Tan, Chieh-Hsiang et al. (2017) International Worm Meeting "The ZIPT-7.1 transporter mediates zinc signaling to promote sperm activation in C. elegans."

Kornfeld, Kerry, Scharf, Andrea, Ellis, Ronald E., Dietrich, Nicholas, Warnhoff, Kurt, Zhao, Yanmei, Tan, Chieh-Hsiang

[International Worm Meeting, 2017]

Sperm activation involves the rapid transition of a round, immotile spermatid into a polarized, motile sperm capable of fertilization. This transition in cell fate does not involve transcriptional changes, because the nucleus is not accessible. Although a large number of genes involved in sperm activation have been characterized in C. elegans, the signaling pathway that mediates this transition is not well-defined. We identified the zipt-7.1 gene, which encodes a ZIP (Zrt/ Irt-like protein), using a reverse genetic approach. A deletion of the gene caused a partially penetrant sterile phenotype in hermaphrodites and reduced the fertility of males. The sterile hermaphrodites laid unfertilized oocytes and had mostly inactive sperm. Since the sterile phenotype of the hermaphrodites could be rescued by crossing with wild-type males, it must have been caused by a sperm defect. A similar phenotype was observed in mutants of the related species C. tropicalis. Finally, WT spermatids could be activated in vitro with pronase, trypsin or zinc, but zipt-7.1(lf) spermatids were less responsive to pronase and trypsin and had almost no response to zinc. Consistent with these results, RNAi showed that ZIPT-7.1 functions in the germ line, and GFP::ZIPT-7.1 was expressed in spermatocytes. Sperm activation in C. elegans depends on two genetic pathways - the spe-8 pathway and the try-5 protease pathway. Epistasis studies suggest that zipt-7.1 functions downstream of spe-6 in the spe-8 pathway. To understand the mechanism of zipt-7.1-dependent sperm activation, we used molecular approaches. Transcription of zipt-7.1 was regulated by environmental zinc levels, and zipt-7.1 mutant sperm displayed reduced amounts of labile zinc, suggesting that it regulates zinc levels in sperm. Expression of ZIPT-7.1 in mammalian cells promoted zinc uptake, showing that it can transport zinc. Homologues of ZIPT-7.1 in other species localize to ER-Golgi, and ZIPT-7.1 expressed in human cell lines localized to the Golgi and lysosomes. Based on these results, we propose that ZIPT-7.1 is localized to vesicles in spermatids, and that a sperm activation signal results in the activation of its transporter activity and the release of zinc stores into the cytoplasm. This cytoplasmic zinc constitutes a second messenger that promotes activation. Our results provide the first identification of a zinc transporter that mediates sperm activation, and may have broad relevance, since zinc has been implicated in sperm function in a variety of animals.

Tan, Chieh-Hsiang et al. (2015) International Worm Meeting "hke-4.1/ ZIP7 encodes a zinc transporter involved in zinc homeostasis and sperm function in Caenorhabditis elegans."

Dietrich, Nicholas, Kornfeld, Kerry, Warnhoff, Kurt, Ellis, Ronald E., Tan, Chieh-Hsiang, Zhao, Yanmei

[International Worm Meeting, 2015]

Zinc is an essential trace element that plays many important roles in biological systems. The homeostasis of zinc in eukaryotes is mediated by two conserved families of transmembrane proteins- Znt/CDF (Cation Diffusion Facilitator) proteins transport zinc out of the cytoplasm, whereas ZIP (Zrt/Irt-like protein) proteins transport zinc into the cytoplasm. Based on the existence of CDF and ZIP homologues for every zinc transporter in humans, the nematode Caenorhabditis elegans is a promising model for the study of zinc homeostasis. While important progress has been made in understanding CDF proteins in worms, there is very little information about the function of ZIP proteins. Studies of human cells suggest that ZIP7 functions as a key component of intracellular zinc signaling by mediating release of ER-stored zinc molecules after being activated by phosphorylation. There are two homologues of human ZIP7 in C. elegans, hke-4.1 and hke-4.2. To investigate the function of hke-4.1, we analyzed a deletion mutation that creates a null allele. Loss of hke-4.1 caused worms to be hypersensitive to low dietary zinc induced by using a zinc chelator. Moreover, the level of hke-4.1 mRNA was increased by low dietary zinc. Thus, hke-4.1 is induced by low dietary zinc and necessary for growth in these conditions, suggesting it plays a role in zinc trafficking and homeostasis. Loss of hke-4.1 also caused a partially penetrant, cold-sensitive sterile phenotype in hermaphrodites and reduced the fertility of males. The sterile phenotype of the hermaphrodites was rescued by mating to wild-type males. These results indicate hke-4.1 is necessary for sperm function. We are currently analyzing the sperm defects in hke-4.1 mutant animals and investigating the hypothesis that hke-4.1 mediates a zinc signal during C. elegans sperm development.

