The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Xenobiotic biotransformation is an important modulator of anthelmintic drug potency and a potential mechanism of anthelmintic resistance. Both the free-living nematode Caenorhabditis elegans and the ruminant parasite Haemonchus contortus biotransform benzimidazole drugs by glucose conjugation, likely catalysed by UDP-glycosyltransferase (UGT) enzymes. To identify C. elegans genes involved in benzimidazole drug detoxification, we first used a comparative phylogenetic analysis of UGTs from humans, C. elegans and H. contortus, combined with available RNAseq datasets to identify which of the 63 C. elegans ugt genes are most likely to be involved in benzimidazole drug biotransformation. RNA interference knockdown of 15 prioritized C. elegans genes identified those that sensitized animals to the benzimidazole derivative albendazole (ABZ). Genetic mutations subsequently revealed that loss of ugt-9 and ugt-11 had the strongest effects. The “ugt-9 cluster” includes these genes, together with six other closely related ugts. A CRISPR-Cas-9 deletion that removed seven of the eight ugt-9 cluster genes had greater ABZ sensitivity than the single largest-effect mutation. Furthermore, a double mutant of ugt-22 (which is not a member of the ugt-9 cluster) with the ugt-9 cluster deletion further increased ABZ sensitivity. This additivity of mutant phenotypes suggest that ugt genes act in parallel, which could have several, not mutually exclusive, explanations. ugt mutations have different effects with different benzimidazole derivatives, suggesting that enzymes with different specificities could together more efficiently detoxify drugs. Expression patterns of ugt-9, ugt-11 and ugt-22 gfp reporters differ and so likely act in different tissues which may, at least in part, explain their additive effects on drug potency. Overexpression of ugt-9 alone was sufficient to confer partial ABZ resistance, indicating increasing total UGT activity protects animals. In summary, our results suggest that the multiple UGT enzymes have overlapping but not completely redundant functions in benzimidazole drug detoxification and may represent “druggable” targets to improve benzimidazole drug potency.

Microtubule severing plays important role in cell structure and cell division. The microtubule severing protein katanin, composed of the MEI-1/MEI-2 subunits in Caenorhabditis elegans, is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. Katanin activity is regulated by 2 ubiquitin-based protein degradation pathways. Another ubiquitin ligase, HECD-1, the homolog of human HECTD1/HECT domain E3 ubiquitin protein ligase 1, regulates katanin activity without affecting katanin levels. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase complex, which affects cell proliferation and a variety of signaling pathways. Here we conducted a systematic screen of how mutations in striatin-interacting kinase phosphatase components affect katanin function in C. elegans. Striatin-interacting kinase phosphatase core components (FARL-11, CASH-1, LET-92, and GCK-1) were katanin inhibitors in mitosis and activators in meiosis, much like HECD-1. By contrast, variable components (SLMP-1, OTUB-2) functioned as activators of katanin activity in mitosis, indicating they may function to alter striatin-interacting kinase phosphatase core function. The core component CCM-3 acted as an inhibitor at both divisions, while other components (MOB-4, C49H3.6) showed weak interactions with katanin mutants. Additional experiments indicate that katanin may be involved with the centralspindlin complex and a tubulin chaperone. HECD-1 shows ubiquitous expression in the cytoplasm throughout meiosis and early development. The differing functions of the different subunits could contribute to the diverse functions of the striatin-interacting kinase phosphatase complex in C. elegans and other organisms.

Parasitic nematodes are major human and agricultural pests, and benzimidazoles are amongst the most important broad-spectrum anthelmintic drug class used for their control. Benzimidazole resistance is now widespread in many species of parasitic nematodes in livestock globally and an emerging concern for the sustainable control of human soil-transmitted helminths. β-tubulin is the major benzimidazole target, although other genes may influence resistance. Among the 6 Caenorhabditis elegans β-tubulin genes, loss of ben-1 causes resistance without other apparent defects. Here, we explored the genetics of C. elegans β-tubulin genes in relation to the response to the benzimidazole derivative albendazole. The most highly expressed β-tubulin isotypes, encoded by tbb-1 and tbb-2, were known to be redundant with each other for viability, and their products are predicted not to bind benzimidazoles. We found that tbb-2 mutants, and to a lesser extent tbb-1 mutants, were hypersensitive to albendazole. The double mutant tbb-2 ben-1 is uncoordinated and short, resembling the wild type exposed to albendazole, but the tbb-1 ben-1 double mutant did not show the same phenotypes. These results suggest that tbb-2 is a modifier of albendazole sensitivity. To better understand how BEN-1 mutates to cause benzimidazole resistance, we isolated mutants resistant to albendazole and found that 15 of 16 mutations occurred in the ben-1 coding region. Mutations ranged from likely nulls to hypomorphs, and several corresponded to residues that cause resistance in other organisms. Null alleles of ben-1 are albendazole-resistant and BEN-1 shows high sequence identity with tubulins from other organisms, suggesting that many amino acid changes could cause resistance. However, our results suggest that missense mutations conferring resistance are not evenly distributed across all possible conserved sites. Independent of their roles in benzimidazole resistance, tbb-1 and tbb-2 may have specialized functions as null mutants of tbb-1 or tbb-2 were cold or heat sensitive, respectively.

