in seam cells cause abnormal vulval morphogenesis in ipla-1 mutants. ipla-1 functions cell-autonomously to regulate the terminal asymmetric divisions of seam cells The lateral seam cells are specialized epithelial cells. During each larval stage, seam cells divide asymmetrically in a stem cell-like manner producing an anterior daughter cell that fuses with hyp7 and loses the expression of the seam cell marker (scm::gfp, transgene wIs51), and a posterior daughter cell that assumes the seam cell fate again and continues to express scm::gfp (Figure 2A and D). To understand the nature of seam cell defects in ipla-1 mutants, we first analysed the number of seam cells using scm::gfp. Wild-type adult hermaphrodites usually contain evenly spaced 16 scm::gfp-positive nuclei on each side of the animals, derived from the 10 embryonically derived blast cells H0, H1, H2, V1-6 and T (Figures 1A and 2A). ipla-1 mutants, by contrast, contained more than 16 unevenly spaced seam cell nuclei (Figure 1B–D; Table I, see group A), some of which lay outside a row of seam cells (Figure 1C, arrow), and some of which lay close to each other (Figure 1C, arrowhead). Furthermore, scm::gfp-positive nuclei were occasionally lost (Figure 1D, arrowheads), and in such area seam cells were separated by gaps as indicated by a break in ajm-1::gfp, cdh-3::gfp and lateral alae (Supplementary Figure 3). To determine the cause of the missing or additional seam cells in ipla-1 mutants, we then followed the development of seam cells during each larval stage. Although ipla-1 mutants showed normal number and alignment of seam cells during the L2 and L3 stages, they exhibited aberrant number and alignment of seam cells at the L4 stage (Figure 1E). This suggests that ipla-1 expression is required for the terminal asymmetric divisions of seam cells that occur at the beginning of the L4 stage, which we call hereafter the ‘S4 divisions’ (Seam cell divisions at the L4 stage). To confirm this idea, we expressed ipla-1 cDNA by the heat-shock promoter at specific developmental stages. We found that heat-shock treatments before but not after the S4 divisions (even at the end of the L3 stage) restored proper number and alignment of seam cells to ipla-1 mutants (Figure 1F). Thus, presence of IPLA-1 protein during the S4 divisions is sufficient for proper number and alignment of seam cells at the adult stage. Figure 1.ipla-1 mutants have aberrant number and alignment of seam cells. (A–D) Seam cells in adult hermaphrodites were visualized by a seam cell marker::gfp (scm::gfp) fusion. (A) Wild-type. Evenly spaced 16 scm::gfp-positive nuclei are observed. (B–D) ipla-1(xh13). More than 16 seam cell nuclei align unevenly. scm::gfp-positive nuclei are occasionally observed outside the row of seam cells (C, arrow). ipla-1(xh13) mutants show loss of scm::gfp-positive nuclei (D, delimited by arrowheads). (E) The number of scm::gfp-positive nuclei in each developmental stage. On average, more scm::gfp-positive nuclei were observed in ipla-1(xh13) than in wild-type animals at the late L4 stage and the young adult stage (YA). L2 cell counts were often high by one cell because of the comparatively late division of cells of the T lineage, causing both daughters to be scored. The numbers in parentheses indicate the number of animals scored. (F) Stage-specific rescue of seam cell defects of ipla-1(xh13). Synchronized ipla-1(xh13) animals carrying hsp::ipla-1 were heat shocked at various developmental times. Rescue was obtained by heat-shock treatments before the L4 stage. The number above each point is the number of animals scored. Scale bars are 50 μm in (A–D). Download figure Download PowerPoint Figure 2.ipla-1 mutants disrupt the orientation and polarity of