Khan, Munzareen et al. (2021) International Worm Meeting "

Khan, Munzareen, Sengupta, Piali, Dwyer, Noelle, Hartmann, Anna, Bargmann, Cori, Piccione, Madeline

[International Worm Meeting, 2021]

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Riga, Amalia et al. (2019) International Worm Meeting "The role of basolateral polarity regulators in epithelial tissue homeostasis."

Boxem, Mike, Pires, Helena, Riga, Amalia

[International Worm Meeting, 2019]

Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, loss of LET-413/Scrib and DLG-1/Dlg results in defects in epithelial polarization and junction formation during embryonic development, resulting in embryonic lethality. However, their importance in larval epithelia is less well understood. To study the role of LET-413 in larval epithelia, we generated an allele that allows inducible protein degradation using the auxin-inducible system recently developed for C. elegans (1). We find that ubiquitous degradation of LET-413 in larval tissues results in a developmental arrest. Tissue-specific degradation of LET-413 in the larval intestine does not cause defects in development. In contrast, degradation of LET-413 in the seam cells and hypodermis mimics the developmental arrest seen upon ubiquitous LET-413 degradation. Live-cell imaging of seam cells shows a severe effect on outgrowth and reattachment following the L1 asymmetric division. In addition, we observed that loss of LET-413 results in punctate and discontinuous localization pattern of DLG-1, similar to previous observations in C. elegans embryos. We are currently following the effects of LET-413 inactivation on cell recognition and migration pathways to understand the outgrowth defects of the seam cells. (1) Zhang L, Ward JD, Cheng Z, Dernburg AF. Development. 2015 Dec 15;142(24):4374-84.

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.

Gilleard JS et al. (1995) International C. elegans Meeting "REGULATION OF THE CUTICULAR COLLAGEN GENE dpy-7"

Barry JD, Gilleard JS, Johnstone IL

[International C. elegans Meeting, 1995]

We are interested in identifying trans acting factors which control cuticular collagen gene expression. Sequences sufficient for regulated expression of dpy-7 appear to reside relatively close to the initiator ATG since the wild type gene only requires 310bp of 5' flanking sequence for transformation repair of phenotype. We have analysed sequences necessary and sufficient for dpy-7 expression using lac-Z fusions and we have identified a 95bp fragment, encompassing the transcritional start sites, which is sufficient to produce lac-Z expression predominantly, or possibly exclusively, in hypodermal cells. This fragment includes a region in which 65 out of 72bp are conserved in the 5' flank of the C.briggsae dpy-7 homolog and interspecies transformation rescue and lac-Z fusion experiments have shown that the dpy-7 cis regulatory elements are functionally conserved. A 15bp deletion at the 5' end of this homology which removes a AGATAA motif, also present in C.briggsae, completely ablates lac-Z expression . We are attempting to identify transcription factors which bind to this motif by South Western expression library screening. We are also searching for new C.elegans GATA factor homologs with a particular interest in any which are expressed in hypodermal cells postembryonically.

Zhang, L. et al. (2015) International Worm Meeting "An auxin-inducible degradation (AID) system for precise temporal and spatial control of protein depletion."

Dernburg, A., Cheng, Z., Zhang, L., Ward, J.

[International Worm Meeting, 2015]

Established approaches for protein depletion in C. elegans have primarily relied on disrupting genes or inhibiting their expression. A method for posttranslational depletion of proteins has recently been developed (Armenti et al., 2014), but this approach has some limitations: In particular, it cannot be used in the germline or in early embryos, and conditional depletion involves induction of a ubiquitin ligase, requiring both heat shock and lag time. As an alternative method for rapid, inducible protein depletion, we have implemented the auxin-inducible degradation (AID) system in C. elegans. This system relies on expression of a plant-specific F-box protein, TIR1, which interacts with endogenous Skp1 and Cul1 proteins. This SCF ubiquitin ligase complex recognizes substrates only in the presence of auxin (indole acetic acid, or IAA). We expressed an optimized TIR1 transgene and found that it mediates rapid, auxin-dependent degradation of nuclear or cytoplasmic proteins tagged with a short (44-amino acid) degron sequence. The degron can be fused to endogenous genes or transgenes, with or without a fluorescent protein or other tag as a reporter for expression and degradation. CRISPR/Cas9 editing with pha-1(ts) co-conversion (Ward, 2015) allowed efficient introduction of degron-3xFLAG tags in 8-9 days. TIR1 expression or IAA alone had no detectable effects on worm viability or fertility. The AID system mediates inducible protein degradation in somatic tissues, embryos, and the mitotic and/or meiotic germline. Degron-tagged proteins are depleted within 30 minutes in the soma or an hour in the germline. Using MosSCI, we have engineered TIR1 transgenes with various promoter and 3'UTR sequences to restrict expression to specific somatic tissues or subregions within the germline. We have harnessed the system both to explore the role of dynamically expressed nuclear hormone receptors in molting and to analyze meiosis-specific roles for proteins required for germline proliferation. Thus, the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in C. elegans.References: Armenti, S.T., Lohmer, L.L., Sherwood, D.R., and Nance, J. (2014). Development 141: 4640-7.