Actin and myosin mediate the epidermal cell contractions that elongate the Caenorhabditis elegans embryo from an ovoid to a tubular-shaped worm. Contraction occurs mainly in the lateral epidermal cells, while the dorsoventral epidermis plays a more passive role. Two parallel pathways trigger actinomyosin contraction, one mediated by LET-502/Rho kinase and the other by PAK-1/p21 activated kinase. A number of genes mediating morphogenesis have been shown to be sufficient when expressed either laterally or dorsoventrally. Additional genes show either lateral or dorsoventral phenotypes. This led us to a model where contractile genes have discrete functions in one or the other cell type. We tested this by examining several genes for either lateral or dorsoventral sufficiency. LET-502 expression in the lateral cells was sufficient to drive elongation. MEL-11/Myosin phosphatase, which antagonizes contraction, and PAK-1 were expected to function dorsoventrally, but we could not detect tissue-specific sufficiency. Double mutants of lethal alleles predicted to decrease lateral contraction with those thought to increase dorsoventral force were previously shown to be viable. We hypothesized that these mutant combinations shifted the contractile force from the lateral to the dorsoventral cells and so the embryos would elongate with less lateral cell contraction. This was tested by examining 10 single and double mutant strains. In most cases, elongation proceeded without a noticeable alteration in lateral contraction. We suggest that many embryonic elongation genes likely act in both lateral and dorsoventral cells, even though they may have their primary focus in one or the other cell type.

BACKGROUND:Peripheral neuropathies are often caused by disruption of genes responsible for myelination or axonal transport. In particular, impairment in mitochondrial fission and fusion are known causes of peripheral neuropathies. However, the causal mechanisms for peripheral neuropathy gene mutations are not always known. While loss of function mutations in MYH14 typically cause non-syndromic hearing loss, the recently described R941L mutation in MYH14, encoding the non-muscle myosin protein isoform NMIIC, leads to a complex clinical presentation with an unexplained peripheral neuropathy phenotype. METHODS:Confocal microscopy was used to examine mitochondrial dynamics in MYH14 patient fibroblast cells, as well as U2OS and M17 cells overexpressing NMIIC. The consequence of the R941L mutation on myosin activity was modeled in C. elegans. FINDINGS:We describe the third family carrying the R941L mutation in MYH14, and demonstrate that the R941L mutation impairs non-muscle myosin protein function. To better understand the molecular basis of the peripheral neuropathy phenotype associated with the R941L mutation, which has been hindered by the fact that NMIIC is largely uncharacterized, we have established a previously unrecognized biological role for NMIIC in mediating mitochondrial fission in human cells. Notably, the R941L mutation acts in a dominant-negative fashion to inhibit mitochondrial fission, especially in the cell periphery. In addition, we observed alterations to the organization of the mitochondrial genome. INTERPRETATION:As impairments in mitochondrial fission cause peripheral neuropathy, this insight into the function of NMIIC likely explains the peripheral neuropathy phenotype associated with the R941L mutation. FUND: This study was supported by the Alberta Children's Hospital Research Institute, the Canadian Institutes of Health Research and the Care4Rare Canada Consortium.

The cytoskeleton is the basic machinery that drives many morphogenetic events. Elongation of the C. elegans embryo from a spheroid into a long, thin larva initially results from actomyosin contractility, mainly in the lateral epidermal seam cells, while the corresponding dorsal and ventral epidermal cells play a more passive role. This is followed by a later elongation phase involving muscle contraction. Early elongation is mediated by parallel genetic pathways involving LET-502/Rho kinase and MEL-11/MYPT myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase in another. While the LET-502/MEL-11 pathway appears to act primarily in the lateral epidermis, here we show that FEM-2 can mediate early elongation when expressed in the dorsal and ventral epidermis. We also investigated the early elongation function of FHOD-1, a member of the formin family of actin nucleators and bundlers. Previous work showed that FHOD-1 acts in the LET-502/MEL-11 branch of the early elongation pathway as well as in muscle for sarcomere organization. Consistent with this, we found that lateral epidermal cell-specific expression of FHOD-1 is sufficient for elongation, and FHOD-1 effects on elongation appear to be independent of its role in muscle. Also, we found that fhod-1 encodes long and short isoforms that differ in the presence of a predicted coiled-coil domain. Based on tissue-specific expression constructions and an isoform-specific CRISPR allele, the two FHOD-1 isoforms show partially specialized epidermal or muscle function. Although fhod-1 shows only impenetrant elongation phenotypes, we were unable to detect redundancy with other C. elegans formin genes.

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen’s design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10−4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.

Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10–12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.

After fertilization, rapid changes of the Caenorhabditis elegans cytoskeleton occur in the transition from meiosis to mitosis, requiring precise regulation. The MEI-1/MEI-2 katanin microtubule-severing complex is essential for meiotic spindle formation but must be quickly inactivated to allow for proper formation of the mitotic spindle. MEI-1/MEI-2 inactivation is dependent on multiple redundant pathways. The primary pathway employs the MEL-26 substrate adaptor for the CUL-3/cullin-based E3 ubiquitin ligase, which targets MEI-1 for proteosomal degradation. Here, we used quantitative antibody staining to measure MEI-1 levels to determine how other genes implicated in MEI-1 regulation act relative to CUL-3/MEL-26. The anaphase-promoting complex/cyclosome, APC/C, the DYRK (Dual-specificity tyrosine-regulated kinase), MBK-2, and the CUL-2-based E3 ubiquitin ligase act together to degrade MEI-1, in parallel to MEL-26/CUL-3. CUL-2 is known to keep MEL-26 low during meiosis, so CUL-2 apparently changes its target from MEL-26 in meiosis to MEI-1 in mitosis. RFL-1, an activator of cullin E3 ubiquitin ligases, activates CUL-2 but not CUL-3 for MEI-1 elimination. HECD-1 (HECT/Homologous to the E6AP carboxyl terminus domain) E3 ligase acts as a MEI-1 activator in meiosis but functions as an inhibitor during mitosis, without affecting levels of MEI-1 or MEI-2. Our results highlight the multiple layers of MEI-1 regulation that are required during the switch from the meiotic to mitotic modes of cell division.

