Politi, Kristin A. et al. (2009) International Worm Meeting "Three dimensional structure of the Ward body."

Derr, KD, Rice, William J., Politi, Kristin A., Fisher, Kevin, Crocker, Chris, Hall, David H.

[International Worm Meeting, 2009]

The plasma membrane of the nematode hypodermis features a unique set of stacked membrane invaginations that we presume to be associated with cuticle deposition, although there is still no experimental evidence to prove it. These structures were first noted by TEM in C. elegans by Sam Ward in the early 1970s, and briefly mentioned in WormBook I by John White (1). Here we present electron microscopic evidence for their organization in the wild type adult, combining thin sections, freeze fracture and electron tomography to view their organization in groupings along the body wall. The so-called "Ward body" resembles a Golgi body that has been rotated on end so that each succeeding membrane leaflet can fuse directly to the plasma membrane. Ward bodies are often clustered nearby to one another, but the orientation of each Ward body seems to be independent of its neighbors, and seemingly has no fixed angle with respect to the body axis. In some cases their leaflets lie virtually parallel to the plasma membrane. Similar membrane infoldings have also been seen in the excretory duct''s luminal membrane, but are not known to occur in seam cells or other epithelia in the nematode. We hypothesize that large extended proteins, such as collagens, may co-assemble within the protected environment of a Ward body leaflet prior to deposition in the cuticle. Supported by NIH RR 12596 to DHH. 1. W. Wood (Ed), The nematode C. elegans. Chapter 4, "The Anatomy", Fig 2a, 1988.

L'Hernault SW et al. (1995) International C. elegans Meeting "HOW DO SPERM FIND EGGS AND KNOW WHAT TO DO WHEN THEY FIND THEM?"

Mercer KB, L'Hernault SW

[International C. elegans Meeting, 1995]

Unlike many other organisms, fertilization in C. elegans is an extraordinarily efficient process; normally every sperm produced by a hermaphrodite successfully fertilizes an egg within the spermatheca (Ward, S. and J. Carrel, 1979 Dev. Biol. 73: 304). Many mutants that affect spermatogenesis and, consequently, disrupt fertilization have been selected by both ours and the Ward laboratory. Most of these mutants have obvious cytological abnormalities, so their defects in fertilization are perhaps not surprising. We have been studying mutants that complete spermatogenesis and form spermatozoa with superficially normal cytology but are defective in hermaphrodite self-fertilization. So far, our studies have been confined to mutants that map to three genes: spe-9, spe-13 and spe-16. Mutant males were assessed for sperm transfer by using spe-8 hermaphrodites as recipients; spe-8 hermaphrodites are self-sterile except when they receive "competent" seminal fluid from a male, which allows self-fertility (Shakes, D. & Ward, S. 1989 Dev. Biol. 134: 189). In such tests, none of the examined mutant males had defects in seminal fluid transfer to hermaphrodites. However, spe- 13 or spe-16 males cannot sire progeny in cross- fertilization tests while spe-9 males (either of two ts alleles) can sire cross progeny. Two recently recovered nonconditional spe-9 mutants are being examined to determine if stronger loss of function mutants also exhibit this phenotype. Double mutants that are spe-9ts and spe- 13ts are nonconditionally sterile, suggesting some form of synergism between these two mutations. We are in the process of examining spe-13 and spe-16 in order to determine if male derived spermatozoa can target properly to the hermaphrodite spermatheca, even though they cannot fertilize eggs.

Starr, D. (2015) International Worm Meeting "Cloning the male sterile, sperm defective genes fer-2 and fer-3."

Starr, D.

[International Worm Meeting, 2015]

We all know that the sperm of our favorite model organism are weird. But, did you know they do not have a membranous nuclear envelope? Wolf et al (1978) showed using EM that sperm nuclei are surrounded by a diffuse perinuclear halo that encapsulates the condensed chromatin and the centriole. The peri-nuclear halo is hypothesized to consist of RNA and protein. Sam Ward and his lab performed forward genetic screens for fertilization-defective mutants that disrupted spermatogenesis (Argon and Ward 198). Their ultrastructural analyses identified an interesting disruption of the perinuclear halo in fer-2, fer-3, or fer-4 mutant sperm. The mutant sperm lack the perinuclear halo. Instead, tubular complexes ~50 nm in diameter seem to originate from near the nucleus and radiate away from the nucleus (Ward et al 1981). Given my long-term interest in the nuclear envelope, I set out to clone these fer mutants with the goal of identifying components of the perinuclear halo as an alternative to the nuclear envelope that normally circles eukaryotic nuclei. Using whole-genome sequencing, I have identified possible molecular lesions for fer-2 and fer-3. For fer-2, I identified a nonsense mutation near the beginning of the mib-1 gene. RNAseq and microarray experiments deposited in Wormbase show that mib-1 expression is enriched in sperm. The MIB-1 protein is a predicted RING Zn-finger E3 Ubiquitin ligase with ankyrin repeats that is conserved with fly and vertebrate Mindbomb. fer-3 had a nonsense mutation in the eri-3 gene. Mutations in eri-3 were previously shown to cause temperature-sensitive male sterility. ERI-3 is a nematode-specific accessory component of DICER.

