Z Chen et al. (1999) International C. elegans Meeting "abi-1, a gene required for vulval morphogenesis, encodes a nuclear hormone receptor homolog"

M Han, Z Chen

[International C. elegans Meeting, 1999]

Vulval cell fate specification causes three out of six vulval precursor cells (VPCs) to undergo some characteristic divisions, followed by specific cell migration and fusion events. Here we report the study of abi-1(ku217) and another gene defined by the ku285 allele that disrupt these processes. In a WT vulva, the induced VPCs, P(5-7).p, divide twice in a longitudinal orientation. In the third and final round of division, the four granddaughters of P5.p undergo longitudinal(L), longitudinal(L), transverse(T) and no(N) divisions respectively. P7.p divides in a mirror-symmetrical way to P5.p, and the granddaughters of P6.p undergo only transverse(T) divisions. In ku217 animals, the "T" divisions of vulval cells are disrupted specifically, resulting in no division. However, the cell fates of these nondivided "T" cells may not be the same as those of the "N" cells, based on the study of the expression of some vulval cell-specific marker genes. In addition to the division defects, cell migration and fusion processes are affected in abi-1(ku217) animals. These morphogenetic defects are also displayed in the pseudovulvae of let-60(gf);abi-1 and lin-1(lf);abi-1 double mutants, indicating that the abi-1 gene functions in an event downstream of the vulval cell fate induction. The abi-1(ku217) is a TS, recessive mutation, and dosage analysis suggests that it is a loss-of-function mutation. We have cloned this gene using DNA-mediated microinjection transformation. The ABI-1 protein, which is 572 a.a. long, is homologous to members of the nuclear hormone receptor superfamily. A missense mutation (L32F) in the DNA-binding domain is associated with the ku217 allele. An abi-1::gfp translational fusion gene is expressed extensively in the worm, including in the 12 Pn.p cells and hypodermal cells, but not in the AC or any other somatic gonadal cells. However, RNAi of the abi-1 gene displays a highly penetrate gonadal phenotype. The ku285 is another mutant which disrupts the transverse divisions of the vulval cells. Lineage studies of ku285 animals show that, unlike in the abi-1(ku217) mutant, there is a transformation from transverse division to longitudinal division, with no defects in other division patterns. The ku285 is also a loss-of-function allele, and the molecular cloning is in process."

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

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

[International Worm Meeting, 2021]

<div id="i4c-draggable-container" style="position: fixed; z-index: 1499; width: 0px; height: 0px;">

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.

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.

Frederick J Tan et al. (2004) West Coast Worm Meeting "Functions of the Outer Mitochondrial Membrane Protein CED-9"

R Blake Hill, Andrew Z Fire, Frederick J Tan

[West Coast Worm Meeting, 2004]

CED-9 (CEll Death abnormal) negatively regulates the programmed cell death pathway in C. elegans . This membrane associated pro-survival protein prevents the activation of the CED-3 caspase. Loss of function ced-9 animals arrest at early embryogenesis, presumably dying from ectopic cell deaths. Conversely, gain of function animals appear healthy, albeit with extra cells (Hengartner, Ellis and Horvitz, 1992). These effects have been ascribed to the ability of CED-9 to sequester the CED-4/CED-3 caspase complex at the mitochondria (Chen, et al. , 2000). We hope to understand the structural features of CED-9 that participate in its function. To this end, we are assaying the activity of various CED-9 mutants and derivatives for ability to prevent cell death and respond to appropriate modulating signals. A question that we consider to be central is whether the C-terminal transmembrane domain of CED-9 serves only as a targeting sequence to the mitochondrion, or plays a more active role. We have demonstrated that this domain is essential for mitochondrial localization and plays some role in the function of CED-9. Distinction between localization and other structural and functional roles is in progress. Hengartner MO, Ellis RE and HR Horvitz. 1992. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356 , 494-499. Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, and HR Horvitz. 2000. Translocation of C. elegans CED-4 to Nuclear Membranes During Programmed Cell Death. Science 287 , 1485-1489.

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.

D Omura et al. (1999) International C. elegans Meeting "A screen for mutations that affect the localization of CED-4"

F Chen, B Horvitz, B Hersh, D Omura

[International C. elegans Meeting, 1999]

In the programmed cell death pathway, ced-4 is a key activator of the C. elegans caspase ced-3 . Genetically, ced-4 is upstream of ced-3 and is necessary for normal programmed cell death to occur. egl-1 and ced-9 act upstream of ced-4 in the genetic pathway and are involved in activating and inhibiting ced-4 activity, respectively. Previous work has shown that the localization of ced-4 seems intimately tied to its activity. In cells that are not undergoing programmed cell death, CED-4 is localized to mitochondrial membrane surfaces. When these cells are induced to die by overexpressing egl-1 or by ced-9 loss-of-function, CED-4 is localized to the perinuclear membrane 1 . CED-4 localization appears to correlate with the life-or-death decision of a cell, but how and why the localization occurs is unknown. When overexpressed, a CED-4::GFP fusion protein appears to localize to the mitochondria and perinuclear region of cells that are not undergoing programmed cell death. To identify factors important for the localization of CED-4, we will screen for mutants in which this CED-4::GFP fusion protein fails to localize to the perinucleus of viable cells. 1 Hersh, B., Chen, F., Conradt, B., Zhou, Z., and Horvitz, HR (1999) 12th International C. elegans Meeting