Dolinski CM et al. (1997) International C. elegans Meeting "PHYLOGENETIC IMPLICATION OF BUCCAL CAPSULE DEVELOPMENT IN SOME BACTERIAL FEEDING NEMATODES"

Baldwin JG, Dolinski CM, Thomas WK

[International C. elegans Meeting, 1997]

Bacterial feeding nematodes including Zeldia punctata (Rhabditida: Nematoda), and Caenorhabditis elegans differ profoundly in feeding adaptations and specifically in the rhabdions (cuticular thickenings), and associated cells lining in the buccal capsule. We are carrying out a range of tests to determine what rhabdions are evolutionarly homologous between the two species. Reconstruction of the buccal capsule and procorpus (pharynx) with transmission electron microscopy indicates that the lining of the buccal capsule of Z. punctata includes four sets of muscular radial cells ma, mb, mc, md in contrast the same region in C. elegans which has two sets of epithelial radial cells (e1, e3) and two sets of radial muscle cells (m1, m2). In Z. punctata ma is a set of three radial muscle cells each with 2 nuclei. In C. elegans m1 has the identical arrangement. Similarly, in Z. punctata mb is a set of 3 muscle cells each with one nucleus, similar to m1 in C. elegans. These findings could contradict all previous hypotheses of homology, and suggest instead that ma and mb in Z. punctata are homologous respectively with m1 and m2 in C. elegans. Ongoing additional tests of homology include comparative cell lineages and screening mutants to recognize genes involved in buccal capsule expression.

Mi-Mi, Lei et al. (2013) International Worm Meeting ""Ultrastructure analysis of the sarcomeres in worms that lack Z-line formins"."

Pruyne, David, Mi-Mi, Lei

[International Worm Meeting, 2013]

Even with extensive studies that revolve around actin cytoskeleton, the detailed mechanism as to how actin filaments are recruited into sarcomeres, the smallest units of striated muscle contractile lattice, is yet to be elucidated. The barbed ends of actin filaments anchor at the Z-line structures (dense bodies in C. elegans) that define the sarcomere ends. Net actin polymerization favorably occurs at the barbed end that is known to have unique association with formins, one of the highly conserved and most widespread families of actin nucleating factors. We have shown in our previous study that two nematode formins, CYK-1 and FHOD-1 are Z-line-associated proteins that work together to organize and maintain sarcomeric organization in C. elegans striated muscle - the first evidence in an intact organism. In this study, we have carried out the ultrastructure analysis on sarcomeric structures of worms lacking CYK-1 and/or FHOD-1. Not only do the current electron microscopy results complement our previous conclusions, they also suggest that CYK-1 and/or FHOD-1 may contribute in achieving normal Z-line structures in C. elegans sarcomeric organization.

Britton C et al. (1997) International C. elegans Meeting "Regulation of the gut cysteine protease gene gcp-1"

Johnstone IL, Britton C, McKerrow JH

[International C. elegans Meeting, 1997]

Nematode proteolytic enzymes are often expressed in a stage- and tissue-specific manner to carry out defined functions. We have been examining the regulatory mechanisms controlling expression of a cysteine protease gene, gcp-1, of C. elegans. Northern blot analysis and in situ hybridisation have shown that gcp-1 is expressed specifically in the gut of all stages except the embryo; this is also demonstrated by gcp-1/lac Z reporter constructs. Promoter deletion analysis has indicated that 210bp upstream of the transcription start site is sufficient for gut-specific expression. Within this region are three GATA motifs, mutation of which individually results in a reduction in reporter gene expression. To examine the role of GATA motifs in gcp-1 regulation, lac Z reporter constructs were generated containing multimerised copies of this motif linked to a 100bp region upstream of the gcp-1 start site. In contrast to the native gcp-1/lac Z constructs, this concatamer-containing construct resulted in strong expression in the embryonic gut and in adult hypodermal cells, as well as larval gut cells. More constructs are currently being examined to try to determine the possible mechanisms involved in this alteration in stage and tissue specificity.

