Jud, Molly et al. (2019) International Worm Meeting "A screen for temperature-sensitive morphogenesis-defective mutants."

Fennell, Francesca, Yang, Yuqi, Bowerman, Bruce, Jud, Molly, Miller, Alex, Lowry, Josh, Bao, Zhirong, Tran, Nhah, Padilla, Thalia, Shao, Hong

[International Worm Meeting, 2019]

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs. Cell adhesion and the cytoskeleton are important for morphogenesis, but the signaling pathways involved remain poorly understood. As genes required for morphogenesis often have earlier roles in development, temperature-sensitive embryonic lethal (TS-EL) alleles are useful tools for investigating morphogenesis. From a collection of ~1,000 TS-EL mutants, we have classified the terminal phenotypes of 191 with normal early embryogenesis, after upshifts from the permissive (15 deg C) to the restrictive temperature (26 deg C) at the L4 stage, to identify 79 with highly penetrant elongation defects. We identify causal mutations using whole genome sequencing (WGS) and genetic complementation tests. So far, we have identified 30 alleles representing 21 genes. Many of these genes likely regulate gene expression globally with roles in cell fate specification (e.g. glp-1, which encodes a Notch receptor involved in blastomere specification) or differentiation (e.g. chaf-1, which encodes a chromatin assembly factor subunit), while others appear to be weak alleles of cell division genes (e.g. zwl-1, which encodes a kinetochore protein). To identify mutants that are specifically defective in morphogenesis, we upshifted mutant embryos just prior to the bean stage, after most cell division, cell fate specification and differentiation has occurred, and identified 12 with penetrant elongation defects. These include two with causal mutations in rib-1 and rib-2, which encode heparin sulfate synthesis proteins that regulate morphogenesis in other organisms, although how post-translational sugar modifications influence morphogenesis is still unclear. We are now using WGS to identify genes for 6 more of these 12 late-upshift mutants. We also are characterizing morphogenetic defects in fixed mutant embryos, using antibodies against epidermal cell membrane proteins to assess cell shape dynamics. Our long term goal is to advance our understanding of morphogenesis by identifying more genes that influence this process.

Molly Jud et al. (2007) International Worm Meeting "RNP foci in C. elegans oocytes contain stress granule proteins and are conserved in Caenorhabditis nematodes."

Jamie Razelun, Jeremy Bickel, Megan Wood, Molly Jud, Sarah Fausett, Jennifer Schisa, Michael Czerwinski

[International Worm Meeting, 2007]

In the arrested oocytes of old-aged Caenorhabditis elegans hermaphrodites that are depleted of sperm, giant ribonucleoprotein (RNP) foci form. The RNP foci are composed of putative RNA-binding proteins and translationally repressed maternal mRNAs. Based on their components, we hypothesize that RNP foci function to maintain maternal mRNA stability, protect the integrity of oocytes, and/or regulate translation of maternal mRNAs while a worm waits for sperm. Because hermaphroditism is rare in the animal kingdom, we tested whether arrested ovulation and RNP foci formation occur in gonochoristic nematodes ancestral to C. elegans: C. remanei, C. sp. PS1010, C. sp. CB5161, and Rhabditella axei. We demonstrate that in multiple Caenorhabditis species, ovulation arrests in unmated females and RNP foci form in the arrested oocytes. These data suggest that a mechanism for preserving oocytes has been conserved from an ancestral gonochoristic state. We are currently expanding our studies to include additional nematode species outside the Caenorhabditis clade. Our hypothesis for the function of RNP foci in oocytes is similar to that for mammalian RNPs called stress granules. We are currently investigating whether orthologs of mammalian stress granule proteins co-localize to C. elegans RNP foci. Our results to date indicate that poly(A) binding protein (PABP-1) co-localizes with the RNA-binding protein, MEX-3, in oocyte foci. We also have preliminary data that large foci of MEX-3 form in non-arrested oocytes in response to heat shock, similar to the induction of stress granules in response to heat stress. We are currently characterizing the heat-shock-induced foci to determine how similar they are to the RNP foci that form in oocytes when sperm are absent.