Nicholas J D Gower et al. (2001) International C. elegans Meeting "Dissection of the upstream regulatory elements of the Inositol 1,4,5-trisphosphate receptor gene itr-1"

Gillian R Temple, Marco Marra, Nicholas J D Gower, Denise S Walker, Jacqueline E Schein, Howard A Baylis

[International C. elegans Meeting, 2001]

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a central role in the organisation of calcium signals by regulating intracellular calcium flux. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-1 (1) and dec-4 (2) ). In previous work (3), we characterised the gene structure in detail and identified three putative transcriptional start sites and three alternative splice sites. Sequence comparisons to the C. briggsae itr-1 homologue support the hypothesis that the itr-1 gene has three promoters (pA, pB and pC), each of which gives rise to an alternative mRNA and hence unique protein. The pattern of expression directed by each promoter was determined using GFP reporter gene fusions. pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the amphid socket cells, the PDA motor neuron and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine (4). Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different IP3R isoforms. Analysis of the sequence conversation between promoter A of C. elegans and C. briggsae identified four short regions (pA-A to pA-D) of conservation. Deletion analysis of pA demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C contains sequences which show homology to binding sites for known C. elegans transcription factors. We are currently identifying factors that regulate the promoter using a yeast one-hybrid screen. 1. Clandinin et al (1998) Cell: 92, 523-533. 2. Dal Santo et al (1999) Cell: 98 757-767. 3. Baylis et al (1999) J. Mol Biol: 294, 467-476. 4. Gower et al (2001) J. Mol Biol: 306, 145-157.

Takashi Murayama et al. (2006) East Asia C. elegans Meeting "Chemotactic response toward basic pH in C. elegans"

Ichiro Maruyama, Takashi Murayama

[East Asia C. elegans Meeting, 2006]

C. elegans responds to water-soluble chemicals such as ions and amino acids. We are especially interested in the response to OH^- as basic pH. In C. elegans, acid pH (H⁺) is sensed as a nociceptive stimuli, and basic pH (OH^-) is sensed as an attractive stimuli (Ward, 1973; Dusenbery, 1974). Sambongi et al. (2000) have shown some of amphid chemosensory neurons are responsible for acid pH avoidance by ablating these neurons. In general, however, a neural mechanism underlying OH^- response in any animals still remains to be explored. To investigate the OH^- chemosensation, we have developed an assay system. On the assay plate with a pH gradient from pH 7.0 to pH 10.0, wild-type animals move toward basic pH along the gradient. When some chemotaxis mutants were analyzed with this assay, osm-6, a sensory cilium-defective mutant, did not show any response to basic pH. In addition, some chemotaxis mutants have normal sensory cilia are found to be insensitive to the pH gradient. Now, we are analyzing some other chemotaxis mutants, and trying to isolate mutants showing irregular response to basic pH. Ward (1973), PNAS 70, 817. Dusenbery (1974), J. Exp. Zool. 188, 41. Sambongi et al. (2000), Neuroreport 11, 2229.

Minniti AN et al. (1995) International C. elegans Meeting "THE HERMAPHRODITE-INITIATED SPERMIOGENESIS PATHWAY."

Minniti AN, Nance JF, Sadler C, Ward S

[International C. elegans Meeting, 1995]

During spermiogenesis, round nonmotile spermatids are rapidly transformed into asymmetrical crawling spermatozoa. In addition to spe-8 and spe-12, we found two new genes, spe-27 and spe-29, that when mutated, disrupt spermiogenesis in an identical manner: mutant hermaphrodites are self-sterile, while mutant males are fertile. Sperm from both sexes activate abnormally in vitro, suggesting that these gene products are expressed in both sperm, but only hermaphrodites require them for activation. The hermaphrodite's spermatids, however, can be activated by mating. This phenotype has been explained by a model that has two distinct pathways of activation, one for males and one for hermaphrodites (Shakes and Ward, 1989). The four genes are needed only for the hermaphrodite pathway. spe-27 and spe-12 have been cloned. Their mRNA is found exclusively in the germline during spermatogenesis. Their predicted protein sequences show no similarities to known proteins. We are currently cloning spe-29. To further understand how these genes act during spermiogenesis we are determining the subcellular localization of their gene products. In addition, suppressor analysis is under way (see abstract by Paul Muhlrad and Samuel Ward) to look for other genes in this pathway and for interactions between these genes.