Cao, Mengyi et al. (2021) International Worm Meeting "Developing Steinernema hermaphroditum as a model system to study symbiosis"

Cao, Mengyi, Sternberg, Paul, Schwartz, Hillel, Tan, Chieh-Hsiang

[International Worm Meeting, 2021]

Entomopathogenic nematodes (EPNs, which include Steinernema and Heterorhabditis) seek out insect hosts that they infect and rapidly kill with the assistance of their mutualistic symbiotic bacteria (Xenorhabdus and Photorhabdus), with which they exist in species-specific partnerships. The development of genetic tools in Xenorhabdus and Photorhabdus bacteria have proved these symbiotic systems to be highly valuable for the study of both mutualism and parasitism. However, hopes that EPNs could be genetic models have been frustrated for decades by inconsistent in vitro growths, variable success in cryopreservation, and low mating efficiency in the laboratory. We obtained a recently described Indian strain of Steinernema hermaphroditum (Bhat et al., 2019) and optimized its in vitro growth with a generation time of three days on a thin layer of its native symbiotic bacteria Xenorhabdus griffiniae on transparent growth media (NGM). We developed a simple and efficient cryopreservation method by freezing mixed juvenile stages of nematodes. Previously, S. hermaphroditum was described as first generation hermaphroditic and second generation gonochoristic (male-female) when isolated from its insect hosts. We discovered that at least when growing in vitro, this species produced exclusively self-reproducing hermaphrodites for consecutive generations, except for rare spontaneous males. We performed EMS mutagenesis screens in S. hermaphroditum which generated multiple mutant lines with visible phenotypes. Mating of recessive mutant hermaphrodites with wild-type males produced wild-type hermaphrodite F1 cross-progeny that produced mutant and wild-type F2 self-progeny with Mendelian ratios, consistent with self-fertilization. We also acquired X-linked recessive mutants that showed sex determination is chromosomal. We report S. hermaphroditum-India as the first case of a true consistently hermaphroditic species of EPN known to date, and find it to be highly tractable in the laboratory. We will present our ongoing progress in developing this species into a genetic model for symbiosis research.

Zhao, Yanmei et al. (2015) International Worm Meeting "The zinc transporter HKE-4.1 controls sperm activation."

Ellis, Ronald, Tan, Chieh-Hsiang, Zhao, Yanmei, Kornfeld, Kerry

[International Worm Meeting, 2015]

After males make sperm, they store them in an inactive state. The activation process only occurs at mating, and involves extensive changes that confer mobility. In it, chemical signals in the ejaculate act through redundant pathways to trigger changes in the cytoskleton. Genetic studies using C. elegans revealed that the TRY-5 protease activates male sperm by acting through the membrane protein SNF-10. A second pathway functions in both sexes, and is mediated by SPE-8, SPE-12, SPE-19, SPE-27 and SPE-29. What happens after these events at the plasma membrane remains a mystery. However, analyses of weak spe-6 alleles suggest that it encodes a critical downstream kinase.Because C. elegans sperm can be activated by zinc in vitro, we looked for genes that regulate zinc metabolism and are required for fertility. These criteria led us to HKE-4.1, one of two members of the ZIP7 family of zinc transporters. An hke-4.1 deletion produces sterile hermaphrodites that lay many oocytes, a phenotype associated with sperm defects. As expected, the mutant oocytes are normal and can be fertilized by wildtype sperm. However, most of the hermaphrodite sperm remain inactive and are soon lost. The hke-4.1 males also have reduced fertility and show sperm activation and mobility defects. Although normal spermatids can be activated in vitro by a variety of methods, mutations in hke-4.1 lower the response to pronase or trypsin, and almost completely eliminate the response to zinc. Furthermore, the mutations lower the amount of zinc present inside of sperm, as judged by staining with the dye zinpyr-1. These results suggest that HKE-4.1 regulates sperm activation by controlling zinc transport.Weak alleles of spe-6 cause male sperm to activate prematurely. Surprisingly, sperm fail to activate in spe-6(hc163); hke-4.1 double mutants, which suggests that HKE-4.1 is the most downstream player known in the activation process. RT-PCR analyses show that hke-4.1 is predominantly expressed in germ cells, and preliminary studies of HKE-4.1::FLAG produced by genome editing suggest that it is found in the cytoplasm of developing spermatocytes and concentrates near the nucleus of spermatids. Thus, we propose that HKE-4.1 regulates sperm activation by controlling the release of internal zinc stores. As a result, this gene provides an ideal model for studying the regulation of biological events by zinc.

