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1.  Histone Methylation Restrains the Expression of Subtype-Specific Genes during Terminal Neuronal Differentiation in Caenorhabditis elegans 
PLoS Genetics  2013;9(12):e1004017.
Although epigenetic control of stem cell fate choice is well established, little is known about epigenetic regulation of terminal neuronal differentiation. We found that some differences among the subtypes of Caenorhabditis elegans VC neurons, particularly the expression of the transcription factor gene unc-4, require histone modification, most likely H3K9 methylation. An EGF signal from the vulva alleviated the epigenetic repression of unc-4 in vulval VC neurons but not the more distant nonvulval VC cells, which kept unc-4 silenced. Loss of the H3K9 methyltransferase MET-2 or H3K9me2/3 binding proteins HPL-2 and LIN-61 or a novel chromodomain protein CEC-3 caused ectopic unc-4 expression in all VC neurons. Downstream of the EGF signaling in vulval VC neurons, the transcription factor LIN-11 and histone demethylases removed the suppressive histone marks and derepressed unc-4. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit. Thus, epigenetic mechanisms help establish subtype-specific gene expression, which are needed for optimal activity of a neural circuit.
Author Summary
As neurons differentiate they express specific genes that give them their distinctive shapes, activities, and functions. Much of this differentiation is controlled by the expression of transcription factors, proteins that turn on the expression of other genes. We find, however, that another aspect of terminal neuronal differentiation is the removal of inhibitory constraints on gene expression. These constraints often involve the modification of DNA or of general DNA binding proteins such as histones. This modification, referred to as epigenetic regulation, can activate or inactive genes without changing the genetic material. We found that the differentiation of nematode motor neurons was affected by genes involved in histone modification. Specifically, a gene that is needed in a subset of the motor neurons is initially turned off in all cells by histone modification. Mutation of histone modification genes causes the gene to be on in all cells. Normally, however, this removal of the inhibition is triggered by an external signal that only affects the specific cells.
PMCID: PMC3861114  PMID: 24348272
2.  Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy 
Nature cell biology  2012;15(1):10.1038/ncb2639.
Although single gene loss of function analyses can identify components of particular processes, important molecules are missed due to the robustness of biological systems. Here we show that large scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 C. elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologs. We experimentally confirmed functional roles predicted by the network and characterised through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.
PMCID: PMC3836181  PMID: 23242217
4.  Identification of DSB-1, a Protein Required for Initiation of Meiotic Recombination in Caenorhabditis elegans, Illuminates a Crossover Assurance Checkpoint 
PLoS Genetics  2013;9(8):e1003679.
Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.
Author Summary
For most eukaryotes, recombination between homologous chromosomes during meiosis is an essential aspect of sexual reproduction. Meiotic recombination is initiated by programmed double-strand breaks in DNA, which have the potential to induce mutations if not efficiently repaired. To better understand the mechanisms that govern the initiation of recombination and regulate the formation of double-strand breaks, we use the nematode Caenorhabditis elegans as a model system. Here we describe a new gene, dsb-1, that is required for double-strand break formation in C. elegans. Through analysis of the encoded DSB-1 protein we illuminate an important regulatory pathway that promotes crossover recombination events on all chromosome pairs to ensure successful meiosis.
PMCID: PMC3749324  PMID: 23990794
5.  A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans 
eLife  2013;2:e00429.
