An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos.

In Caenorhabditis elegans, the MEI-1-katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3 untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1

pop-1 Encodes an HMG box protein required for the specification of a mesoderm precursor in Early C. elegans embryos

AbstractIn C. elegans embryogenesis, the MS blastomere produces predominantly mesodermal cell types, while its sister E generates only endodermal tissue. We show that a maternal gene, pop-1, is essential for the specification of MS fate and that a mutation in pop-1 results in MS adopting an E fate. Previous studies have shown that the maternal gene skn-1 is required for both MS and E development and that skn-1 encodes a transcription factor. We show here that the pop-1 gene encodes a protein with an HMG box similar to the HMG boxes in the vertebrate lymphoid-specifictranscriptional regulators TCF-1 and LEF-1. We propose that POP-1 and SKN-1 function together in the early embryo to allow MS-specific differentiation

The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7α and CeGLC-7β, abolishes the restricted localization pattern of AIR-2. In Ceglc-7α/β(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7α/β(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7α/β, directly or indirectly, antagonize AIR-2 activity

Multiple RNA-binding proteins function combinatorially to control the soma-restricted expression pattern of the E3 ligase subunit ZIF-1

AbstractIn C. elegans embryos, transcriptional repression in germline blastomeres requires PIE-1 protein. Germline blastomere-specific localization of PIE-1 depends, in part, upon regulated degradation of PIE-1 in somatic cells. We and others have shown that the temporal and spatial regulation of PIE-1 degradation is controlled by translation of the substrate-binding subunit, ZIF-1, of an E3 ligase. We now show that ZIF-1 expression in embryos is regulated by five maternally-supplied RNA-binding proteins. POS-1, MEX-3, and SPN-4 function as repressors of ZIF-1 expression, whereas MEX-5 and MEX-6 antagonize this repression. All five proteins bind directly to the zif-1 3′ UTR in vitro. We show that, in vivo, POS-1 and MEX-5/6 have antagonistic roles in ZIF-1 expression. In vitro, they bind to a common region of the zif-1 3′ UTR, with MEX-5 binding impeding that by POS-1. The region of the zif-1 3′ UTR bound by MEX-5/6 also partially overlaps with that bound by MEX-3, consistent with their antagonistic functions on ZIF-1 expression in vivo. Whereas both MEX-3 and SPN-4 repress ZIF-1 expression, neither protein alone appears to be sufficient, suggesting that they function together in ZIF-1 repression. We propose that MEX-3 and SPN-4 repress ZIF-1 expression exclusively in 1- and 2-cell embryos, the only period during embryogenesis when these two proteins co-localize. As the embryo divides, ZIF-1 continues to be repressed in germline blastomeres by POS-1, a germline blastomere-specific protein. MEX-5/6 antagonize repression by POS-1 and MEX-3, enabling ZIF-1 expression in somatic blastomeres. We propose that ZIF-1 expression results from a net summation of complex positive and negative translational regulation by 3′ UTR-binding proteins, with expression in a specific blastomere dependent upon the precise combination of these proteins in that cell

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation, results in delayed degradation of maternal proteins and embryonic lethality

AbstractIn vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos

Transmission Dynamics of Heritable Silencing Induced by Double-Stranded RNA in Caenorhabditis elegans

Heritable silencing effects are gene suppression phenomena that can persist for generations after induction. In the majority of RNAi experiments conducted in Caenorhabditis elegans, the silencing response results in a hypomorphic phenotype where the effects recede after the F1 generation. F2 and subsequent generations revert to the original phenotype. Specific examples of transgenerational RNAi in which effects persist to the F2 generation and beyond have been described. In this study, we describe a systematic pedigree-based analysis of heritable silencing processes resulting from initiation of interference targeted at the C. elegans oocyte maturation factor oma-1. Heritable silencing of oma-1 is a dose-dependent process where the inheritance of the silencing factor is unequally distributed among the population. Heritability is not constant over generational time, with silenced populations appearing to undergo a bottleneck three to four generations following microinjection of RNA. Transmission of silencing through these generations can be through either maternal or paternal gamete lines and is surprisingly more effective through the male gametic line. Genetic linkage tests reveal that silencing in the early generations is transmitted independently of the original targeted locus, in a manner indicative of a diffusible epigenetic element