Trisomy, the presence of a third copy of one chromosome, is deleterious and results in inviable or defective progeny if passed through the germ line. Random segregation of an extra chromosome is predicted to result in a high frequency of trisomic offspring from a trisomic parent. Caenorhabditis elegans with trisomy of the X chromosome, however, have far fewer trisomic offspring than expected. We found that the extra X chromosome was preferentially eliminated during anaphase I of female meiosis. We utilized a mutant with a specific defect in pairing of the X chromosome as a model to investigate the apparent bias against univalent inheritance. First, univalents lagged during anaphase I and their movement was biased toward the cortex and future polar body. Second, late-lagging univalents were frequently captured by the ingressing polar body contractile ring. The asymmetry of female meiosis can thus partially correct pre-existing trisomy.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Trisomy, the presence of a third copy of one chromosome, is deleterious and results in inviable or defective progeny if passed through the germ line. Random segregation of an extra chromosome is predicted to result in a high frequency of trisomic offspring from a trisomic parent. Caenorhabditis elegans with trisomy of the X chromosome, however, have far fewer trisomic offspring than expected. We found that the extra X chromosome was preferentially eliminated during anaphase I of female meiosis. We utilized a mutant with a specific defect in pairing of the X chromosome as a model to investigate the apparent bias against univalent inheritance. First, univalents lagged during anaphase I and their movement was biased toward the cortex and future polar body. Second, late-lagging univalents were frequently captured by the ingressing polar body contractile ring. The asymmetry of female meiosis can thus partially correct pre-existing trisomy. https://doi.org/10.7554/eLife.06056.001 eLife digest Inside cells, DNA is found packaged into structures called chromosomes. Most human and animal cells contain two sets of chromosomes, one inherited from each parent. Chromosomes from one set pair up with the equivalent chromosome from the other set. However, egg and sperm cells only contain one copy of each chromosome, so that when the egg is fertilized, the resulting cell again has two sets of chromosomes. If there are either more or fewer than two copies of a chromosome in the fertilized cell, this can cause birth defects and conditions such as Down syndrome. An egg cell develops from a cell called an oocyte via a process called meiosis. The oocyte first duplicates its DNA so that it contains four copies of each chromosome. The oocyte then divides, and the resulting cells divide again, to produce four cells that each contains one copy of each chromosome. Only one of these cells is an egg cell: the other three are called polar bodies, and these normally self-destruct. The tiny roundworm C. elegans is a model organism used to study meiosis. Worms can be hermaphrodites or males; the hermaphrodites normally have a pair of ‘X’ sex chromosomes. However, sometimes problems with meiosis can produce hermaphrodite worms with three X chromosomes in each of their cells. In these cells, two of the X chromosomes pair with each other as normal, and one X chromosome remains unpaired. Cortes et al. examined meiosis in mutant worms that had an extra copy of the X chromosome by marking all the chromosomes with a fluorescent tag. This allowed the movement of the chromosomes to be tracked through images taken using a microscope. This revealed that an unpaired X chromosome moves more slowly than a normal paired set. Furthermore, the unpaired chromosomes tend to move toward the region of the oocyte that will develop into a polar body. Thus, when the oocyte divides, the unpaired chromosomes are placed in the polar body and eliminated. This mechanism improves the chance that the correct number of chromosomes will end up in the egg cell. Women with three X chromosomes are often fertile and in most cases produce normal offspring. Further work is needed to see whether human oocytes remove extra chromosomes by a mechanism similar to that seen in the roundworms. https://doi.org/10.7554/eLife.06056.002 Introduction During female meiosis, a G2 oocyte containing four genome copies undergoes two asymmetric cell divisions depositing one genome in a single haploid egg, while the other three genomes are segregated into polar bodies. These divisions are mediated by meiotic spindles that are asymmetrically positioned against the oocyte cortex with the pole-to-pole axis of the spindle perpendicular to the cortex. Both the inheritance of only one of the four genome copies and the distinct perpendicular positioning of the meiotic spindle are remarkably conserved among animal phyla suggesting a selective advantage (Maro and Verlhac, 2002; Fabritius et al., 2011a; Maddox et al., 2012). Several advantages of asymmetric meiosis have been suggested previously, yet none are applicable to all animals. Asymmetric meiotic spindle positioning maximizes the volume of a single egg, helps prevent interference with the meiotic spindle by the sperm aster (McNally et al., 2012), and preserves predetermined embryonic polarity gradients. Here, we suggest a previously unrecognized advantage of asymmetric meiosis, the ability of meiotic spindles to correct trisomy by preferentially depositing the extra chromosome copy into a polar body. Accurate segregation of homologous chromosomes to opposite spindle poles depends on a physical attachment, or chiasma, between homologous chromosomes. A chiasma consists of a crossover, which holds the two homologous chromosomes together in a bivalent so that kinetochores can be properly oriented toward opposite poles (Moore and Orr-Weaver, 1998; Miller et al., 2013). When a chiasma does not form, univalent chromosomes may maintain sister cohesion and move to poles independent of their homologs at anaphase I as can occur in Saccharomyces cerevisiae (Buonomo et al., 2000). If a univalent chromosome biorients, loses cohesion, and segregates sister chromatids at anaphase I (e.g., Nicklas and Jones, 1977; Lemaire-Adkins and Hunt, 2000; Kouznetsova et al., 2007), the resulting single chromatid will segregate randomly at anaphase II. Random segregation of homologs at anaphase I or single chromatids at anaphase II should result in equal frequencies of haplo and diplo ova in the case of a trisomy (Figure 1A) and equal frequencies of nullo and diplo ova in the case of a crossover failure. Figure 1 Download asset Open asset Trisomy correction during meiosis I. (A) Illustration showing expected outcomes of female meiosis in XXX wild-type worms, assuming the extra univalent X (red) does not lose cohesion (yellow) between sister chromatids during anaphase I and assuming random segregation. (B) Illustration of a spindle with chromosomes at the metaphase plate with poles marked ‘P’ (left) and a projection of the cross-sectional view down the pole-to-pole axis at the metaphase plate (right). (C–E) Z projections of fixed meiotic embryos viewed down the pole-to-pole spindle axis. Meiotic embryos from XXX wild-type mothers were stained with DAPI and anti-tubulin antibody. (C) Metaphase I spindles with 7 chromosomes; right images of X-fluorescence in situ hybridization (FISH) show two X chromosomes on the spindle. See also Supplementary file 2. (D) Metaphase II spindles with 6 chromosomes; right images of X-FISH show one X on the spindle and 2–3 foci in the polar body. See also Supplementary file 3. (E) Metaphase II spindle with 7 chromosomes. (F) Frequency of each spindle class among the progeny of XXX wild-type mothers. Insets show polar bodies, marked by asterisks, which were used to identify metaphase II spindles. Bar = 5 μm. https://doi.org/10.7554/eLife.06056.003 Deviations from random segregation are suggested by observations of X chromosomes in Caenorhabditis elegans. In C. elegans, the single unpaired X chromosome from an XXX mother is inherited with unexpectedly low frequency with twice as many haploX ova produced as diploX ova (Hodgkin et al., 1979). HIM-8 is a zinc finger protein that binds to specific DNA sequences that are enriched on the X chromosome. him-8 mutants have a pairing defect that is completely specific for the X chromosome, resulting in two X univalents and five autosomal bivalents in 95% of diakinesis oocytes (Phillips et al., 2005). If the two X univalents segregated randomly, him-8 mutants would be expected to produce equal frequencies of nulloX ova and diploX ova. However, Hodgkin et al. (1979) demonstrated a fivefold preponderance of nulloX ova over diploX ova in him-8. Using sex-reversed him-8 XX males, these authors showed the opposite effect in spermatogenesis. Rather than producing the 50% haploX, 25% diploX, 25% nulloX sperm expected from random segregation, him-8 XX males produced 86% haploX, 3% diploX, 11% nulloX sperm, indicating symmetric distribution of univalents during male meiosis. Thus, achiasmate maternal X chromosomes are inherited with unexpectedly low frequency in worms. Five mechanisms might reduce the frequency of trisomic offspring from trisomic or him-8 mothers. First, trisomic embryos might die during embryonic development resulting in undercounting of XXX offspring. This is unlikely in C. elegans because both XXX and him-8 mutant mothers produce a very low frequency of dead embryos (Hodgkin et al., 1979; Supplementary file 1). A second possibility is that mitotic non-disjunction in the XXX mother results in a mosaic gonad that contains both diploX and triploX oocytes. Selective apoptosis of XXX germ line cells (Bhalla and Dernburg, 2005) would then enrich for XX germ line cells. This does not contribute to the segregation bias in C. elegans, as the most mature diakinesis oocytes in him-8 and wild-type XXX worms have 7 rather than 6 DAPI-staining bodies (Phillips et al., 2005; this study). A third possibility is that a univalent present during metaphase I