Regulation of animal development in response to nutritional cues is an intensely studied problem related to disease and aging. While extensive studies indicated roles of the Target of Rapamycin (TOR) in sensing certain nutrients for controlling growth and metabolism, the roles of fatty acids and lipids in TOR-involved nutrient/food responses are obscure. Caenorhabditis elegans halts postembryonic growth and development shortly after hatching in response to monomethyl branched-chain fatty acid (mmBCFA) deficiency. Here, we report that an mmBCFA-derived sphingolipid, d17iso-glucosylceramide, is a critical metabolite in regulating growth and development. Further analysis indicated that this lipid function is mediated by TORC1 and antagonized by the NPRL-2/3 complex in the intestine. Strikingly, the essential lipid function is bypassed by activating TORC1 or inhibiting NPRL-2/3. Our findings uncover a novel lipid-TORC1 signaling pathway that coordinates nutrient and metabolic status with growth and development, advancing our understanding of the physiological roles of mmBCFAs, ceramides, and TOR.
eLife digest
Animals require nutrients, including carbohydrates, lipids, and amino acids, for development and growth, and to maintain the normal functioning of cells. However, in most natural environments, the availability of food tends to fluctuate. Some animals have therefore acquired the ability to dramatically reduce their metabolic activity, and thus their energy and nutrient needs to survive fasting conditions.
Caenorhabditis elegans is a transparent nematode worm that is used extensively as a model organism. When C. elegans larvae hatch in a food-free environment, they enter a quiescent state in which they suspend growth and cell division to conserve energy. However, the mechanisms that underlie this ability are not fully understood.
Here, Zhu et al. reveal that a type of lipid called a sphingolipid is required for C. elegans larvae to begin postembryonic development. When this lipid is absent in the environment and not synthesized internally, the larvae remain in a state of arrested development, which can be overcome by resupplying the lipid. Zhu et al. show that the lipid acts through a signaling pathway involving an enzyme complex called TORC1 and that the effect of the lipid can be blocked by another protein complex called NPRL-2/3. TORC1 is well known for its role in sensing amino acids and growth factors, but this is the first time that it has been shown to be involved in detecting lipids. Strikingly, Zhu et al. also show that, in the absence of the lipid, postembryonic growth and development can be initiated by activating TORC1 or inhibiting NPRL-2/3.
The work of Zhu et al. thus reveals a novel regulatory function of a specific fatty acid and sphingolipid variant that is used by C. elegans to coordinate its growth and development with its metabolic status or the availability of nutrients. Since all components of the pathway are conserved in mammals, the results could help to improve our understanding of how caloric restriction influences human health and aging.
PMCID: PMC3660743  PMID: 23705068
branched-chain fatty acid; growth arrest; nutrient sensing; NPRL; glucosylceramide; target of rapamycin; C. elegans
6.  Duplication and Retention Biases of Essential and Non-Essential Genes Revealed by Systematic Knockdown Analyses 
PLoS Genetics  2013;9(5):e1003330.
When a duplicate gene has no apparent loss-of-function phenotype, it is commonly considered that the phenotype has been masked as a result of functional redundancy with the remaining paralog. This is supported by indirect evidence showing that multi-copy genes show loss-of-function phenotypes less often than single-copy genes and by direct tests of phenotype masking using select gene sets. Here we take a systematic genome-wide RNA interference approach to assess phenotype masking in paralog pairs in the Caenorhabditis elegans genome. Remarkably, in contrast to expectations, we find that phenotype masking makes only a minor contribution to the low knockdown phenotype rate for duplicate genes. Instead, we find that non-essential genes are highly over-represented among duplicates, leading to a low observed loss-of-function phenotype rate. We further find that duplicate pairs derived from essential and non-essential genes have contrasting evolutionary dynamics: whereas non-essential genes are both more often successfully duplicated (fixed) and lost, essential genes are less often duplicated but upon successful duplication are maintained over longer periods. We expect the fundamental evolutionary duplication dynamics presented here to be broadly applicable.