Warnhoff, Kurt et al. (2015) International Worm Meeting "nhr-33 encodes a nuclear receptor that is regulated by high zinc and mediates transcriptional activation to maintain zinc homeostasis in C. elegans."

Kornfeld, Kerry, Tan, Chieh-Hsiang, Schneider, Daniel, Roh, Hyun, Kocsisova, Zuzana, Croswell, Damari, Warnhoff, Kurt, Morrison, Andrew

[International Worm Meeting, 2015]

Zinc is an essential element involved in many biological processes and human disorders. Excess zinc is deleterious, and therefore animals have evolved sophisticated mechanisms to maintain zinc homeostasis in response to variable zinc availability. However, mechanisms of zinc sensing and detoxification are not well defined. Caenorhabditis elegans responds to excess dietary zinc by activating transcription of critical zinc-homeostasis genes, such as the zinc exporters cdf-2 and ttm-1b. We demonstrated that the transcriptional response to high dietary zinc is mediated by the high zinc activated (HZA) element, a 14 bp DNA enhancer that is present in multiple genes that are induced by high zinc (Roh et al., NAR, 2014). To identify genes that are necessary for the response to excess dietary zinc, we conducted a forward mutagenic screen for animals with defects in zinc-induced transcription. We identified five loss-of-function mutations and one gain-of-function mutation in nhr-33, which encodes an uncharacterized nuclear receptor. Nuclear receptors are transcription factors that have a DNA binding domain and a ligand binding domain. nhr-33(lf) mutations abrogate the induction of zinc-regulated genes and dramatically impair zinc homeostasis, as documented by reduced growth and development when mutant animals were cultured with excess dietary zinc. By contrast the nhr-33(gf) mutation caused constitutive induction of zinc-regulated genes. Thus, nhr-33 is necessary and sufficient for high zinc-activated transcription. To characterize the mechanism of action of nhr-33, we analyzed the expression pattern; NHR-33 is expressed in intestinal cells and accumulates in nuclei in response to high dietary zinc. Biochemical experiments demonstrated that NHR-33 binds the HZA element with high specificity. These results indicate that nhr-33 is the master regulator of high zinc homeostasis in C. elegans. High dietary zinc causes NHR-33 to accumulate in the nucleus where it binds the HZA element and activates transcription of genes that promote zinc homeostasis. These findings establish a new paradigm for environmental sensing by identifying a novel and unexpected role for a nuclear receptor in metal homeostasis.

Scharf, Andrea et al. (2021) International Worm Meeting "A population state shift supports aging as a cause of adult death"

Tan, Chieh-Hsiang, Scharf, Andrea, Brady, Brian, Wilson, Andrea, Schneider, Daniel, DiAntonio, Gabe, Sanchez, Francesca, Ram, Natasha, Jin, He, Armstead, Brinda, Kocsisova, Zuzana, Kornfeld, Kerry

[International Worm Meeting, 2021]

