Slone, R. Daniel et al. (2010) Worm Breeder's Gazette "A C. elegans distal tip cell-specific RNAi screen"

Schwarzbauer, Jean E., Kimble, Judith, Slone, R. Daniel, Byrd, Dana

[Worm Breeder's Gazette, 2010]

(2024) Development "The people behind the papers - Nadia Manzi and Daniel Dickinson."

[Development, 2024]

The mitotic kinase Aurora A has been shown to regulate the anterior-posterior polarity in developing Caenorhabditis elegans embryos. In a new study, Daniel Dickinson and colleagues find that Aurora A has temporally distinct roles in coordinating the localization of Partitioning defective (PAR) proteins to establish cell polarity during development. To find out more about the story behind the paper, we caught up with first author Nadia Manzi and corresponding author Daniel Dickinson, Assistant Professor at the University of Texas at Austin.

(2019) Development "The people behind the papers - Daniel Osorio, Elaine Chan, Joana Saramago and Ana Carvalho."

[Development, 2019]

Animal cytokinesis is driven by an actomyosin ring that assembles at the cell equator and constricts to physically separate the two daughters. Although myosin is known to be essential for cytokinesis in multiple systems, whether this requirement reflects its motor or actin crosslinking activities has recently been a matter of contention. A new paper in Development now addresses this problem using the first divisions of the <i>Caenorhabditis elegans</i> embryo as a model. We caught up with the paper's three first authors Daniel Osorio, Elaine Chan and Joana Saramago, and their supervisor Ana Carvalho, Principal Investigator at the University of Porto's i3S consortium, to find out more about the story.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "The core apoptotic executioner proteins CED-3 and CED-4 promote neuronal regeneration in Caenorhabditis elegans."

Slone, Daniel, Hulme, Elizabeth, Shevkoplyas, Sergey, Gabel, Christopher, Samuel, Aravinthan, Driscoll, Monica, Weisberg, Sarah, Pinan-Lucarre, Berangere, Xue, Jian, Whitesides, George

[International Worm Meeting, 2009]

How neurons in their native environments respond to, and recover from, localized physical disruptions such as axon severing is poorly understood. Exciting breakthrough developments in femtosecond laser microsurgery allow precise cutting of individual axons within living Caenorhabditis elegans. Fantastically, some severed neurons have the ability to regenerate, making C. elegans a powerful model for dissecting the genetic requirements of in vivo axonal regeneration. We applied this technology to investigate the role of apoptotic and necrotic genes in the neuronal response to laser severing of ALM touch neurons visualized by pmec-4GFP in the adult C. elegans. Using ced-3 mutants including ced-3(n2433), which encodes a single amino acid substitution that disrupts the caspase active site, we showed that CED-3 caspase, extensively characterized for its role as the essential core executioner protease in apoptosis, promotes efficient regeneration of ALM as well as D-type motor neurons as assessed 24h following axotomy. Time-lapse studies using a microfluidics device further revealed that CED-3 is needed early. We expressed the caspase inhibitor P35 specifically in touch neurons and observed reduced regenerating capacity, indicating that the caspase activity is required inside the severed neuron for its regeneration. In a complementary genetic approach, we found that the regeneration defect caused by ced-3(n2433) was rescued by specific expression of ced-3 in the touch neurons, demonstrating that CED-3 acts cell autonomously for neuronal regeneration. The apoptotic caspase activator CED-4 is required for efficient axonal regeneration, but the upstream apoptotic regulators CED-9 and EGL-1 are dispensable, revealing regulation mechanistically distinct from developmental apoptosis. Regeneration also depends on caspase-related genes csp-1, csp-2 and csp-3 as well as crt-1, a critical necrotic gene encoding the Ca2+-storing ER chaperone calreticulin, which appears to act with CED-3 in the same biological pathway. Our work reveals an unexpected reconstructive role for proteins known to orchestrate cell death. We will present more data about the mechanistic regulation of this regeneration pathway at the meeting. This work was supported in part by the New Jersey Commission on Spinal Cord Research.

Vo, An A. et al. (2021) MicroPubl Biol

Ward, Jordan D., Ragle, James Matthew, Levenson, Max T., Vo, An A.

[MicroPubl Biol, 2021]

The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223

Astrid Kleinert et al. (2006) European Worm Meeting "Analysis of Insulin-Like Peptides in C. elegans by Mass Spectrometry"

Astrid Kleinert, Daniel Hess, Joy Alcedo, Jan Hofsteenge

[European Worm Meeting, 2006]

Astrid Kleinert, Daniel Hess, Jan Hofsteenge and Joy Alcedo. Genetic analyses in C. elegans have implicated an endocrine signaling pathway, the insulin/IGF-1 pathway, in regulating lifespan. Mutations in the gene daf-2, which encodes an insulin/IGF-1 receptor homologue, can increase worm lifespan by more than 100% (1). In addition, the insulin and IGF-1 pathways have been shown to influence fly and mouse lifespan (2-5).. Although downstream components of the DAF-2 pathway have been studied in detail, far less is known about the putative DAF-2 ligands predicted to be encoded by 38 insulin-like genes in the worm. It remains unknown which of the predicted insulin-like peptides are present in wild type or in longevity mutants. In order to determine this, we have initiated a project to analyze the polypeptide subfractions of the C. elegans proteome by mass spectrometry. Worm lysates will be prepared from mixed stage, wild-type worms, excluding membrane-bound proteins. Acid-ethanol extraction and gel filtration will be tested as methods to obtain a subfraction enriched in small polypeptides. Peptide identification will be done by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data analyses will be carried out using a MASCOT search database, which contains a subset of the UNIPROT database and a database generated of relevant C. elegans polypeptides. Finally, using the SILAC method for quantification (6), we plan to compare differences in the levels of polypeptides, e.g., insulin-like peptides, between wild-type worms and longevity mutants. . References: (1) Kenyon et al., 1993. Nature 366: 461-4. (2) Clancy et al., 2001. Science 292: 104-6. (3) Tatar et al., 2001. Science 292: 107-110. (4) Holzenberger et al., 2003. Nature 421: 182-7. (5) Blher et al., 2003. Science 299:572-4. (6) Krijgsveld et al., 2003. Nature Biotech. 21: 927-931.