POP-1 and Anterior–Posterior Fate Decisions in C. elegans Embryos

AbstractBlastomeres in C. elegans embryos execute lineage programs wherein the fate of a cell is correlated reproducibly with the division sequence by which that cell is born. We provide evidence that the pop-1 gene functions to link anterior–posterior cell divisions with cell fate decisions. Each anterior cell resulting from an anterior–posterior division appears to have a higher level of nuclear POP-1 protein than does its posterior sister. Genes in the C. elegans Wnt pathway are required for this inequality in POP-1 levels. We show that loss of pop-1(+) activity leads to several types of anterior cells adopting the fates of their posterior sisters. These results suggest a mechanism for the invariance of blastomere lineages

Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection

Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of C. elegans with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection

Uncoupling Different Characteristics of the <i>C. elegans</i> E Lineage from Differentiation of Intestinal Markers

<div><p>In the 4-cell <i>C. elegans</i> embryo, a signal from P₂ to its anterior sister, EMS, specifies the posterior daughter of EMS, E, as the sole founder cell for intestine. The P₂-to-EMS signal restricts high level zygotic expression of the redundant GATA transcription factors, END-1 and END-3, to only the E lineage. Expression of END-1 or END-3 in early blastomeres is sufficient to drive intestinal differentiation. We show here that a number of E lineage characteristics, which are also regulated through P₂-EMS signaling, can be uncoupled from intestine development, and each with a different sensitivity to specific perturbations of the P₂-EMS signal. For example, we show that the extended cell cycle in Ea and Ep depends on the P₂-induced high level expression of the cell cycle regulator, WEE-1.1, in E. A mutation in <i>wee-1.1</i> results in shortened Ea and Ep cell cycles, but has no effect upon intestinal differentiation or embryogenesis. Furthermore, it has been shown previously that the total number of E lineage cell divisions is regulated by a mechanism dependent upon E being specified as the intestinal founder cell. We now show, however, that cell division counting can be uncoupled from intestine differentiation in the E lineage. Many mutations in P₂-EMS signal genes exhibit nonfully-penetrant defects in intestinal differentiation. When embryos with those mutations generate intestinal cells, they often make too many intestinal cells. In addition, at the level of individual embryos, expression of <i>end-1</i> and <i>end-3</i>, and another very early E-specific zygotic gene, <i>sdz-23</i>, exhibit stochastic and discordant defects in P₂-EMS signaling mutants. We show here that <i>sdz-23</i> is expressed close to wildtype levels in embryos deleted of both <i>end-1</i> and <i>end-3</i>. <i>sdz-23</i> does not appear to function in intestine development, raising the intriguing possibility that the P₂-EMS interaction has downstream molecular consequences within the E lineage independent of <i>end-1/3</i> and intestinal development.</p></div

Single embryo gene expression analysis.

<p>Total cDNA was PCR amplified from poly-A+ RNA prepared from individual wildtype or mutant 12-cell stage embryos (indicated above each blot compilation), separated briefly on an agarose gel side-by-side with like-staged samples of the same genotype, and blotted to membrane ('pseudo Northern' blot; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106309#s4" target="_blank">Materials and Methods</a>). Replicate blots were hybridized with ³²-P labeled probes prepared from the <i>end-1</i>, <i>end-3</i>, and <i>sdz-23</i> genes, and the <i>tba-1</i> (alpha tubulin) gene as a loading control. Exposures were to x-ray film which was then scanned. All <i>end-1</i>, <i>end-3</i> and <i>sdz-23</i> exposures were for a similar length of time (18–20 hours), whereas the <i>tba-1</i> exposures were much shorter (10–15 minutes). <i>itDf2</i> is a deletion that removes the genomic region containing both <i>end-1</i> and <i>end-3</i> (along with a number of other genes). Dots below lane numbers denote samples from individual <i>mom-1(or10)</i>, <i>mom-2(or42)</i>, <i>mom-3(or78)</i> and <i>mom-4(ne19)</i> mutant embryos that express greatly reduced combined levels of <i>end-1</i> and <i>end-3</i>.</p