Author Summary
Duplicate genes occur in all organisms. It has been found that mutations in duplicate genes cause defects much less often than when single copy genes are mutated. It is widely believed that this is due to functional redundancy—that is, the two genes can carry out similar functions so that the non-mutated duplicate gene can cover for or “mask” the phenotype of the mutation in the first duplicate. To determine whether this hypothesis is true, it is necessary to test systematically whether defects indeed occur in the organism when both duplicate genes are inhibited. We have for the first time carried out such an analysis in a multicellular organism, the nematode Caenorhabditis elegans. In contrast to expectations, we observed that when both copies of duplicate genes are inhibited deleterious effects are very rare. We show that this is because duplicate genes are much more often non-essential compared to genes where there is only a single copy. Non-essential genes are also lost from the genome much more often than essential genes. However, when essential genes are duplicated, they remain present in the genome over longer periods. Our results give a framework to explain the evolutionary dynamics of duplications in the genome.
PMCID: PMC3649981  PMID: 23675306
8.  Neurons Refine the Caenorhabditis elegans Body Plan by Directing Axial Patterning by Wnts 
PLoS Biology  2013;11(1):e1001465.
In Caenorhabditis elegans, the axons of specific neurons help direct the location and strength of Wnt signaling that patterns the epidermis.
Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior–posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.
Author Summary
How a limited number of conserved growth factors such as Wnts generate diverse bodies throughout the animal kingdom is a fundamental question in developmental and evolutionary biology. Diversity is thought to arise in part through variations in the strength and location of growth factor signaling. How the signaling properties of growth factors are precisely tuned at specific locations to generate distinct tissue patterns is not well understood. Here, we present evidence that the axons of two specific neurons that span the anterior–posterior axis help pattern the epidermis of the nematode Caenorhabditis elegans. When the posteriorly directed axons of these neurons fail to grow to their normal length, the symmetry of the mid-body vulva is altered, and additional vulval tissue inappropriately forms in the posterior epidermis. We further present evidence that these neurons direct epidermal patterning by binding and sequestering posteriorly derived Wnts, thereby refining the strength and location of Wnt signaling along the anterior–posterior axis. We postulate that the evolution of neurons not only improved animals by endowing them with complex behaviors, but also by helping expand the diversity of body patterns generated by growth factors.
PMCID: PMC3539944  PMID: 23319891
9.  H4K20me1 Contributes to Downregulation of X-Linked Genes for C. elegans Dosage Compensation 
PLoS Genetics  2012;8(9):e1002933.
The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.
Author Summary
In many animals, males have one X chromosome and females have two. However, the same amount of gene expression from X chromosomes is needed in the two sexes. The process of dosage compensation (DC) globally regulates X-chromosome gene expression to make it equal between the sexes, and it occurs in different ways in different animals. In mammals, one X chromosome in females is randomly inactivated, leaving one active X chromosome. In contrast, in the nematode worm C. elegans, the two X chromosomes in hermaphrodites are repressed two-fold to match gene expression to the single X chromosome in males. Previous work in C. elegans identified proteins required for DC that bind to the X chromosome, but their mode of action is not known. Here we show that DC proteins lead to higher levels of histone H4 lysine 20 monomethylation (H4K20me1) on hermaphrodite X chromosomes and that H4K20me1 functions in repressing X-chromosome gene expression. This shows that histone modification is an important aspect of the mechanism of dosage compensation. Together with previous work linking H4K20me1 to chromatin structure regulation, our results suggest that dosage compensation might lower gene expression on hermaphrodite X chromosomes by compacting them.
PMCID: PMC3441679  PMID: 23028348
10.  Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types 
Developmental biology  2007;305(1):347-357.
During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the C. elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.
PMCID: PMC3330270  PMID: 17383625
C. elegans; cell migration; polarity; vulva; anchor cell; gonad; Cdc42; Par3; Par6; aPKC
11.  An assessment of histone-modification antibody quality 
We report testing of the specificity and utility of over 200 antibodies raised against 57 different histone modifications, in Drosophila melanogaster, Caenorhabditis elegans and human cells. While most antibodies performed well, over 25% failed specificity tests by dot blot or western blot. Among specific antibodies, over 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use and provide a website for posting new test results.