or a single chromatid present during metaphase II would be broken or otherwise degraded during anaphase. A fourth possibility is that many XXX progeny look normal because of the stochastic nature of dosage compensation and thus are undercounted. A fifth possibility is that univalent chromosomes present at metaphase I are preferentially placed in the first polar body. Here, we demonstrate that indeed biased deposition of univalent X chromosomes into the first polar body reduces the frequency of trisomic zygotes resulting from oocytes with unpaired X chromosomes. Results XXX wild-type oocytes preferentially lose the achiasmate X chromosome between metaphase I and metaphase II Elimination of the extra chromosome from an oocyte starting with a trisomy would result in rescue to a euploid state. It has previously been shown that C. elegans XXX wild-type oocytes have a paired bivalent X and an unpaired univalent X chromosome in pachytene (Goldstein, 1984). We picked wild-type XXX adult hermaphrodites from the progeny of an XXX strain (AV494, Mlynarczyk-Evans et al., 2013) based on their characteristic dumpy morphology as described by Hodgkin et al. (1979). Meiotic embryos from XXX mothers were fixed and stained for microtubules and DNA. Chromosomes are well separated by bundles of microtubules during C. elegans female meiotic metaphase. This unique morphology facilitates counting of individual chromosomes on the metaphase plate when viewed down the pole-to-pole axis of the spindle (Figure 1B). We found that 100% of metaphase I meiotic embryos from XXX wild-type worms had 7 DAPI-staining bodies on the spindle (Figure 1C,F), consistent with 6 bivalents and a single univalent X. Two chromosomes were labeled with an X-specific fluorescence in situ hybridization (FISH) probe in these spindles (Figure 1C, Supplementary file 2). This result shows that a mosaic gonad resulting from mitotic nondisjunction cannot explain the low frequency of XXX offspring from XXX worms. If the univalent segregated randomly during anaphase I, 50% of metaphase II spindles should have 6 DAPI-staining bodies (6 bivalents) and 50% should have 7 DAPI-staining bodies (6 bivalents and 1 univalent). Instead, 71% of metaphase II spindles contained only 6 DAPI-staining bodies and only 29% contained 7 DAPI-staining bodies (Figure 1D–F). These frequencies match the 2:1 ratio of X to XX ova previously interpreted from genetic studies (Hodgkin et al., 1979) and are significantly different than the 50% expected from random segregation (one-tailed p = 0.004, Pearson's chi-squared test). This result eliminates the possibilities that XXX mothers have many XXX offspring that are undercounted due to incomplete penetrance of the XXX dumpy phenotype or that hermaphrodite nulloX sperm contributes significantly to the low frequency of XXX self-progeny. The finding that all assayed metaphase I spindles had 7 chromosomes also indicates that our method of identifying XXX worms as dumpy individuals is accurate and the high frequency of metaphase II spindles with 6 chromosomes is not a result of misidentifying diploid worms as XXX worms. FISH with an X-specific probe revealed that in 6/6 metaphase II embryos with only 6 DAPI-staining bodies, a single hybridization signal was present in the spindle and 2–3 hybridization signals were present in the polar body (Figure 1D; Supplementary file 3). Because only a single X-hybridization signal was observed in the first polar body in 5/5 spindles from diploids, these results demonstrate that single X univalents are deposited in the first polar body with greater than 50% frequency. X and V univalents are frequently deposited in the first polar body To further investigate the mechanism leading to preferential loss of univalents during meiosis I, we utilized him-8 worms as a more tractable model. It has previously been shown that diakinesis stage him-8 oocytes have 5 autosomal bivalents and two X univalents (Phillips et al., 2005). If segregation of the two X univalents was random, these worms should produce equal numbers of nulloX and diploX ova. Instead, him-8 mutants produce a fivefold higher frequency of nulloX ova over diploX ova (Hodgkin et al., 1979), indicating that both maternal X univalents are lost at some time after diakinesis in a large fraction of embryos. To determine when maternal X univalents are preferentially lost, we imaged both live embryos within him-8 worms expressing GFP::tubulin and mCherry::histone (Figure 2A–E) and also fixed embryos stained with DAPI and anti-tubulin antibodies (Figure 2F–J). We assayed the number of chromosomes (defined here as DAPI-staining or mCherry:histone-positive bodies that would include univalents and bivalents) present at metaphase of meiosis I and II (Figure 2K). At meiosis I metaphase, 7 chromosomes were present in 96% of him-8 embryos (Figure 2B,K), with the remainder having 6 chromosomes. If the two univalents segregated randomly without losing cohesion, 25% of metaphase II spindles would be expected to have 5 autosomes and no X, 50% would have 5 autosomes and 1 X, and 25% would have 5 autosomes and 2 X chromosomes. Instead, 40% of him-8 metaphase II embryos had 5 chromosomes, 55% had 6 chromosomes, and only 5% had 7 chromosomes (Figure 2K). These frequencies differ significantly from those expected from unbiased segregation (chi-square test, two-tailed p < 0.0001), closely match the ratio of nulloX to diploX ova inferred by Hodgkin et al. (1979) and support the hypothesis that the majority of X univalents are eliminated between metaphase I and metaphase II. These maternal chromosome counts are also unaffected by nulloX or diploX sperm that might contribute to phenotype-based progeny counts. Figure 2 with 1 supplement see all Download asset Open asset X univalents are preferentially lost between metaphase I and metaphase II in him-8 mutants. Z projections of living (A–E) and fixed (F–J) C. elegans meiotic embryos viewed down the pole-to-pole spindle axis at metaphase I (A, B, F, G) or metaphase II (C, D, E, H, I, J). mCherry::Histone H2B and GFP::tubulin label the chromosomes and spindle, respectively, in live embryos. Fixed embryos were stained with DAPI, anti-tubulin antibody, and a LacO FISH probe that recognizes a LacO array integrated on the X chromosome. Asterisks indicate polar bodies. Insets show polar bodies that did not fit in the image frame. In (E), ‘s’ denotes a sperm outside of the embryo. Percentages are shown for each outcome (K, L). Bar = 5 μm. https://doi.org/10.7554/eLife.06056.004 To confirm that the two chromosomes lost between these stages were indeed the X univalents, we used FISH with a lac operator probe to detect a multi-copy lac operator array integrated on the X chromosomes in a him-8 background (Figure 2F–J). FISH revealed two X univalents and 7 total DAPI-staining bodies on 93% of all the him-8 metaphase I plates (Figure 2G,L). At metaphase II, FISH revealed at least two X hybridization foci in the first polar body and none on the metaphase plate when the spindle had 5 DAPI-staining bodies (Figure 2H). When 6 DAPI-staining bodies were present on the metaphase II plate, we always observed one X hybridization focus each on the metaphase plate and in the first polar body (Figure 2I). Finally, metaphase II embryos containing 7 DAPI-staining bodies had two X hybridization foci on the metaphase plate and none in the first polar body (Figure 2J). Together, these results demonstrate that both achiasmate X univalents are deposited into the first polar body in 40% of him-8 embryos as compared with the 25% expected from random segregation. To test whether achiasmate autosomes are also placed in the first polar body with higher than random frequency, we analyzed a strain with a lac operator array integrated on chromosome V and bearing a loss of function mutation in the him-8 homolog zim-2, which contributes to chromosome V pairing (Phillips and Dernburg, 2006). Unlike the situation with him-8 and pairing of the X, redundancy between ZIM proteins may contribute to chromosome V pairing. Phillips and Dernburg reported only 72% of diakinesis oocytes with 7 rather than 6 DAPI-staining bodies in a zim-2 mutant, and our zim-2 strain with lacO(V) had only 62% of diakinesis oocytes with 7 DAPI-staining bodies (Figure 2—figure supplement 1F). FISH revealed two distinct chromosome V hybridization foci and 7 DAPI-staining bodies on 41% of metaphase I spindles (Figure 2—figure supplement 1A,E). Starting with 41% achiasmate V's, random segregation should yield 10% of metaphase II embryos with both V's in the first polar body (25% of 41%). Instead, FISH revealed 27% of metaphase II embryos had five DAPI-staining bodies on the metaphase plate and chromosome V hybridization foci only in the first polar body (Figure 2—figure supplement 1B,E). Likewise, random segregation of achiasmate V's should yield 10% metaphase II spindles with 7 DAPI-staining bodies on the metaphase II spindle, two distinct chromosome V hybridization foci on the spindle, and none in the first polar body. Only 5% of this embryo class was observed (Figure 2—figure supplement 1D,E). These frequencies are significantly different than those expected from random segregation (chi-square test, two-tailed p < 0.0002). The discrepancy in the fraction of zim-2 oocytes with 7 DAPI-staining bodies at diakinesis vs metaphase I raises the possibility that chromosomes might be systematically undercounted in zim-2 metaphase I spindles (but not in wild-type, him-8, or XXX metaphase I spindles). If this is the case, the two V univalents must be positioned close together on the spindle because 0/57 zim-2 metaphase I plates with 6 DAPI-staining bodies had two widely spaced lacO(V) FISH foci and the deviation between expected and observed nulloV metaphase II spindles would be even greater. Two results strongly indicate that the same mechanisms acting on univalent X's in him-8 mutants also act on V univalents in the zim-2 mutant. First, the fivefold preponderance of metaphase II spindles with 5 DAPI-staining bodies over those with 7 DAPI-staining