the S4 divisions. (A) A schematic drawing of seam cell lineages. The V lineage seam cells are shown. Lineage branches expressing scm::gfp are shown in green. The dotted box marks the lineages analysed in this study; V5.pppp lineage (a), V6.papp lineage (b) and V6.pppp lineage (c). Grey circles, anterior daughters that fuse with the hyp7; green squares, posterior daughters that assume the seam cell fate again. Red and blue lines indicate the developmental stages corresponding to those of (B, C), and (D, E), respectively. (B, C) Fluorescent images of seam cells during or just after the S4 divisions visualized by scm::gfp. Three pairs of daughter cells are shown with brackets for each panel. (B) Wild-type. The three seam cells divide parallel to the A–P axis. (C) ipla-1(xh13). Seam cells, as shown by asterisks, divide in various directions. (D, E) Seam cells of late L4 hermaphrodite visualized by scm::gfp. (D) Wild-type. The posterior daughters assume the seam cell fate in all the three lineages. (E) ipla-1(xh13). The seam cell fates are adopted by either or both of the anterior and posterior daughters and the mitotic spindle is misoriented. The seam cell lineages are shown in the images. The letters (a), (b) and (c) correspond to those of (A). Results are summarized in (I). The characters indicate the directions of the S4 divisions; a, anterior; p, posterior; av, anterior-ventral; ad, anterior-dorsal; pv, posterior-ventral and pd, posterior-dorsal. (F) Schematic for measurement of spindle orientation. A, anterior; P, posterior; D, dorsal; and V, ventral. (G, H) Quantification of spindle orientation of the S4 divisions. The percentages are determined from a random sample of angles from wild-type (G) or ipla-1(xh13) (H) seam cells measured as depicted in (F). (I) Abnormal V-cell lineages in ipla-1(xh13) after the S4 divisions. The numbers of seam cells that showed the lineages are indicated below the diagrams. The letters (a), (b) and (c) correspond to those of (A). Spindle orientation is defective in types IV–VI (indicated with diagonal lines). Additional cell divisions occur just after the first divisions in types III and VI. The S4 divisions do not occur in types VII and VIII. The dotted line in type VIII indicates that the scm::gfp fluorescence disappears during the L4 stage. Anterior is oriented towards the left. Scale bars in (B–E) are 20 μm. Download figure Download PowerPoint Table 1. Seam cell phenotypes Group Genotype No. of seam cell nuclei High Low scm::gfp alignmenta N P-Values A N2 15.9 17 15 N (0) 19 — ipla-1(xh13) 19.1 26 16 A (100) 29 <0.0001b ipla(xh13);Ex[dpy-7p::ipla-1] 16.1 18 14 N (8) 13 <0.0001c ipla(xh13);Ex[scm::ipla-1] 16.3 17 16 N (0) 12 <0.0001c ipla(xh13);Ex[scm::ipla-1 S489A] 19.4 24 14 A (100) 25 0.6489c ipla(xh13);Ex[Pe15*2::ipla-1]d 17.2 23 13 A (100) 21 0.0050c B mon-2(xh22) 16.0 17 15 N (0) 21 — ipla-1(xh13);mon-2(xh22) 16.2 17 14 N (7) 18 <0.0001c ipla-1(xh13);mon-2(RNAi)e 16.2 18 14 N (7) 28 <0.0001c ipla-1(xh13);mon-2(xh22);Ex[scm::mCherry::mon-2] 18.2 23 15 A (94) 18 <0.0001f N2; Ex[scm::mCherry::mon-2] 16.0 17 15 N (0) 26 0.7588b C tbc-3(xh23) 16.0 17 16 N (0) 30 — ipla-1(xh13);tbc-3(xh23) 15.9 18 15 N (4) 24 <0.0001c ipla-1(xh13);tbc-3(RNAi)e 16.2 17 15 N (0) 16 <0.0001c ipla-1(xh13);tbc-3(xh23);Ex[scm::mCherry::tbc-3a] 16.0 17 15 N (0) 18 0.7709g ipla-1(xh13);tbc-3(xh23);Ex[scm::mCherry::tbc-3b] 18.5 21 15 A (89) 19 <0.0001g N2; Ex[scm::mCherry::tbc-3b] 16.0 17 15 N (0) 29 0.7588b D sid-1(qt2) 16.1 15 17 N (0) 31 — ipla-1(xh13);sid-1(qt2) 20.4 26 14 A (100) 27 — ipla-1(xh13);sid-1(qt2);Ex[scm::tat-5 RNAi] 15.8 17 15 N (0) 14 <0.0001h ipla-1(xh13);sid-1(qt2);Ex[Pe15*2::tat-5 RNAi]d 18.5 21 15 A (100) 18 0.0012h ipla-1(xh13);sid-1(qt2);Ex[scm::pad-1 