PMCID: PMC3017233  PMID: 21131980
12.  Systematic bias in high-throughput sequencing data and its correction by BEADS 
Nucleic Acids Research  2011;39(15):e103.
Genomic sequences obtained through high-throughput sequencing are not uniformly distributed across the genome. For example, sequencing data of total genomic DNA show significant, yet unexpected enrichments on promoters and exons. This systematic bias is a particular problem for techniques such as chromatin immunoprecipitation, where the signal for a target factor is plotted across genomic features. We have focused on data obtained from Illumina’s Genome Analyser platform, where at least three factors contribute to sequence bias: GC content, mappability of sequencing reads, and regional biases that might be generated by local structure. We show that relying on input control as a normalizer is not generally appropriate due to sample to sample variation in bias. To correct sequence bias, we present BEADS (bias elimination algorithm for deep sequencing), a simple three-step normalization scheme that successfully unmasks real binding patterns in ChIP-seq data. We suggest that this procedure be done routinely prior to data interpretation and downstream analyses.
PMCID: PMC3159482  PMID: 21646344
13.  MosSCI and Gateway Compatible Plasmid Toolkit for Constitutive and Inducible Expression of Transgenes in the C. elegans Germline 
PLoS ONE  2011;6(5):e20082.
Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3′UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.
PMCID: PMC3102689  PMID: 21637852
14.  The caenorhabditis elegans CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells 
Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified.
We have characterised CDT-2's role during vulva development and found that it is a novel attenuator of LET-23 signalling. CDT-2 acts redundantly with negative modulators of LET-23 signalling and CDT-2 or CUL-4 downregulation causes persistent expression of the egl-17::cfp transgene, a marker of LET-23 signalling during vulva development. In addition, we show that CDT-2 physically interacts with SEM-5 (GRB2), a known negative modulator of LET-23 signalling that directly binds LET-23, and provide genetic evidence consistent with CDT-2 functioning at or downstream of LET-23. Interestingly, both SEM-5 and CDT-2 were identified independently in a screen for genes involved in receptor-mediated endocytosis in oocytes, suggesting that attenuation of LET-23 by CDT-2 might be through regulation of endocytosis.
In this study, we have shown that CDT-2 and CUL-4, members of the CUL-4/DDB-1/CDT-2 E3 ubiquitin ligase complex attenuate LET-23 signalling in vulval precursor cells. In future, it will be interesting to investigate the potential link to endocytosis and to determine whether other signalling pathways dependent on endocytosis, e.g. LIN-12 (Notch) could be regulated by this ubiquitin ligase complex. This work has uncovered a novel function for the CUL-4/DDB-1/CDT-2 E3 ligase that may be relevant for its mammalian oncogenic activity.
PMCID: PMC2984460  PMID: 20977703
15.  Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans 
PLoS Biology  2010;8(8):e1000450.
Several hundred proteins become insoluble and aggregation-prone as a consequence of aging in Caenorhabditis elegans. The data indicate that these proteins influence disease-related protein aggregation and toxicity.
Aberrant protein aggregation is a hallmark of many age-related diseases, yet little is known about whether proteins aggregate with age in a non-disease setting. Using a systematic proteomics approach, we identified several hundred proteins that become more insoluble with age in the multicellular organism Caenorhabditis elegans. These proteins are predicted to be significantly enriched in β-sheets, which promote disease protein aggregation. Strikingly, these insoluble proteins are highly over-represented in aggregates found in human neurodegeneration. We examined several of these proteins in vivo and confirmed their propensity to aggregate with age. Different proteins aggregated in different tissues and cellular compartments. Protein insolubility and aggregation were significantly delayed or even halted by reduced insulin/IGF-1-signaling, which also slows aging. We found a significant overlap between proteins that become insoluble and proteins that influence lifespan and/or polyglutamine-repeat aggregation. Moreover, overexpressing one aggregating protein enhanced polyglutamine-repeat pathology. Together our findings indicate that widespread protein insolubility and aggregation is an inherent part of aging and that it may influence both lifespan and neurodegenerative disease.