bodies is similar to him-8. Second, the presence of lacO FISH signal only in the first polar body of metaphase II embryos with 5 DAPI-staining bodies on the spindle is the same in him-8 and zim-2. Thus, achiasmate autosomes, like achiasmate X chromosomes, are preferentially deposited into the first polar body. Univalents biorient at metaphase I and tend to lag during anaphase I To understand the mechanism by which univalent X chromosomes are preferentially deposited in polar bodies, we examined their orientation and position in the spindle. Antibodies specific for the cohesin subunit, REC-8, label a cruciform on metaphase I bivalents (Figure 3A,B) and a single band on metaphase II chromosomes (Figure 3A,C). The single REC-8 bands on wild-type metaphase II chromosomes and on him-8 metaphase I univalents were both oriented perpendicular to the pole-to-pole axis of the spindle (Figure 3D), indicating that him-8 X univalents biorient at metaphase I. him-8 worms expressing GFP::KNL-2, which labels the C. elegans cup-shaped meiotic kinetochores (Dumont et al., 2010), were also analyzed for biorientation and yielded the same conclusion as analysis by REC-8 antibody (Figure 3—figure supplement 1). We also examined the localization of GFP:AIR-2, the aurora B kinase that is essential for the loss of cohesion at anaphase I and which is loaded between homologs of wild-type bivalents in a chiasma-dependent fashion (Rogers et al., 2002). The fluorescence intensity of GFP::AIR-2 was 2.3 times higher on bivalents than univalents at metaphase I of him-8 embryos (Figure 3—figure supplement 2B,C). AIR-2 is normally re-loaded between sister chromatids at metaphase II. GFP::AIR-2 on metaphase II chromosomes was 1.7 times higher than on him-8 metaphase I univalents (Figure 3—figure supplement 2C), indicating that the reduced amount of AIR-2 on metaphase I univalents was not simply a consequence of the smaller size of a univalent relative to a bivalent. AIR-2 is required for the crossover dependent, prometaphase, partial removal of REC-8 from between homologs in a wild-type bivalent, an event proposed to be essential for loss of cohesion at anaphase I (Severson and Meyer, 2014). Consistent with the low levels of AIR-2, him-8 univalents had 1.7 ± 0.3 times the intensity of REC-8 staining as the inter-homolog region of bivalents in the same spindle (Figure 3B; n = 8 embryos, two-tailed p = 0.04, chi square relative to expected 1.0). Figure 3 with 2 supplements see all Download asset Open asset X univalents biorient at metaphase I in him-8 embryos. (A) Cartoon diagram of REC-8 staining on bivalents and univalents. (B and C) Anti-REC-8 staining of metaphase I and metaphase II embryos with bivalents (yellow arrow head) and univalents (white arrow head). In him-8 embryos, univalents at metaphase I have a single band of REC-8 with the same orientation seen on normal chromosomes at metaphase II. (D) Quantification of the orientation of univalents by offset angle from the metaphase plate, 0° corresponds to perfect biorientation and 90° corresponds to perfect mono-orientation. Cortical pole is on the left in all images. Bar = 5 μm. https://doi.org/10.7554/eLife.06056.006 Because X univalents biorient at metaphase I but load half as much AIR-2, which is required for loss of cohesion at anaphase I in C. elegans (Kaitna et al., 2002; Rogers et al., 2002), and retain twice as much REC-8, we hypothesized that bioriented univalents might be pulled toward both spindle poles and lag on the anaphase spindle as they fail to lose cohesion. To test this possibility, we did time-lapse imaging of him-8 embryos expressing GFP::tubulin and mCherry::histone, focusing specifically on the events of anaphase I. 90% of him-8 embryos at anaphase I had one or two lagging chromosomes (n = 119) compared to 2% of wild-type embryos (n = 52) (Figure 4A,B). In 51% of living him-8 embryos with lagging chromosomes at anaphase I, two discrete lagging chromosomes could be resolved. Each lagging chromosome eventually moved as a single unit either toward the cortex or into the embryo in 98% of embryos (n = 179) (Figures 4B, 5A), indicating that cohesion between sister chromatids is maintained and that univalents are not broken or destroyed during anaphase. At anaphase II, only 10% of him-8 embryos exhibited lagging chromosomes (n = 60) and 0/22 wild-type embryos had lagging chromosomes, suggesting that lagging chromosomes are caused by the presence of univalents at meiosis I. Figure 4 Download asset Open asset X univalents lag at anaphase I. (A) Time-lapse images of a living wild-type embryo undergoing anaphase I show chromosomes separating as two distinct masses. (B) Time-lapse images of a living him-8 embryo show a lagging chromosome at anaphase I. (C–E) Z projections of fixed anaphase I embryos. (C) LacO FISH labeling of a wild-type strain with a LacO array integrated on the X chromosome shows normal segregation of two X homologs from one X bivalent. (D) LacO FISH shows that a lagging chromosome in him-8 is the X. (E) LacO FISH labeling of a zim-2 strain with a LacO array integrated on chromosome V showing a univalent V lagging at anaphase I. Cortical pole is to the left in all images. Bar = 5 μm. https://doi.org/10.7554/eLife.06056.009 Figure 5 Download asset Open asset The contractile ring moves inward past the lagging chromosomes of him-8 embryos. (A) Time-lapse sequence of anaphase I in a him-8 strain with GFP::PH, GFP::Tubulin, and mCherry::Histone H2B. The plasma membrane ingresses past the lagging chromosomes to engulf them in the polar body. (B) Fraction of him-8 anaphase I embryos in which a lagging chromosome eventually resolved toward the cortex or eventually resolved into the embryo (interior). Lagging univalents resolved more frequently toward the cortex during both early and late anaphase. Depletion of NMY-2, the myosin required for polar body formation, eliminated only the late anaphase bias. Pairwise two-tailed p values by Fisher's exact test: him-8 late vs him-8 nmy-2(RNAi) late = 0.02, him-8 early vs him-8 nmy-2(RNAi) early = 0.80, him-8 early vs him-8 late = 1.0, him-8 nmy-2(RNAi) early vs him-8 nmy-2(RNAi) late = 0.26. p values from Pearson's chi-squared test: him-8 late vs 50% = 0.003, him-8 nmy-2(RNAi) late vs 50% = 0.32, him-8 early vs 50% = 0.02, him-8 nmy-2(RNAi) early vs 50% = 0.38. (C) Top, diagram illustrating how the position of scission by the contractile ring along the pole-to-pole spindle axis was scored. Bottom, representative images from time-lapse sequences showing scission at different positions along the length of the spindle. (D) Average position of contractile ring scission along the pole-to-pole spindle axis in wild-type embryos and in him-8 embryos with no lagging chromosomes, lagging chromosomes that end up at the cortex (cortical), or lagging chromosomes that end up in the embryo (interior). Bar = 5 μm. https://doi.org/10.7554/eLife.06056.010 To confirm that the lagging chromosomes are bioriented X univalents, we used LacO(X) FISH. We found that the X-specific FISH probe labeled one or two lagging chromosomes at anaphase I of him-8 embryos, indicating that lagging chromosomes are X univalents (13/14) (Figure 4D). 36% of fixed him-8 anaphase I embryos with lagging chromosomes had two distinct FISH-positive chromosomes lagging. Another 21% had a single FISH-positive lagging body, but no other FISH-positive chromosomes on the spindle indicating that the two X univalents were likely too close to resolve in these embryos. The remaining 36% had one FISH-positive lagging chromosome and one FISH-positive chromosome in one of the main chromosome masses (one embryo had a lagging chromosome that was a bivalent). These results suggest that one or both X univalents lag in up to 90% of him-8 embryos. Similar results were obtained for chromosome V in zim-2 mutants, where 40% of metaphase I embryos have univalent V's (Figure 2—figure supplement 1). 27% (4/15 or over half of anaphase I spindles expected to have V univalents) had a lagging chromosome. 100% (4/4) of these lagging chromosomes were chromosome V as assayed by LacO(V) FISH (Figure 4E). These results indicate that achiasmate autosomes lag at anaphase I, just like achiasmate X chromosomes. The meiotic contractile ring captures lagging X univalents in the first polar body After establishing that lagging chromosomes were univalents, we next asked if univalents that lagged were subject to biased segregation at anaphase I. To analyze this, we conducted time-lapse imaging of embryos from him-8 worms expressing GFP::tubulin and mCherry::histone, as well as embryos from him-8 worms expressing these along with GFP::PH (plextrin homology domain) to label the plasma membrane (Figure 5A). Our time-lapse analysis revealed that 65% of lagging chromosomes eventually moved toward the cortex and the forming polar body of him-8 embryos during anaphase I (n = 181) (Figure 5B), indicating that preferential expulsion of lagging univalents into the first polar body could contribute to the higher than random frequency of metaphase II spindles with 5 autosomes and no X. Because the polar body contractile ring ingresses inward toward the midpoint of the late anaphase spindle where it normally scissions, we hypothesized that the preferential resolution of lagging chromosomes toward the cortex might result from inhibition of contractile ring scission until the ring ingresses past lagging chromosomes (Figure 5C). In wild type, ingression of the polar body contractile ring initiates when homologs have separated by 2.3 μm and polar body scission completes when homologs have separated by 5.6 μm (Fabritius et al., 2011b). The bias of univalents that moved toward the cortex before initiation of contractile ring ingression could not be caused by engulfment by the polar body. Therefore, we separated lagging univalents into two categories, early-resolving univalents that moved to one pole while the main chromosome masses were separated by less than 4.0 µm and late-resolving univalents that moved to one pole only after the main chromosome