RNAi] 15.9 16 15 N (0) 11 <0.0001h ipla-1(xh13);sid-1(qt2);Ex[Pe15*2::pad-1 RNAi]d 18.8 23 14 A (100) 15 0.0141h ipla-1(xh13);sid-1(qt2);Ex[scm::vps-35 RNAi] 16.1 18 15 N (7) 30 <0.0001h ipla-1(xh13);sid-1(qt2);Ex[Pe15*2::vps-35 RNAi]d 18.7 23 15 A (92) 25 0.0012h a Percentage of animals with aberrant alignment is shown in parentheses; N, normal; A, aberrant. b Compared with N2. c Compared with ipla-1(xh13). d Pe15*2 represents the tandem egl-15 enhancer elements (Huang and Stern, 2004). e Feeding RNAi. f Compared with ipla-1(xh13); mon-2(xh22). g Compared with ipla-1(xh13); tbc-3(xh23). h Compared with ipla-1(xh13); sid-1(qt2). All strains contain seam cell marker wIs51(scm::gfp). The number of scm::gfp-positive nuclei was scored in young adult hermaphrodites. We next conducted cell-specific rescue experiments to confirm the cell autonomy of ipla-1 function. The seam cell defects were fully rescued by expression of ipla-1 under the dpy-7 promoter and the seam cell-specific scm promoter. No rescue was obtained when ipla-1 was expressed with the hyp7-specific egl-15 enhancer elements (Table I, see group A). Furthermore, expression of catalytically inactive mutant IPLA-1 (ipla-1 S489A, see Supplementary data, Supplementary Figure 1A and C, for details) by the scm promoter did not rescue the seam cell defects of ipla-1 mutants (Table I, see group A). These results indicate that ipla-1 functions cell-autonomously in seam cells to regulate their S4 divisions through its enzymatic activity. ipla-1 is required for spindle orientation and cell-fate determination in the S4 divisions We next analysed seam cells undergoing the S4 divisions and performed lineage analyses. In wild-type animals, the S4 divisions invariably occurred parallel to the anterior–posterior axis (A–P axis) (Figure 2B). In ipla-1 mutants, however, orientation of the S4 divisions was essentially randomized relative to the A–P axis (asterisks in Figure 2C). To quantify this defect, we measured the angle between a line connecting the two daughter nuclei and the A–P axis as depicted in Figure 2F (see Supplementary data). In wild-type animals, the measured angle was always less than 10° (n=110; Figure 2G). In ipla-1 mutants, the majority of seam cells underwent the S4 divisions in various directions (52%, n=107; Figure 2H). These results suggest that ipla-1 mutants have defects in spindle orientation during the S4 divisions. Furthermore, detailed lineage analyses revealed that in ipla-1 mutants, the asymmetry of the divisions was often disrupted (62%, n=53), leading to the transformation of the anterior daughters from hyp7 cells to seam cells or to the reversal of these cell fates (Figure 2E and I). It is noted that seam cells that divided normally in the A–P direction did have defects in the asymmetry of the divisions (Figure 2I; types I and II), indicating that defects in cell-fate determination are not secondary to the spindle orientation phenotype. We conclude that ipla-1 mutants are defective in both spindle orientation and cell-fate determination in the S4 divisions. ipla-1 regulates the cortical asymmetry of β-catenin wrm-1 prior to the S4 divisions In C. elegans, many asymmetric divisions are controlled by the Wnt/β-catenin asymmetry pathway, including β-catenin wrm-1 and TCF pop-1 transcription factor (Mizumoto and Sawa, 2007b). In several asymmetric divisions, including those of seam cells at the L1 stage, WRM-1 localizes asymmetrically to the anterior cortex in mother cells (Nakamura et al, 2005; Takeshita and Sawa, 2005; Mizumoto and Sawa, 2007a). After the asymmetric divisions, the anterior daughters have more nuclear GFP::POP-1 than posterior daughters. This phenomenon has been dubbed ‘POP-1 asymmetry’ (Lin et al, 1998). To