Jiang LI et al. (1997) International C. elegans Meeting "STUDY OF NEUROGENESIS DURING C. ELEGANS MALE SPICULE DEVELOPMENT"

Sternberg PW, Jiang LI

[International C. elegans Meeting, 1997]

SPD, SPV and SPC are three spicule associated neurons in C. elegans males responsible for multiple steps during male mating behaviors including spicule insertion and sperm transfer. These three neurons are generated from b and z sublineages, descendants of the male B blast cell. b and z sublineages share similarity with the Drosophila sensory organ precursor lineage, where one cell divides twice to generate neuron and non-neuronal structural cells, sheath and socket. We are interested in how these neuronal precursors are generated, and how the neuronal vs. non-neuronal fates are determined. Previous studies have shown that b and z fates are specified via multiple cell-cell interactions. We are using a spicule neuron-specific molecular marker, gpa-1, to further analyze these lineages. Cell ablation experiments have shown that gpa-1::lacZ is expressed in SPD neurons specifically, while gpa-1::GFP is expressed in all three spicule associated neurons. Using this molecular marker, we have started to examine neurogenesis during male spicule develop-ment in several types of mutants with either defective spicule morphology or defective mating behavior. We have also started an F1 clonal screen to look for mutants with misexpression of this marker. From a pilot screen of 3,200 gametes, several classes of mutants have been isolated, including those that lack gpa-1 expression and those with additional expression specifically in the spicule region.

Cockrum, Chad et al. (2017) International Worm Meeting "Transmission of an epigenetic "MESsage" for germline development in C. elegans."

Strome, Susan, Kreher, Jeremy, Cockrum, Chad

[International Worm Meeting, 2017]

During C. elegans development, two primordial germ cells (PGCs) must acquire germ fate and proliferate into hundreds of germ cells to form a complete and fertile adult germline. How PGCs acquire and maintain germ cell fate is poorly understood. Two important contributors are the chromatin regulators MES-4, a histone methyltransferase, and MRG-1, a chromodomain-containing protein. Without maternal delivery of these proteins to embryos, germ cells in early larvae fail to proliferate and die, resulting in sterile adults. We hypothesize that MES-4 and MRG-1 transmit the memory of a germline gene expression program from parent germ cells to progeny germ cells to ensure that PGCs express the appropriate genes for their survival and proliferation. Our working model is that 1) during embryogenesis, MES-4 maintains a memory of which genes were previously expressed in the parental germline by maintaining H3K36me3 on nucleosomes that package those genes, and 2) MRG-1 effects the memory by binding to H3K36me3 generated by MES-4 and activating an appropriate germline transcription program in PGCs. To test our model, I am determining whether PGCs from L1s that lack MES-4 or MRG-1 fail to launch an appropriate germline gene expression program. Due to the previous limitation in gathering enough PGCs for mRNA profiling, I first profiled mRNAs from mutant adult germlines that inherited maternally provided MES-4 and MRG-1, but cannot express one of the gene products (M+Z-). mes-4 M+Z- and mrg-1 M+Z- mutant adult germlines misexpress a similar set of genes that are normally repressed in germ cells: somatic genes and genes on the X chromosome. Thus, MES-4 and MRG-1 are required for a proper germline gene expression program and they regulate similar genes in adult germlines. We predict that PGCs in L1 progeny of these mutant adults, which completely lack MES-4 or MRG-1 (M-Z-), fail to acquire germ cell fate because they fail to launch a proper germline transcription program and more severely misexpress somatic genes and genes on the X chromosome. To test this prediction, I developed a method to isolate PGC pairs from wild-type and mutant L1 larvae to profile their mRNAs. I showed that this approach can successfully profile the mRNA population from single sister pairs of wild-type PGCs. This dissection approach offers advantages over FACS sorting large populations of PGCs: I can analyze mutant PGCs instead of RNAi PGCs, PGCs are of a uniform age, and PGCs spend minimal time in buffer before RNA preparation. I am currently analyzing the transcriptome of PGCs isolated from mes-4 and mrg-1 M-Z- mutant larvae, to learn how maternal MES-4 and MRG-1 ensure PGC survival and proliferation.