Markussen F-H et al. (2000) Midwest Worm Meeting "Establishing the Gonad Primordium"

Kimble JE, Blelloch RH, Henderson ST, Markussen F-H, Mathies LD

[Midwest Worm Meeting, 2000]

The gonad in all organisms contains both somatic and germline tissues that must be united to produce a functional organ. In C. elegans, the somatic and germline precursor cells coalesce during embryogenesis to form the gonad primordium. Specifically, the somatic gonadal precursors (Z1 and Z4) migrate toward and flank the germline precursors (Z2 and Z3). The 4-celled gonad primordium is arranged with two-fold rotational symmetry in both sexes. This symmetry is maintained throughout hermaphrodite development generating a two-armed gonad, but is broken in males to generate a single-armed gonad. We have set out to dissect the genetic controls of gonad primordium formation. Using genetic screens and RNAi, we have identified at least 4 genes that are required for proper assembly of the gonad primordium. In each of these mutants, the symmetry of the gonad primordium is disrupted resulting in aberrantly-shaped adult gonads. Thus, the proper organization of the gonad primordium is essential for establishing organ shape. In the simplest case, the gonad primordium of gnd-2 RNAi animals is frequently missing one of the two somatic precursor cells, giving rise to an adult gonad with a single arm. gnd-1 and gnd-3 mutants can have asymmetric gonad primordia that lead to a more severely disorganized gonad and sterility. We have also examined previously existing mutants and find that mutations in the sex determination gene tra-1 result in an asymmetric gonad primordium. We will report our progress in analyzing these mutants phenotypically and molecularly.

Markussen F-H et al. (1998) Midwest Worm Meeting "A screen for mutations affecting gonad polarity and somatic gonad formation in C. elegans"

Kimble JE, Friedman LC, Siegfried KR, Markussen F-H, Mathies LD, Kroll-Conner PL

[Midwest Worm Meeting, 1998]

We are studying the problem of organogenesis using the C. elegans gonad as a model. The gonad develops postembryonically from a primordium of two somatic and two germ line cells. The four cells are in an arrangement of two-fold rotational symmetry in either sex. In hermaphrodites this polarity is maintained throughout development generating a symmetrical two-armed gonad. In males, the symmetry is broken after the first round of cell division establishing a non-symmetrical single armed gonad. The somatic gonad develops in three phases. An initial round of cell divisions generates 12 somatic cells in hermaphrodites and 10 cells in males. Second, rearrangement of the somatic cells generates the somatic gonadal primordium. This phase consolidates the gonad, laying down a !blueprint! of the final arrangement of tissues. Lastly, a phase of cell division, differentiation, and morphogenesis shapes the final organ. We are doing an F2 screen for mutations disrupting the somatic gonad in either sex. From 6660 mutagenized haploid genomes we have isolated 58 independent mutations specific to somatic gonad development. Based on our current understanding of gonadogenesis, we are classifying the mutants in three broad classes. 1) Gon; gonad-less: absence of the L1 gonad primordium or arrested development at the L1 gonadal primordium stage. 2) Spf; somatic gonadal primordium formation defective. This includes polarity defects, early lineage defects, and defects in primordium consolidation. 3) Sgo; somatic gonad defective: the over-all polarity and organization of the gonad is normal indicating that a somatic gonadal primordium did form. Development fails during the last phase of gonadogenesis often resulting in malformed or absent tissues such as uterus and spermatheca. The number of complementation groups in each class is still unknown. Representative phenotypes of the different classes will be presented.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Friedman LC et al. (2000) Midwest Worm Meeting "Sexual dimorphism of organ polarity in gonad formation"

Markussen F-H, Mathies LD, Kroll-Conner PL, Kimble JE, Siegfried KR, Friedman LC

[Midwest Worm Meeting, 2000]

When the L1 larva hatches, the gonad primordium consists of 4 cells arranged with two-fold rotational symmetry in both sexes. Two of these cells, Z1 and Z4, are somatic and their development determine the shape of the organ. During L1, cell divisions generate 8 somatic cells in hermaphrodite gonads and 6 in males. In the hermaphrodite, the symmetrical arrangement of somatic cells is maintained during L1. The symmetry is continued throughout later development, producing the two-armed gonad of hermaphrodites. In males, symmetry is broken during L1, and most somatic cells coalesce in an asymmetric cluster at the anterior of the developing gonad. This event underlies the asymmetrical one-armed gonad of males. We have set out to dissect the genetic and cellular basis of sexual dimorphism during early gonadogenesis. To this end, we have isolated two mutants with defects primarily in male gonadogenesis. One mutant, tentatively called man-1 for male asymmetric gonad defective, fails to break gonad symmetry in L1 males. Another similar mutant has defects in early male gonadogenesis. We will report our progress in characterizing these mutants and our efforts to describe cellular anatomy of the gonad primordium at a molecular level.