Birth and death drive population dynamics and determine whether a population survives or is doomed for extinction. Individual traits that influence development, diapause, reproduction, aging and lifespan must impact the age structure and survival of a population. However, the relationships between individual traits and population dynamics are still a major challenge in the emerging field of ecology-development (eco-devo), because wild populations exist in complex ecosystems that are challenging to investigate and undesirable to manipulate. Here, we introduce a laboratory ecosystem based on the model organism C. elegans that can be used to measure and manipulate worm populations over hundreds of generations. To complement this experimental system, we developed a computational simulation that realistically models the ecosystem and makes it possible to monitor the life history of every single worm in the population. With this integrated systems approach, we investigated the role of aging in population dynamics. The first question we addressed was why some populations support old animals whereas, in other populations, all animals die young. We discovered that old age as a cause of death is influenced by three conditions: maximum lifespan, rate of adult culling, and progeny number/food stability. More specifically, the populations displayed an unexpected tipping point for aging as the primary cause of adult death. In populations with high progeny survival and regular starvation almost all adults died young. In contrast, a reduction of progeny survival over the tipping point caused a dramatic shift in the population with the consequence that nearly all adults died of old age. By defining these conditions, we establish a conceptual framework that explains why animals as different as mayflies and elephants die of old age in the wild. In conclusion, we created a powerful experimental platform to investigate the relationships between individual developmental processes, developmental plasticity, and population dynamics including extinction.

Catherine A Mastroieni et al. (2004) West Coast Worm Meeting "ANALYSIS OF MAP KINASE PHOSPHORYLATION SITES IN THE C. ELEGANS TRANSCRIPTION FACTOR LIN-31"

Corey A Morris, Catherine A Mastroieni, Leilani M Miller

[West Coast Worm Meeting, 2004]

Complex signaling pathways are required for the proper specification of cell fates. In the C. elegans hermaphrodite, six cells of initially equivalent developmental potential respond differentially to a Ras/MAP kinase signaling pathway, correctly choosing their respective fates to form the adult vulva. Activation of the Ras/MAP kinase pathway culminates in the phosphorylation of one or more transcription factors by MAP kinase, resulting in the induction of fate-specific genes. One of the transcription factors phosphorylated by MAP kinase is LIN-31, a winged-helix transcription factor (Miller et al., Genes Dev. 7:933, 1993) that contains four consensus MAP kinase phosphorylation sites. Co-immunoprecipitation experiments by Tan et al. (Cell 93:569, 1998) showed that LIN-31 forms a heterodimer with the LIN-1 ETS transcription factor and that this heterodimer is disrupted upon phosphorylation of LIN-31 by MAP kinase. These results support the following model in which LIN-31 has two functions: 1) in its unphosphorylated, dimerized form LIN-31 promotes non-vulval cell fates, and 2) in its phosphorylated, undimerized form LIN-31 promotes vulval cell fates. Consistent with the model that phosphorylation of the MAP kinase consensus sites is necessary for LIN-31 function in promoting vulval cell fates, previous studies (Tan et al., 1998) have shown that deletion of all four phosphorylatable sites in LIN-31 results in defective vulval formation. Since the core sequence for MAP kinase consensus sites (S/T-P) consists of only two amino acids, many "consensus" sites identified by sequence alone may not actually be phosphorylated by MAP kinase. Although the first MAP kinase phosphorylation site in the LIN-31 protein is a "full" site (P-X-S/T-P) and Tan et al. (1998) showed that removal of this site resulted in a 50% decrease in overall phosphorylation of the LIN-31 protein, the other three sites contain only the "core" sequence. We are using site-directed mutagenesis, protein expression and purification, and phosphorylation experiments to investigate the phosphorylation pattern of the putative MAP kinase phosphorylation sites in the LIN-31 protein.

Ewbank JJ et al. (2000) European Worm Meeting "Using C. elegans to identify Serratia marcescens virulence factors"

Ewbank JJ, Kurz CL, Finlay BB, Celli J

[European Worm Meeting, 2000]

Tan and Ausubel, working with the enterobacterium Pseudomonasaeruginosa, have established C. elegans as a model for the study of pathogenesis and host defences. Clearly, the use of the worm as a model for studying pathogenicity will be limited to those pathogens that are able to infect the worm. Luckily, in this respect, its susceptibility to P. aeruginosaappears not to be an isolated case. A second opportunistic human pathogen, Serratia marcescens, is also capable of infecting C. elegans. Like P. aeruginosa, S. marcescens is able to infect a broad range of plant and animal hosts and has been used as a model pathogen in studies of Drosophila innate immunity. Using a strain of S. marcescensthat expresses GFP, we have been able to follow the infection process. The bacteria are able to survive within the usually hostile environment of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens. We are currently screening these mutant bacterial clones individually for those showing reduced virulence.Of the first 2000 mutants screened, 18 showing markedly reduced virulence have been retained for further study. The molecular characterization of these mutants may reveal novel virulence factors that represent potential drug targets. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. S: 29-34.