Johnsen, Holly et al. (2013) International Worm Meeting "Assisted Suicide: a Caspase- and Engulfment-Dependent Cell Death."

Horvitz, Bob, Johnsen, Holly

[International Worm Meeting, 2013]

Programmed cell death occurs during the development of many organisms. The C. elegans cell-death pathway has been extensively studied and is evolutionarily conserved. During programmed cell death, caspases are activated in the dying cell. The cell corpse is engulfed by a neighboring cell and degraded. Almost all cell deaths are "suicides"-they are cell-autonomous, caspase-dependent and can occur even in engulfment-defective animals. In the C. elegans male, the cells B.alapaav and B.arapaav are generated during the late L3 stage. During the early L4 stage one of these cells dies, and the other survives and adopts an epithelial fate. These two cells form an equivalence group; the decision of which cell dies and which survives is stochastic and takes place during the L3/L4 lethargus. The cell that dies is engulfed by the neighboring cell P12.pa. In contrast to most C. elegans cell deaths, the B.al/rapaav death is engulfment-dependent; if engulfment is blocked by a mutation in one of the genes in the engulfment pathway, both B.alapaav and B.arapaav survive. Furthermore, we have found that if the engulfing cell P12.pa is ablated, the B.al/rapaav death fails to occur in approximately 60% of animals. These observations suggest that cell interactions between B.alapaav and B.arapaav as well as between B.al/rapaav and P12.pa are involved in this cell death, leading some to suggest that P12.pa "murders" B.al/rapaav. We are investigating the control and execution of the B.al/rapaav cell death. When the B.al/rapaav cell death is blocked by engulfment defects or P12.pa ablation, the undead cell still initiates the cell-death pathway. Similar to other dying cells, the undead cell looks round by Nomarski and EM and exposes phosphatidylserine on its surface. egl-1 and ced-3 are required for the B.al/rapaav cell death and are expressed in the undead cell, suggesting that the core cell-death pathway is required but not sufficient for this cell death, i.e. that this death is an assisted suicide. We hope our studies will provide insight into new mechanisms of programmed cell death, cell-cell signaling, and fate determination within equivalence groups.

Qiang Liu et al. (2004) East Coast Worm Meeting "CaM KII Regulates Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Zhao-Wen Wang, Qiang Liu

[East Coast Worm Meeting, 2004]

Ca 2+ /calmodulin-dependent kinase II (CaM KII) plays an important role in synaptic plasticity and development of the nervous system. Malfunction of CaM KII is associated with a variety of diseases including epilepsy. In the nervous system, CaM KII is expressed at both pre- and post-synaptic sites. Little is known about the potential role of CaM KII in neurotransmitter release. We have embarked on a project to study the function of CaM KII in neurotransmitter release using C. elegans as model system. C. elegans has a single CaM KII gene, unc-43 . Both gain-of-function and loss-of-function mutants are available for analysis. To evaluate the function of CaM KII in neurotransmitter release, miniature and evoked postsynaptic currents (mPSCs and ePSCs) were recorded at the neuromuscular junction with the postsynaptic body-wall muscle cell clamped at -60 mV. We initially analyzed mPSCs and ePSCs in unc-43(n498) , a gain-of-function mutant. Compared with the wild-type, the mutant showed a significant reduction in the amplitude of ePSCs (wild-type 1579 +/- 129 pA, n = 8; n-498 721 +/- 79 pA, n = 12) and in the frequency of mPSCs (wild-type 50.6 +/- 3.4 Hz, n = 22; n-498 22.6 +/- 3.4 Hz, n = 6). Postsynaptic receptor sensitivity appeared normal in the mutant since the amplitude of mPSCs did not change (wild-type 27.4 +/- 1.6 pA, n = 22; n-498 28.4 +/- 4.3 pA, n = 6). These results suggest that a normal function of CaM KII is to regulate neurotransmitter release. Experiments are under way to determine 1) whether unc-43 loss-of-function mutants show opposite changes of mPSCs and ePSCs compared with the gain-of-function mutant; 2) whether the apparent effect on neurotransmitter release is due to an action of presynaptic CaM KII, or a retrograde signal generated by postsynaptic CaM KII; and 3) what are the phosphorylation targets of CaM KII in regulating neurotransmitter release.