Hong M et al. (2008) Biochem Biophys Res Commun "The anesthetic action of ethanol analyzed by genetics in Caenorhabditis elegans."

Lee J, Choi MK, Hong M

[Biochem Biophys Res Commun, 2008]

Acute exposure to ethanol causes paralysis at high concentrations in the nematode Caenorhabditis elegans. We set out to elucidate the mechanism of the anesthetic action of ethanol by genetic approaches. We identified nine mutations that conferred reduced sensitivity to ethanol after chemical, irradiation, or transposon insertion mutagenesis. Of these nine, we further characterized five mutations that defined four genes, jud-1-jud-4. Analysis of the phenotypes of the animals heterozygous for two unlinked genes revealed that jud-1 and jud-3 act synergistically in a gene dose-dependent manner. We cloned jud-4 and found that it encodes a protein with limited homology to human Homer proteins. jud-4 was expressed in the hypodermis and vulva muscles, suggesting that this gene acts in tissues directly exposed to the external environment. Characterization of the other mutations identified in this study will facilitate the elucidation of the molecular mechanism for the anesthetic action of ethanol.

Choi M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "PTR-6, a C. elegans Patched related gene, regulates the anesthetic effect of ethanol"

Lee J, Choi M, Hong M

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Acute exposure to ethanol causes paralysis at high concentrations in the nematode Caenorhabditis elegans. We set out to elucidate the mechanism of the anesthetic action of ethanol by genetic approaches. We identified nine mutations that conferred reduced sensitivity to ethanol after chemical, irradiation, or transposon insertion mutagenesis. In the previous study, molecular nature of the mutant made by transposon insertion, jud-4, was identified. jud-4 encodes a C. elegans homolog of Homer proteins, which are important for synaptic protein localization in other organisms. jud-4 was expressed in the hypodermis and vulva muscles, suggesting that this gene acts in tissues directly exposed to the external environment. In this study, we wanted to characterize other mutations affecting ethanol sensitivity. Among them, jud-1 mutations, most strong mutants, were identified by SNP mapping and whole-genome sequencing. We found that jud-1 encodes a C. elegans homolog of PATCHED-related receptor, PTR-6. PTR-6 has an SSD (Sterol-sensing domain) and a PATCHED family motif which has an essential role for hedgehog signal transduction. Although hedgehog signaling is not fully conserved in C. elegans, it has been reported that homologs of core components in the hedgehog pathway have many biological significance. Detailed analysis of ptr-6 should elucidate a novel mechanism for the anesthetic effect of ethanol.

Mingi Hong et al. (2001) International C. elegans Meeting "Ethanol Sensitivity Genes in the nematode Caenorhabditis elegans"

Min Sung Choi, Mingi Hong, Junho Lee

[International C. elegans Meeting, 2001]

Alcohol has many effects in higher organisms, especially in the nervous system. The mechanisms and sites of action of ethanol are not fully understood. In the hope of understanding the mechanisms of ethanol, we identified genes that control sensitivity to ethanol and anesthetics in the invertebrate system Caenorhabditis elegans. 15 mutations that confer resistance to the anesthetic effect of ethanol were identified, either by chemical mutagenesis or transposon insertion mutagenesis. We have cloned two of the genes responsible for the resistant phenotypes by mapping and rescue, and by transposon tagging. One of these, jud-1, encodes a protein similar to contactin. The cDNA analysis of the jud-1 gene shows that it is alternatively spliced. The shorter transcript has a single Ig domain, while the longer transcript has three Ig domains and one fibronectin type III domain. The functions of the two alternatively spliced transcripts are not known at this moment. In the mammalian system, contactin is known to be a cell adhesion/recognition molecule in the nervous system and has roles in the formation of axon projections in the developing nervous system. In C. elegans, jud-1 is also expressed in some neurons as shown by GFP expression analysis. jud-5, an ethanol sensitivity gene cloned by transposon tagging, encodes a novel protein. From the expression pattern of its GFP fusion protein, it could be seen that jud-5 is expressed in most muscles including pharyngeal, body wall, tail and vulva muscles. Further studies of these mutants are under way.

Fenker, Kristin et al. (2013) International Worm Meeting "The role of an SLC6 family transporter in C. elegans sperm activation."