Wei-meng Woo et al. (2004) West Coast Worm Meeting "The extracellular matrix protein F-spondin is essential for muscle attachment and epidermal elongation"

Chris Suh, Yishi Jin, Andrew D Chisholm, Mei Ding, Christie Emigh, Wei-Meng Woo, Jian Cao

[West Coast Worm Meeting, 2004]

In C. elegans epidermal intermediate filaments (IFs) and their associated structures, the trans-epidermal attachments, are essential for embryonic epidermal elongation (Woo et al 2004). The formation of muscle contractile units and trans-epidermal attachments are mutually dependent during epidermal elongation. To understand how the connection between epidermis and muscle is established and how the two tissues communicate during organogenesis, we performed a screen for epidermal elongation-defective mutants. One locus identified in this screen was defined by three lethal alleles and mapped to the cluster of LG II. Subsequent analysis showed that these mutations were allelic to vab-13 and ven-3 . By genetic mapping and allele sequencing we showed that all these mutations affect F10E7.4, which encodes the C. elegans member of the F-spondin family of secreted proteins. F-spondin has been shown to play roles in axon guidance, cell migration, and angiogenesis. Our genetic analysis shows that in C. elegans F-spondin is required for epidermal elongation and muscle attachment, as well as for proper positioning of neuronal processes. Using GFP reporters, we found that F-spondin is expressed in body muscle cells and is a secreted protein. Thus, F-spondin may function in embryogenesis in communication between muscle and epidermis. Immunostaining of F-spondin mutants suggest that F-spondin may indirectly affect the organization of epidermal actin microfilaments and trans-epidermal attachments . We are examining the expression patterns of muscle and basement membrane components in F-spondin mutants. To study the signaling pathways regulated by F-spondin, we are testing mutations in candidate receptor genes for genetic interactions with F-spondin mutations.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Bonnie Kaas et al. (2008) C.elegans Neuronal Development Meeting "Regulation of Levels of UNC-13 at Synapses"

Rebecca Lawson, Rebecca Kohn, Thomas Limouze, Ryan Wennell, Bonnie Kaas

[C.elegans Neuronal Development Meeting, 2008]

Release of neurotransmitters from neurons is highly regulated. Several proteins play roles in this process, including UNC-13, and decreased release of neurotransmitters in unc-13 mutants results in paralysis. We identified an F-box protein that interacts with UNC-13. F-box proteins participate in ubiquitin ligase complexes and in Drosophila, DUNC-13 is degraded via the ubiquitin proteasome pathway. This UNC-13/F-box interaction may therefore indicate that UNC-13 is tagged for proteasomal degradation with ubiquitin by the ligase complex in C. elegans. The C. elegans knockout consortium isolated a strain with a large deletion in the coding region of the gene that codes for the F-box protein. If the F-box protein is indeed involved in the degradation of UNC-13, this strain would be expected to have higher levels of UNC-13, which could result in changes in phenotypes. We characterized the F-box deletion mutant by assaying brood size, developmental rate, and body bends per minute. Aldicarb assays were used to determine whether a deletion in the gene coding for the F-box protein alters the response to inhibitors of acetylcholinesterase. We found that the deletion resulted in some changes in developmental rate and in aldicarb sensitivity. We are continuing to study strains with mutations in both the gene coding for the F-box protein and in unc-13.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Atsushi Yamanaka et al. (2001) International C. elegans Meeting "Biochemical and functional analysis of SKP1 family in Caenorhabditis elegans"

Yasumi Ohshima, Kei-ichi Nakayama, Masayoshi Yada, Atsushi Yamanaka, Hiroyuki Imaki

[International C. elegans Meeting, 2001]

The ubiuquitin-proteasome pathway is a key mechanism for substrate-specific degradation to control the abundance of a number of proteins. SCF complex, one of ubiquitin-protein ligases (E3s), regulates cell cycle progression, signal transduction, and many other biological systems. The SCF complex consists of invariable components, such as Skp1, Cul-1 and Rbx1, and variable components called F-box proteins that bind to Skp1 through the F-box motif. F-box proteins are substrate-specific adaptor subunits that recruit substrates to the SCF complex. Surprisingly, we found that the genome of Caenorhabditis elegans ( C. elegans ) contains at least 20 Skp1-like sequenses, whereas one or a few Skp1 is present in humans. Therefore, we studied C. elegans Skp1-like proteins (CeSkp1) that are likely to be variable components of SCF complex in addition to F-box proteins. At least, seven CeSkp1s were associated with C. elegans Cul-1 (CeCul-1) in yeast two-hybrid system as well as co-immunoprecipitation assay in mammalian cells, and these expression patterns were different in C. elegans . By RNA interference (RNAi), two of these CeSkp1s showed embyonic lethality and four showed the phenotype of slow growth. There were differences among CeSkp1s in ability to interact with F-box proteins. These results suggest that CeSkp1s, like F-box proteins, act as variable components of SCF complex in C. elegans .