Hill KL et al. (1996) East Coast Worm Meeting "Genetic and Phenotypic Characterization of spe-16, a Gene Required for Fertilization in C. elegans"

Mercer KB, L'Hernault SW, Hill KL

[East Coast Worm Meeting, 1996]

Unlike many other organisms, fertilization in C. elegans is an extraordinarily efficient process; normally every sperm produced by a hermaphrodite successfully fertilizes an egg within the spermatheca (Ward, S. and J. Carrel, 1979 Dev. Biol. 73: 304). Many mutants that affect spermatogenesis and, consequently, disrupt fertilization have been selected by both ours and Sam Ward's laboratory. Most of these mutants have obvious cytological abnormalities, so their defects in fertilization are perhaps not surprising. We have been studying mutants that complete spermatogenesis and form spermatozoa with superficially normal cytology but are defective in hermaphrodite self-fertilization. One of these genes is the spe-16 gene, which is located near tra-1 on the right arm of chromosome III. Mutant males were assessed for sperm transfer by using spe-8 hermaphrodites as recipients; spe-8 hermaphrodites are self-sterile except when they receive "competent" seminal fluid from a male, which allows self-fertility (Shakes, D. & Ward, S. 1989 Dev. Biol. 134: 189). In such tests, spe-16 mutant males were normal in seminal fluid transfer to hermaphrodites, but cannot sire progeny in such cross-fertilization tests. Double mutants that are spe- 9ts and spe-16ts have an enhanced sterile phenotype, suggesting some form of synergism between these two mutations. We recently have developed a fluorescent vital staining procedure and have examined migration of labelled male sperm in the hermaphrodite reproductive tract. Wild type male- derived spermatozoa migrate to and stop at the spermatheca, which is the site of both sperm storage and fertilization. Surprisingly, spe-16 and spe-9 spe-16 double mutant male-derived spermatozoa are similar to wild type in their ability to localize to the spermatheca after sperm transfer during mating. All spe-16 studies have been performed on the one known allele, hc54, which is temperature sensitive. Mutagenesis to obtain new alleles is ongoing and should prove useful in deciphering the specific defect(s) in spe-16 mutant spermatozoa.

Mary Kosinski et al. (2001) International C. elegans Meeting "Control of Oocyte Meiotic Maturation and Gonadal Sheath Cell Contraction by MSP Signaling"

Mary Kosinski, David Greenstein, Michael A Miller

[International C. elegans Meeting, 2001]

Oocyte meiotic maturation and ovulation are essential biological processes required for reproduction. During meiotic maturation, C. elegan s oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in an assembly-line-like fashion. Recently we found that the major sperm cytoskeletal protein (MSP) is the sperm derived signal that induces both oocyte meiotic maturation and gonadal sheath cell contraction (1). Many questions remain about this newly discovered signaling pathway. For example, how does a cytoskeletal protein function as a signaling molecule? How does MSP signal two distinct responses? To examine MSP signaling in vivo , we analyzed extracellular MSP localization. Previous studies reported the intracellular localization of MSP during spermatogenesis (2). We stained mated fog-2 females with anti-peptide antibodies generated to the N- and C- terminal regions of MSP, as well as previously described antibodies (2). In addition to intracellular staining within spermatozoa, we observed an extracellular MSP gradient, with the highest levels of MSP within the spermatheca and lower levels in the proximal gonad arm. The average extent of the observable MSP gradient is approximately 30 m m. Detection of this extracellular MSP gradient is dependent on the number of sperm present and their position in the spermatheca. By mating females with Mr. Vigorous, we have been able to detect the MSP gradient extending as far as 90 m m. These data suggest that a fraction of MSP is released from sperm in vivo, and the most proximal oocyte receives the highest concentration of released MSP. MSP is a bipartite signal: MSP (106-126) is sufficient to promote gonadal sheath cell contraction, and MSP (1-106) is sufficient to promote oocyte maturation (1). In solution MSP exists as a dimer (3). To determine if dimerization is necessary for signaling, we injected dimerization mutants (4) into females and measured oocyte maturation and sheath cell contraction rates. The dimerization mutants tested signal both oocyte maturation and gonadal sheath cell contraction at rates similar to wild-type MSP. To further identify regions responsible for promoting oocyte maturation and gonadal sheath cell contraction, we tested whether Ascaris suum MSP could signal in C. elegans . Ascaris MSP shares about 80% identity with C. elegans MSP at the amino acid level. Our results indicate that Ascaris MSP can signal maturation and sheath cell contraction in C. elegans at rates comparable to C. elegans MSP. We are currently targeting regions for deletion experiments that are conserved between both C. elegans and Ascaris . 1. Miller et al. (2001). Science 291:2144-2147 2. Ward & Klass. (1982). Dev. Bio. 92:203-208 3. Haaf et al.(1996). J. Mol. Biol. 260:251-260 4. Smith & Ward. (1998). J. Mol. Biol. 279:605-619 We would like to thank T. Roberts and P. Griffin for Ascaris clones and S. Ward and H. Smith for dimerization mutants and antibodies.