test whether ipla-1 mutation influences the Wnt/β-catenin asymmetry pathway in the S4 divisions, we determined the subcellular localization of WRM-1::GFP and GFP::POP-1. In wild-type animals, WRM-1::GFP always localized to the anterior cortex in seam cells before the S4 divisions (n=21; Figure 3A, B and D). However, the cortical localization of WRM-1 was randomized in ipla-1 mutants; WRM-1::GFP was symmetrically localized (22%), absent from the cortex (34%) and occasionally enriched posteriorly (6%) (n=34; Figure 3C and D). We also found that ipla-1 mutants had defects in the POP-1 asymmetry just after the S4 divisions; the levels of POP-1 were equally high in the two daughters (32%), and higher in the posterior daughters (36%) in ipla-1 mutants (n=25; Figure 3G and H), whereas the level of GFP::POP-1 was always higher in the anterior daughters in wild-type animals (n=19; Figure 3E, F and H). The frequencies of these mislocalization phenotypes were similar to that of defects in cell-fate determination after the S4 divisions in ipla-1 mutants (62%; Figure 2I). These results indicate that ipla-1 is required for the formation and/or maintenance of cortical asymmetry of WRM-1 prior to the S4 divisions. Figure 3.ipla-1 regulates polarity of the seam cells in the S4 divisions. (A) Schematic for the seam cells before the S4 divisions. WRM-1 localization is shown in green; WRM-1 localizes to the anterior cortex of the seam cells. (B, C) Confocal images showing the localization of WRM-1::GFP in the V6.papp and V6.pppp cell of wild-type (B) and ipla-1(xh13) (C). (D) Frequency of the localization patterns of WRM-1::GFP. The number of samples is shown above each column. The equality and inequality signs indicate relative intensities of cortical WRM-1::GFP. A, intensity of WRM-1::GFP at the anterior cortex; P, intensity of WRM-1::GFP at the posterior cortex. No cortex means that WRM-1::GFP signal is absent from the cortex of the cells. (E) Schematic for the seam cells just after the S4 divisions. POP-1 localization is shown in green; the anterior daughters contain more nuclear POP-1 than posterior daughters. (F, G) Confocal images showing the localization of GFP::POP-1 in wild-type (F) and ipla-1(xh13) (G). Pairs of daughter nuclei are shown with brackets for each panel. The equality and inequality signs indicate relative intensities of GFP::POP-1 levels between two daughters. (H) Frequency of the localization patterns of GFP::POP-1. The number of samples is shown above each column. A, intensity of nuclear GFP::POP-1 in the anterior daughter; P, intensity of nuclear GFP::POP-1 in the posterior daughter. Scale bars are 10 μm in (B, C), or 20 μm in (F, G). Anterior is oriented towards the left; ventral is oriented towards the bottom. Download figure Download PowerPoint A genetic screen for mutations that suppress the ipla-1 seam cell defects To understand the molecular mechanism underlying the regulation of cortical WRM-1 asymmetry mediated by ipla-1, we carried out genetic screens for mutations that suppress the seam cell defects of ipla-1 mutants (see Supplementary data). In a screen of 3000 haploid mutagenized ipla-1(xh13);wIs51 genomes, we isolated two strong suppressor mutations. The two recessive suppressor alleles, xh22 and xh23, mapped to chromosome IV and did complement each other, indicating that they identify two distinct loci. On their own, these suppressor alleles displayed wild-type seam cell number and alignment and had no obvious morphological defects (Table I, see groups B and C; and data not shown). However, they effectively suppressed the seam cell phenotype of ipla-1 mutants with almost complete penetrance (Table I, see group
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