In vivo labeling of endogenous genomic loci in C. elegans using CRISPR/dCas9

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated

miRNAs cooperate in apoptosis regulation during C. elegans development

Programmed cell death occurs in a highly reproducible manner during Caenorhabditis elegans development. We demonstrate that, during embryogenesis, miR-35 and miR-58 bantam family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene egl-1, which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of egl-1 is dependent on binding sites for miR-35 and miR-58 family miRNAs within the egl-1 3\u27 untranslated region (UTR), which affect both mRNA copy number and translation. Furthermore, using single-molecule RNA fluorescent in situ hybridization (smRNA FISH), we show that egl-1 is transcribed in the mother of a cell programmed to die and that miR-35 and miR-58 family miRNAs prevent this mother from dying by keeping the copy number of egl-1 mRNA below a critical threshold. Finally, miR-35 and miR-58 family miRNAs can also dampen the transcriptional boost of egl-1 that occurs specifically in a daughter cell that is programmed to die. We propose that miRNAs compensate for lineage-specific differences in egl-1 transcriptional activation, thus ensuring that EGL-1 activity reaches the threshold necessary to trigger death only in daughter cells that are programmed to die

Twenty million years of evolution: The embryogenesis of four Caenorhabditis species are indistinguishable despite extensive genome divergence

The four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri show more divergence at the genomic level than humans compared to mice (Stein et al., 2003; Cutter et al., 2006, 2008). However, the behavior and anatomy of these nematodes are very similar. We present a detailed analysis of the embryonic development of these species using 4D-microscopic analyses of embryos including lineage analysis, terminal differentiation patterns and bioinformatical quantifications of cell behavior. Further functional experiments support the notion that the early development of all four species depends on identical induction patterns. Based on our results, the embryonic development of all four Caenorhabditis species are nearly identical, suggesting that an apparently optimal program to construct the body plan of nematodes has been conserved for at least 20 million years. This contrasts the levels of divergence between the genomes and the protein orthologs of the Caenorhabditis species, which is comparable to the level of divergence between mouse and human. This indicates an intricate relationship between the structure of genomes and the morphology of animals.publishedVersio

A Complex Regulatory Network Coordinating Cell Cycles During C. elegans Development Is Revealed by a Genome-Wide RNAi Screen

The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development

Pattern formation: Cell focusing in Caenorhabditis elegans

Im C. elegans Embryo basiert die Musterbildung auf dem globalen Sortieren von Zellen. Der Mechanismus wurde von Schnabel et al. als Zellfokussierung bezeichnet. Die Zellposition wird abhängig vom Zellschicksal festgelegt. Diese Arbeit ist dem Problem gewidmet, mit welchen Mechanismen Zellen sortiert werden können und wie sie sich "erkennen". Oder, was ist die molekulare Basis für die Zellfokussierung. Da während der embryonal, als auch während der larvalen Entwicklung Zellteilungen, -wanderungen und -sortierungen stattfinden, kann man erwarten, dass die Funktionen pleiotrop sein können. Um Gene zu identifizieren, die an diesen Prozessen beteiligt sind, wurde eine Mutantenlese nach temperatur-sensitiven Mutanten durchgeführt. Der Vorteil solcher Mutanten ist, dass die Genaktivität durch einen Temperaturshift jederzeit inaktiviert werden kann und so die Pleiotropien ausgeschaltet werden. Um die Mutanten zu sortieren, wurden Embryonen mit Hilfe der 4D-Mikroskopie dokumentiert und kategorisiert. Alle Mutanten mit einem potenziellen Fokussierungsphänotyp wurden mit der Software SIMI°BioCell analysiert. Die Mutante t3091 wurde sorgfältig charakterisiert. In dieser Mutante ist die Zellsortierung defekt, was zu einer Unordnung im Embryo führt. Die Mutation befindet sich im Gen pmm-1, das ein Ortholog zur humanen Phosphomannomutase 2 ist. Das Protein ist ein zentraler Faktor der N-Glykosilierung. Die entsprechenden Glykoproteine haben zell- und entwicklungsbiologische Funktionen. Meine Beobachtungen sind mit der Hypothese konsistent, dass Glykoproteine eine zentrale Rolle bei der Spezifizierung von Zelladressen zur Fokussierung spielen. Nach vorhergehenden Arbeiten scheint es wahrscheinlich, dass jede Zelle im C. elegans Embryo zur Orientierung eine Eigene schicksalsabhängige Adresse besitzt (Schnabel et al., 2006; Bischoff et al., 2006). Spezifische Glykosilierungmuster erscheinen mir eine biochemische Möglichkeit, eine solche Vielfalt an Adressen zu spezifizieren.In C. elegans embryonic pattern formation occurs by global cell sorting. This mechanism was described by Schnabel et al. as "cell focusing" in analogy to the focusing of proteins. The cell position in a C. elegans embryo is correlated to the cell fate. This work addresses the problem by which mechanisms cells recognize each other during the long-range movements through the embryo or what is the molecular basis for cell focusing? However, cell division, migration and sorting are essential during embryonic as well during larval development. Thus, mutations in genes, which are involved in these processes, could have pleoitropic effects. To identify genes that are involved in migration and sorting we performed a screen for temperature sensitive embryonic lethal mutants. The great advantage over non-conditional mutants is, that inactivation of a gene product is induced by a temperature shift which allows to circumvent pleiotropic effects by shifting the organism at different stages of development. To classify the mutants I used 4D microscopy to document the whole embryogenesis. The recordings were analyzed using the Software SIMI°BioCell to classify and to identify mutants with cell focusing defects. I characterized the mutant t3091. The phenotype reflects a strong cell sorting defect, which results in excessively disorganized embryos. The mutation defines the gene pmm-1, which is an ortholog of the human gene phosphomannomutase 2. This protein is a central factor of the N-glycosylation pathway. Glycoproteins are essential for many cell- and developmental processes. The reduction of gene activity leads to multisystemic pathologies in humans. My observations are consistent with the hypothesis, that glycoproteins have a major role in specification of cell addresses, that are needed for a proper sorting/cell focusing. The diversity of glycosylation patterns may be sufficient, to code the potentially large number addresses required for placing cells in the C. elegans embryo