Chong, Conrad, Hansen, Angela, Jud, Molly, Fenker, Kristin, Stanfield, Gillian

[International Worm Meeting, 2013]

Many types of cells must change their morphology to function properly. In C. elegans sperm, morphological changes occur during a regulated process called sperm activation, which culminates in mature sperm that are motile and competent for fertilization. Sperm activation is regulated differently in male and hermaphrodite worms, and we are interested in the signals regulating male sperm activation. Our lab previously identified TRY-5, a serine protease in seminal fluid, as a key regulator. We have identified additional factors, including snf-10, a member of the solute carrier 6 (SLC6) transporter family. Like try-5, snf-10 is not required for fertility and acts in parallel to the spe-8 group of genes that regulate hermaphrodite sperm activation. Males transfer an activator in their seminal fluid during mating; unlike try-5, snf-10 is not required for this transfer. However, without snf-10, sperm are unable to respond to the male activator. These data suggest snf-10 is in the same pathway as try-5, but is unlikely to regulate it. Thus, snf-10 likely acts downstream of try-5 in the male activation pathway. Consistent with this model, SNF-10 functions in sperm, and is polarized to the cell body in activated sperm. We are using in vitro sperm activation assays to address the function of SNF-10. Wild-type sperm can be activated by protease treatment, suggesting a protease may act on a target on the sperm plasma membrane. snf-10 mutant sperm do not activate in response to protease, demonstrating that SNF-10 is biochemically downstream of protease activity. snf-10 mutant sperm do activate in response to treatment with other known activators, such as TEA and DIDS. We also have shown snf-10 mutant sperm are more sensitive than wild-type to treatment with the ionophore monensin. Our results indicate SNF-10 is a good candidate for a target of TRY-5 cleavage. We continue to investigate this relationship, and are pursuing experiments to characterize SNF-10's transporter activity and the role of SNF-10 in transducing signals that mediate changes in cell morphology.

Schisa J A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Dynamics of Novel Nuclear Membrane Structures in Aging and Stressed Oocytes"

Schisa J A, Patterson J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

In several animal species, oocytes arrest in meiosis until they are fertilized. Several cytological studies have reported changes in the cytoplasm of these arrested oocytes, including formation of annulate lamellae and morphological changes in mitochondria. We are focusing on understanding the regulation and function of large ribonucleoprotein (RNP) granules that assemble in the germlines of Caenorhabditis nematodes that are either stressed, or in which ovulation is arrested due to old age or absence of sperm. The large RNP granules are hypothesized to regulate mRNA stability or translation in arrested or stressed oocytes during delays in fertilization (Jud et al., 2008). We have developed fixation and staining procedures for transmission electron microscopy that, along with complementary confocal analyses, allow visualization of novel nuclear membrane structures in arrested and stressed oocytes. We have determined that the number of nuclear pores clustered beneath P granules on the nuclear envelope of pachytene germ nuclei is higher in nematodes in which ovulation is arrested. We have also observed the first evidence for significant blebbing along the nuclear membranes of arrested and heat-stressed oocytes. In addition to the changes in oocyte nuclei, assemblies of annulate lamellae (stacks of nuclear pore-containing membranes) have been detected in the cortical cytoplasm of these oocytes, in close proximity to what we believe are large RNP granules. The cellular responses to aging and stress appear to be conserved, at least from C. remanei to C. elegans. We are currently developing reagents to test a model that proposes functional links between the observed nuclear membrane dynamics and formation of large, cortical RNP granules in arrested and stressed oocytes. Jud et al., 2008. Dev Biol 318: 38-51.