Author Summary
In neurodegenerative diseases, such as Alzheimer's disease and Huntington's disease, specific proteins escape the cell's quality-control system and associate together, forming insoluble aggregates. Until now, little was known about whether proteins aggregate in a non-disease context. In this study, we discovered that the aging process itself, in the absence of disease, leads to the insolubilization and increased aggregation propensity of several hundred proteins in the roundworm Caenorhabditis elegans. These aggregation-prone proteins have distinct structural and functional proprieties. We asked if this inherent age-dependent protein aggregation impacts neurodegenerative diseases. We found that proteins similar to those aggregating in old worms have also been identified as minor components of human disease aggregates. In addition, we showed that higher levels of inherent protein aggregation aggravated toxicity in a C. elegans Huntington's disease model. Inherent protein aggregation is a new biomarker of aging. Understanding how to modulate it will lead to important insights into the mechanisms that underlie aging and protein aggregation diseases.
PMCID: PMC2919420  PMID: 20711477
16.  Differential chromatin marking of introns and expressed exons by H3K36me3 
Nature genetics  2009;41(3):376-381.
Variation in patterns of methylations of histone tails reflects and modulates chromatin structure and function1-3. To provide a framework for the analysis of chromatin function in C. elegans, we generated a genome-wide map of histone H3 tail methylations. We find that C. elegans genes show similarities in distributions of histone modifications to those of other organisms, with H3K4me3 near transcription start sites, H3K36me3 in the body of genes, and H3K9me3 enriched on silent genes. Unexpectedly, we also observe a striking novel pattern: exons are preferentially marked with H3K36me3 relative to introns. H3K36me3 exon marking is dependent on transcription and its level is lower in alternatively spliced exons, supporting a splicing related marking mechanism. We further show that the difference in H3K36me3 marking between exons and introns is evolutionarily conserved in human and mouse. We propose that H3K36me3 exon marking in chromatin provides a dynamic link between transcription and splicing.
PMCID: PMC2648722  PMID: 19182803
17.  A Cell Cycle Timer for Asymmetric Spindle Positioning 
PLoS Biology  2009;7(4):e1000088.
The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC), its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK). Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.
Author Summary
Throughout animal development, and in stem cells, many cell divisions are asymmetric. The one-cell-stage C. elegans embryo divides asymmetrically, as a result of a displacement of the mitotic spindle to one side of the cell. As in other cell divisions, a mitotic progression machinery ensures that all chromosomes are associated with the metaphase plate before anaphase begins. This machinery involves the anaphase-promoting complex and its activator Cdc20/Fizzy, which target proteins for destruction by the proteasome; the cyclin that is targeted for degradation by the proteasome; and a cyclin-dependent kinase. We have asked whether the same machinery has a second function, delaying movement of the spindle to an asymmetric position until spindle assembly is complete. To address this question, we used genetic, reverse genetic, and pharmacological techniques to disrupt the function of elements of the mitotic progression machinery. We find that the mitotic progression machinery does indeed time spindle positioning, acting to delay spindle displacement until spindle assembly completes. This demonstrates a previously unrecognized link between the mitotic progression machinery and asymmetric spindle positioning in an animal cell.
The machinery that times entry into mitotic anaphase has the extra function during an asymmetric cell division of regulating when the mitotic spindle can shift to one side of a cell.
PMCID: PMC2671557  PMID: 19385718
18.  PAR proteins direct asymmetry of the cell cycle regulators Polo-like kinase and Cdc25 
The Journal of Cell Biology  2008;180(5):877-885.