Morphogenesis allows an organism to develop its final body shape. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network in the epidermis mediates the elongation of the worm embryo from a ball of cells into a long, thin worm. This process is controlled by two redundant pathways, one involving the small GTPase RHO-1 and its downstream effectors LET-502/Rho-binding kinase and MEL-11/myosin phosphatase, and another involving PAK-1/p21 activated kinase and FEM-2/PP2c phosphatase. Contraction occurs primarily in the lateral epidermal cells during elongation while the dorsal and ventral epidermal cells have a more passive role, and localized activity of a Rho GEF (guanine exchange factor) could contribute to this asymmetry. We found that loss of the C. elegans Rho GEF encoded by rhgf-2 results in arrest during early elongation. Genetically, rhgf-2 acts as an activator of let-502/Rho-binding kinase, in parallel to fem-2/PP2c phosphatase. Although expressed throughout the embryo, lateral cell-specific RHGF-2 expression can mediate elongation. The Rho GTPase activating protein (GAP) RGA-2 is known to inhibit contraction in the dorsal and ventral epidermis. Although rhgf-2 and rga-2 are individually lethal, the double mutant is viable with elongation still occurring in a let-502 dependent fashion. This indicates that LET-502/Rho-binding kinase has activity independent of the GEF and GAP. Finally, maternal LET-502 and MEL-11 are known to regulate the rate of cleavage furrow ingression in the early embryo and we show that maternal RHGF-2 also influences cleavage but RGA-2 does not. Thus while the LET-502/MEL-11 pathway is employed multiple times during embryogenesis, regulation by GEFs and GAPs differs at different points of the life cycle and fine tunes contractile function.