Beitel GJ et al. (1991) International C. elegans Meeting "CLONING AND SEQUENCING OF THE SYNTHETIC MULTIVULVAL GENE lin-9"

Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype can result from mutations in two separate genes, while each mutation alone results in an apparently wild-type phenotype (Ferguson and Horvitz, Genetics 123:109-121,1989). Mutations that can cause this synthetic Multivulva (syn Muv) phenotype have been grouped into two classes, A and B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. Iin-9(nll2) III is a class B mutation that results in an apparently wild-type phenotype by itself. We previously reported that ZK637 and overlapping cosmids can rescue lin-9 (WBG 11(2 ): p 29,1990). We have now identified an 8 kb subclone with rescuing activity. Using this 8 kb fragment to probe Stuart Kim's cDNA library, six positive plaques representing at least three independent clones were isolated. This 8 kb probe also detects a message of approximately 2.5 kb on Northern blots. The message level appears similar in eggs and mixed-stage populations. We are currently sequencing these cDNAs and looking for evidence that these transcripts do in fact represent the lin-9 gene. This work will be greatly aided by the genomic sequence provided by Molly Craxton, John Sulston and Alan Coulson, as ZK637 was fortuitously chosen as one of the first cosmids to be sequenced in the worm genome sequencing project.

Trombley, Alicia et al. (2015) International Worm Meeting "Membrane Remodeling in the C. elegans Germ Line."

Trombley, Alicia, Schmidt, Kevin, Wood, Megan, Schisa, Jennifer, Davis, Michael

[International Worm Meeting, 2015]

In many animal species, oocytes arrest in meiosis until they are fertilized. As oocytes age, changes occur in their cytoplasm, and fertility diminishes. Our goal is to better understand the regulation and function of ribonucleoprotein (RNP) and membrane remodeling in the oocytes of Caenorhabditis nematodes that are either arrested in meiosis or exposed to stress. The assembly of large RNP granules is hypothesized to maintain oocyte quality by regulating mRNA stability or translation (Jud et al., 2008). Increased blebbing of nuclear membranes also occurs in these oocytes, along with the assembly of annulate lamellae (Patterson et al., 2011). The remodeling of nuclear membranes into blebs in meiotically-arrested or heat-stressed oocytes correlates with the remodeling of RNPs and the formation of annulate lamellae at the oocyte cortex. Our current experiments are leveraging the discovery of large stacks of annulate lamellae in cgh-1 and ifet-1 oocytes. We are assessing the extent of nuclear blebbing in these, and related CGH-1 complex mutants, and so far have discovered increased blebbing in a subset of the mutants. We are also using TEM and the SP12::GFP strain to analyze membranes at the oocyte cortex and throughout the germ line. In addition to stacks of aligned membranes visualized by TEM in the mutant germ lines, we also observe large patches of ER membrane in oocytes and in the distal core. In combination with analyses of RNP granules, our results will address the hypothesis that nuclear blebbing initiates the formation of AL at the oocyte cortex, which in turn promotes the assembly of RNP granules.

Alker, Ashley et al. (2009) International Worm Meeting "Regulation of RNP Granule Assembly in Oocytes."

Hanson, Mariah, Lincoln, Merrick, Goike, Andrew, Aguirre, Cynthia, Schisa, Jennifer, Alker, Ashley

[International Worm Meeting, 2009]

As C. elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) granules form in arrested oocytes that include several groups of RNA-binding proteins: P granule proteins, P body-associated proteins, and stress granule-associated proteins. Large RNP granules are also induced in non-arrested oocytes when worms are exposed to environmental stresses such as heat shock and anoxia (1). One hypothesis for the function of RNP granules in arrested or stressed oocytes is they maintain mRNA stability or prevent precocious translation until fertilization or a stress-free environment resumes. Previous work indicated one branch of the major sperm protein (MSP) pathway regulates the subcellular changes of at least some RNA-binding proteins in arrested or stressed oocytes (1). Our current goal is to better define the pathways regulating the assembly of the different classes of proteins that appear to aggregate into large RNP granules in oocytes. We have undertaken a functional RNAi screen to identify genes required for the RNA-binding protein, MEX-3, to assemble into granules when ovulation is arrested. We have identified 11 genes to date, and we are now determining whether these genes also regulate the assembly of P body and stress granule-associated proteins into granules. In parallel, we are asking if the genes that regulate the response to arrested ovulation are also necessary in effecting the oocyte response to environmental stress. In addition to elucidating the pathways that regulate the assembly of RNP granules in oocytes, we are also characterizing the effects on germline development and RNA metabolism when RNP granule assembly is defective to test our hypothesis for RNP granule function. 1) Jud et al., 2008. Dev Biol 318: 38-51.