Cell cycle lengths vary widely among different cells within an animal, yet mechanisms of cell cycle length regulation are poorly understood. In the Caenorhabditis elegans embryo, the first cell division produces two cells with different cell cycle lengths, which are dependent on the conserved partitioning-defective (PAR) polarity proteins. We show that two key cell cycle regulators, the Polo-like kinase PLK-1 and the cyclin-dependent kinase phosphatase CDC-25.1, are asymmetrically distributed in early embryos. PLK-1 shows anterior cytoplasmic enrichment and CDC-25.1 shows PLK-1–dependent enrichment in the anterior nucleus. Both proteins are required for normal mitotic progression. Furthermore, these asymmetries are controlled by PAR proteins and the muscle excess (MEX) proteins MEX-5/MEX-6, and the latter is linked to protein degradation. Our results support a model whereby the PAR and MEX-5/MEX-6 proteins asymmetrically control PLK-1 levels, which asymmetrically regulates CDC-25.1 to promote differences in cell cycle lengths. We suggest that control of Plk1 and Cdc25 may be relevant to regulation of cell cycle length in other developmental contexts.
PMCID: PMC2265398  PMID: 18316412
19.  A Casein Kinase 1 and PAR Proteins Regulate Asymmetry of a PIP2 Synthesis Enzyme for Asymmetric Spindle Positioning 
Developmental Cell  2008;15(2):198-208.
Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Gα regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP2 synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Gα regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP2. We propose that asymmetric generation of PIP2 by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.
PMCID: PMC2686839  PMID: 18694560
20.  OSM-11 Facilitates LIN-12 Notch Signaling during Caenorhabditis elegans Vulval Development  
PLoS Biology  2008;6(8):e196.
Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.
Author Summary
The classic view of Notch receptor activation involves receptor binding to transmembrane Notch ligands that contain a conserved DSL (Delta, Serrate, and LAG-2) domain. Here, we find that the Caenorhabditis elegans OSM-11 protein is a novel ligand of the well-characterized Notch signal transduction pathway and plays a role in cell fate specification during development. OSM-11 is a secreted, diffusible protein whose loss decreases Notch signaling in vivo. OSM-11 and related C. elegans proteins do not contain a DSL domain, but contain a conserved motif we have named DOS (Delta and OSM-11) that is also found in the extracellular domain of known Notch ligands in organisms other than C. elegans. The functional mammalian homolog of OSM-11 is the secreted protein Deltalike1 (Dlk1), also known as Preadipocyte Factor 1 (PREF1), which plays a poorly defined role in Notch signaling regulating obesity and other developmental decisions. This suggests that Notch ligands are split into two complementary coligand families that act together to regulate Notch signaling in developmental contexts. In addition to regulating development, DOS ligands play roles in osmotic stress and C. elegans behavior, suggesting previously unsuspected roles for Notch signaling across species.
The C. elegans OSM-11 protein acts with DSL ligands to activate Notch signaling in cell fate specification and defines a conserved family of potential Notch co-ligands.
PMCID: PMC2504490  PMID: 18700817
21.  Microtubules are involved in anterior-posterior axis formation in C. elegans embryos 
The Journal of Cell Biology  2007;179(3):397-402.
Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O'Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55–70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89–92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673–684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92–96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527–3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.
PMCID: PMC2064787  PMID: 17967950
22.  Control of Apoptosis by Asymmetric Cell Division 
PLoS Biology  2008;6(4):e84.
Asymmetric cell division and apoptosis (programmed cell death) are two fundamental processes that are important for the development and function of multicellular organisms. We have found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the MIDA1-like protein GlsA of the alga Volvox carteri, as well as the Snail-related proteins Snail, Escargot, and Worniu of Drosophila melanogaster, have previously been implicated in asymmetric cell division. Therefore, C. elegans dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail may be components of a pathway involved in asymmetric cell division that is conserved throughout the plant and animal kingdoms. Furthermore, based on our results, we propose that this pathway directly controls the apoptotic fate in C. elegans, and possibly other animals as well.