Allan Froehlich et al. (2005) International Worm Meeting "An Attempt to Develop Homologous Gene Targeting in C. elegans Using Positive/Negative Selection and a Strain Deficient in Nonhomologous End-joining"

Allan Froehlich, Bob Horvitz

[International Worm Meeting, 2005]

We are attempting to develop a method for gene targeting by homologous recombination in C. elegans by using the worms endogenous DNA repair and recombination machinery to replace a selected region of genomic DNA with any desired exogenous DNA. Double-strand breaks are repaired by two main recombination pathways in eukaryotes, homologous recombination (HR), which requires DNA sequence homology, and non-homologous end-joining (NHEJ), which does not. Genomic integration occurs predominately by the HR pathway in S. cerevisiae but by NHEJ in other organisms, including C. elegans. A new approach to obtain homologous integrants has been recently reported for the filamentous fungus Neurospora crassa. In strains with deletions in KU70 or KU80 (components of the Neurospora NHEJ pathway), homologous integration events increase from 10% to 100% and nonhomologous integrations are eliminated (Y. Ninomiya et al., PNAS 101, 2004). Our method for homologous gene targeting will use a C. elegans strain containing a deletion in the KU80 homologue, cku-80(ok861), together with an extrachromosomal array containing a single transgenic donor construct comprised of two regions of homology to the targeted genomic locus separated by a positive selectable marker and containing a negative selectable marker distal to one of the homologous regions. For positive selection of genomic integration, we are using a neomycin resistance transgene (kindly provided by Jesse Slone and Helen Chamberlin). To select against retention of the extrachromosomal array, we are using a transgene with the unc-54 promoter driving expression of avr-15 (kindly provided by Joe Dent). This transgene confers sensitivity to ivermectin (paralysis) in a strain containing avr-14(ad1302); avr-15(ad1051) glc-1(pk54). We are testing the efficiency of this homologous recombination system by targeting ced-3 in a strain containing an integrated pkd-2::gfp reporter, which expresses in the CEM neurons. Deletion of ced-3 results in the failure of the male-specific CEM neurons to undergo programmed cell death in hermaphrodites. Following ivermectin treatment to select for loss of the extrachromosomal array and neomycin treatment to select for genomic integration, the pkd-2::gfp reporter will enable us to efficiently screen for homologous integration at the ced-3 locus by seeking GFP-positive CEM neurons in hermaphrodites. In addition to generating gene deletions, we hope to use this system of homologous recombination for the precise insertion of exogenous DNA into the C. elegans genome.

Heinze, Svenia.D. et al. (2019) International Worm Meeting "Permissive and instructive functions of LIN-39 during Pn.p cell proliferation."

Heinze, Svenia.D., Hajnal, Prof. Alex

[International Worm Meeting, 2019]

The Caenorhabditis elegans vulva is an excellent model to study how cell proliferation is regulated during animal development. During the first larval stage, six equivalent vulval precursor cells (VPCs) are born. These cells remain arrested in the G1 phase of the cell cycle until the beginning of the third larval stage, when their fates are specified and the three proximal VPCs proliferate to generate exactly 22 vulval cells. It is yet unknown how the exit of the 22 terminally differentiated vulval cells from the cell cycle is established and maintained throughout adulthood. The hox gene lin-39is necessary to maintain the VPCs proliferating by regulating the expression of the two cell-cycle regulators cye-1(cyclin E) and cdk-4 (Roiz et al. 2016). Accordingly, the VPCs cease to divide as soon as the levels of LIN-39 drop below a certain threshold level when the animals reach the fourth larval (L4) stage. We have used the CRISPR-Cas9 system to generate a conditional knock-down allele of lin-39 that allows us to induce LIN-39 protein degradation at different stages during vulval cell proliferation. We observed that secondary (2 deg ) VPCs lacking LIN-39 prematurely exit from the cell cycle as they undergo only two instead of three rounds of cell division. Furthermore, using VPC-specific lin-39 over-expression we observed that Pn.p cells expressing LIN-39 enter the cell cycle prematurely during the L2 stage, generating a hyperplastic phenotype with an excess number of Pn.p cells. These results indicate the LIN-39 is necessary and sufficient to promote VPC proliferation in larvae. Nevertheless, vulval cells overexpressing LIN-39 exit the cell cycle after the L4 stage, indicating that LIN-39 is not sufficient to keep somatic cells proliferating in adults. Exploiting the hyperplastic phenotype of the animals over-expressing LIN-39 as a genetic background we are searching for factors that are required to maintain a prolonged proliferating state in adult somatic cells. References: Roiz, Daniel, et al. "The C. elegans hox gene lin-39 controls cell cycle progression during vulval development." Developmental biology 418.1 (2016): 124-134.