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions--ASPM-1 recruitment to the spindle and microtubule severing--both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.

During the second half of embryogenesis, the ellipsoidal Caenorhabditis elegans embryo elongates into a long, thin worm. This elongation requires a highly organized cytoskeleton composed of actin microfilaments, microtubules and intermediate filaments throughout the epidermis of the embryo. This architecture allows the embryonic epidermal cells to undergo a smooth muscle-like actin/myosin-based contraction that is redundantly controlled by LET- 502/Rho kinase and MEL-11/myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21-activated kinase in a parallel pathway(s). Although actin microfilaments surround the embryo, the force for contraction is generated mainly in the lateral (seam) epidermal cells whose actin microfilaments appear qualitatively different from those in their dorsal/ventral neighbors. We have identified FHOD-1, a formin family actin nucleator, which acts in the lateral epidermis. fhod-1 mutants show microfilament defects in the embryonic lateral epidermal cells and FHOD-1 protein is detected only in those cells. fhod-1 genetic interactions with let-502, mel-11, fem-2 and pak-1 indicate that fhod-1 preferentially regulates those microfilaments acting with let-502 and mel-11, and in parallel to fem-2 and pak-1. Thus, FHOD-1 may contribute to the qualitative differences in microfilaments found in the contractile lateral epidermal cells and their non-contractile dorsal and ventral neighbors. Different microfilament populations may be involved in the different contractile pathways.

Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3(MEL-26)), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3(MEL-26)-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.

The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism.

During the second half of embryogenesis, the ellipsoidal Caenorhabditis elegans embryo elongates into a long, thin worm. This elongation requires a highly organized cytoskeleton composed of actin microfilaments, microtubules and intermediate filaments throughout the epidermis of the embryo. This architecture allows the embryonic epidermal cells to undergo a smooth muscle-like actin/myosin-based contraction that is redundantly controlled by LET- 502/Rho kinase and MEL-11/myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21-activated kinase in a parallel pathway(s). Although actin microfilaments surround the embryo, the force for contraction is generated mainly in the lateral (seam) epidermal cells whose actin microfilaments appear qualitatively different from those in their dorsal/ventral neighbors. We have identified FHOD-1, a formin family actin nucleator, which acts in the lateral epidermis. fhod-1 mutants show microfilament defects in the embryonic lateral epidermal cells and FHOD-1 protein is detected only in those cells. fhod-1 genetic interactions with let-502, mel-11, fem-2 and pak-1 indicate that fhod-1 preferentially regulates those microfilaments acting with let-502 and mel-11, and in parallel to fem-2 and pak-1. Thus, FHOD-1 may contribute to the qualitative differences in microfilaments found in the contractile lateral epidermal cells and their non-contractile dorsal and ventral neighbors. Different microfilament populations may be involved in the different contractile pathways.