Author Summary
Asymmetric cell division and apoptosis (programmed cell death) are two fundamental processes that are important for the development and function of multicellular organisms. Asymmetric cell division creates daughter cells of different fates, and this is critical for the generation of cellular diversity. Apoptosis eliminates superfluous cells from the organism, which is critical for cellular homeostasis. We found that the processes of asymmetric cell division and apoptosis can be functionally linked. Specifically, we show that asymmetric cell division in the nematode Caenorhabditis elegans is mediated by a pathway involving three genes, dnj-11 MIDA1, ces-2 HLF, and ces-1 Snail, that directly control the enzymatic machinery responsible for apoptosis. Interestingly, the role of this pathway in asymmetric cell division and the control of apoptosis might be evolutionarily conserved. Furthermore, it might have an unexpected role in stem cell biology: the process of asymmetric cell division plays an essential role in the ability of stem cells to self-renew, and the mammalian counterparts of two components of the dnj-11 MIDA1, ces-2 HLF, ces-1 Snail pathway have recently been implicated in stem cell function. For this reason, we speculate that a dnj-11 MIDA1, ces-2 HLF, ces-1 Snail–like pathway might function in stem cells to coordinate self-renewal and apoptosis and, hence, the number of stem cells.
A pathway involved in asymmetric cell division in the nematode Caenorhabditis elegans, the dnj-11 MIDA1, ces-2 HLF, ces-1 Snail pathway, directly controls the enzymatic machinery responsible for apoptosis.
PMCID: PMC2288629  PMID: 18399720
23.  Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins 
PLoS Genetics  2007;3(12):e233.
Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.
Author Summary
The mitogen-activated protein (MAP) kinase (MAPK) enzyme is crucial for regulation of both stem cell maintenance and tumorigenesis. Two conserved controls of MAPK include its activation by RAS signaling and a kinase cascade as well as its inactivation by MAPK phosphatases (MKPs). We identify a third mode of conserved MAPK regulation. We demonstrate that PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins repress mRNAs encoding MAPK enzymes in both the Caenorhabditis elegans germline and human embryonic stem cells. PUF proteins have emerged as conserved regulators of germline stem cells in C. elegans, Drosophila, and probably vertebrates. Their molecular mode of action relies on binding to sequence elements in the 3′ untranslated region of target mRNAs. We report that PUF proteins bind and repress mRNAs encoding C. elegans MPK-1 as well as human ERK2 and p38α. We also report that PUF repression and MKP inactivation function redundantly in the C. elegans germline to restrict MPK-1/MAPK activity and prevent germ cell apoptosis. We suggest that this dual regulation of MAPK activity by PUF and MKP proteins may be a conserved mechanism for the control of growth and differentiation during animal development and tissue homeostasis.
PMCID: PMC2323325  PMID: 18166083
24.  Asymmetry of Early Endosome Distribution in C. elegans Embryos 
PLoS ONE  2007;2(6):e493.
Endocytosis is involved in the regulation of many cellular events, including signalling, cell migration, and cell polarity. To begin to investigate roles for endocytosis in early C. elegans development, we examined the distribution and dynamics of early endosomes (EEs) in embryos.
Methodology/Principal Findings
EEs are primarily found at the cell periphery with an initially uniform distribution after fertilization. Strikingly, we find that during the first cell cycle, EEA-1 positive EEs become enriched at the anterior cortex. In contrast, the Golgi compartment shows no asymmetry in distribution. Asymmetric enrichment of EEs depends on acto-myosin contractility and embryonic PAR polarity. In addition to their localization at the cortex, EEs are also found around the centrosome. These EEs move rapidly (1.3um/s) from the cortex directly to the centrosome, a speed comparable to that of the minus end directed motor dynein.
We speculate that the asymmetry of early endosomes might play a role in cell asymmetries or fate decisions.
PMCID: PMC1876258  PMID: 17551574
25.  Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast 
RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.
Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Δ mutant.
These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.
PMCID: PMC1847674  PMID: 17374146

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