Coordination of Cell Proliferation and Cell Fate Determination by CES-1 Snail

<div><p>The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of <i>Caenorhabditis elegans</i> coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene <i>egl-1</i>, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the <i>cdc-25.2</i> gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of <i>cya-1</i>, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the <i>ces-1</i> gene leads to incorrect regulation of this functional ‘module’. Finally, we provide evidence that <i>dnj-11</i> MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.</p></div

<i>ces-1(n703</i>gf<i>)</i>; <i>cya-1(bc416)</i> blocks cell divisions in the ABarp, C and E lineages.

<p>All strains analyzed were homozygous for <i>bcIs66</i>. Lineage analyses were performed for two (wild-type, <i>+/+</i>), three (<i>ces-1(n703</i>gf<i>); cya-1(bc416)</i>) and three (<i>cdc-25.2(RNAi)</i>) embryos raised at 25°C. The ABarp, C and E lineages are shown. Vertical axis indicates approximate time in min after the 1^st round of embryonic division, in which P0 divides into AB and P1. In the case of <i>ces-1(n703</i>gf<i>); cya-1(bc416)</i>, cell division defects observed in three out of three embryos are depicted in red, defects found in two out of three embryos are depicted in blue, and defects found in one out of three embryos are depicted in orange. In the case of <i>cdc-25.2(RNAi)</i>, RNAi was carried out by injection. Since there is some variability of the RNAi effect, the lineage shown here was derived from the embryo with the strongest phenotype (cell division defects observed in this embryo are depicted in green), and the lineages from the other two <i>cdc-25.2(RNAi)</i> embryos are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003884#pgen.1003884.s006" target="_blank">Figure S6</a>. The severe cell division defects in the ABarp, C and E lineages were seen in all three <i>cdc-25.2(RNAi)</i> embryos. The cell death in the ABarp lineage is labeled with the cross. The defects in the C lineage and ABarp lineage result in a defect in the formation of the hypodermis (the mitoses that generate hyp7, hyp5, hyp11, H0, H1, H2, V1, V2, V4, and V6 fail to occur).</p

<i>ces-1(n703</i>gf<i>)</i>; <i>cya-1(bc416)</i> causes temperature-sensitive embryonic lethality.

<p>(A) The percentages of embryonic lethality at 15°C and 25°C. The numbers above the bars represent the percentage of embryonic lethality. For each genotype, around 1000 embryos were scored. DIC images of embryos arrested during the elongation stage of embryogenesis (B, D, E) or during the first larval stage (L1) (C) when grown at 25°C are shown. White arrows point to abnormalities in the hypodermis. All strains analyzed were homozygous for <i>bcIs66</i>. RNAi was performed by injection.</p

<i>ces-1(n703</i>gf<i>); cya-1(bc416)</i> affects the number of ‘NSM-like’ cells.

<p><i>ces-1(n703</i>gf<i>); cya-1(bc416)</i> affects the number of ‘NSM-like’ cells.</p

<i>ces-1</i> Snail represents a functional link between cell cycle progression, cell polarity and apoptosis in the NSM lineage.

<p>Genetic model of <i>ces-1</i> Snail functions in the NSM neuroblast (top), the NSM and the NSM sister cell (bottom). In the NSM neuroblast, <i>ces-1</i> function is negatively regulated by the genes <i>dnj-11</i> MIDA1 and <i>ces-2</i> bZIP. <i>ces-1</i> affects cell cycle progression in the NSM neuroblast by negatively regulating <i>cdc-25.2</i> Cdc25. <i>ces-1</i> also affects the polarity of the NSM neuroblast. However, to date, it is unclear through what mechanism. After the asymmetric division of the NSM neuroblast, the level of <i>ces-1</i> activity is high in the larger NSM (left) and low in the smaller NSM sister cell (right). The activity of <i>ces-1</i> in the NSM is sufficient to block the function of <i>hlh-2/3</i> bHLH, thereby resulting in a level of <i>egl-1</i> BH3-only activity that is too low to induce apoptosis. Conversely, in the NSM sister cell, the activity of <i>ces-1</i> is not sufficient to block the function of <i>hlh-2/3</i>, thereby resulting in a level of <i>egl-1</i> activity that is high enough to induce apoptosis. See text for details and molecular interpretations.</p