C Leopold Kurz et al. (2001) International C. elegans Meeting "Identifying universal Serratia marcescens virulence factors using C. elegans."

Jonathan Ewbank, Jean Celli, Dominique Ferrandon, B Brett Finlay, Emmanuel Andres, Sophie Chauvet, C Leopold Kurz, Mitchell Uh

[International C. elegans Meeting, 2001]

Tan and Ausubel, working with the enterobacterium Pseudomonas aeruginosa , have established C. elegans a s a model for the study of pathogenesis and host defences . A second opportunistic human pathogen, Serratia marcescens , is also capable of infect ing C. elegan s . Like P. aeruginosa , S. marcescens is able to infect a broad range of plant and animal hosts. Using a strain of S. marcescens that expresses GFP, we have been able to follow the infection process. T he bacteria are able to survive within the usually hostile environmen t of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens . We are currently screening these mutant bacterial clones individually for those showing reduced virulence . Of the first 2000 mutants screened, 9 showing markedly reduced virulence have been retained for further study. T he molecular characterization of these mutants has revealed novel virulence factors. In order to determine whether these virulence factors are specific to the infection of the nematode, we have also tested them in a Drosophila infection model and found that 4 of them are attenuated for their virulence. Tests in a mammalian model will reveal whether we have identified virulence factors that are important irrespective of the host. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans : a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 3: 29-34.

Eva L Mora-Blanco et al. (2001) International C. elegans Meeting "SITE-DIRECTED MUTAGENESIS OF LIN-31, A WINGED-HELIX TRANSCRIPTION FACTOR INVOLVED IN CAENORHABDITIS ELEGANS VULVAL DEVELOPMENT"

Leilani M Miller, Eva L Mora-Blanco, David B Doroquez, Yvonne Yang, Jill Haggerty

[International C. elegans Meeting, 2001]

In order to understand cell fate specification during vulval development, we are conducting a structure/function analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans (Miller et al. , Genes and Dev., 7:933, 1993). The LIN-31 protein contains a DNA-binding domain, an acidic region, four MAP kinase consensus phosphorylation sites, and a small region of homology conserved among other winged-helix proteins. We are using site-directed mutagenesis to create plasmids carrying specific mutations in the MAP kinase consensus phosphorylation sites, acidic domain, or the homology region of the LIN-31 protein. These plasmids are then injected into an animal with no functional LIN-31 protein to test for their ability to provide LIN-31 function. LIN-31 is phosphorylated by the MAP kinase MPK-1 in response to an inductive signaling event (Tan et al. , Cell 93: 569, 1998). Tan et al . have already shown that removing all four MAP kinase consensus phosphorylation sites inactivates one of LIN-31's functions, but the individual contribution of each site is not known. Four individual clones are being created, with each one containing a different disrupted MAP kinase consensus phosphorylation site. In addition, it is appealing to imagine that the small acidic domain (six consecutive aspartic acid residues) adjacent to the DNA-binding domain is functioning as a transcriptional activator, as is the case with acidic regions in some other transcription factors. There is no proof, however, that this rather small acidic domain in LIN-31 is even required for function. Five mutant clones, in which different portions of the acidic domain have been removed or replaced will address this question. Finally, the carboxy-terminus of LIN-31 contains a small region of homology that shows similarity to a subset of winged-helix proteins. The function of this homology region is also unknown and is being explored in this project. This structure/function study is especially appealing since the current model (Miller et al. , 1993; Tan et al. , 1998; and Miller et al. , Genetics 156: 1595, 2000) proposes that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p, and P7.p and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. Microinjection of these clones into lin-31(null) animals will allow us to test if each specific mutation disrupts one, both, or none of LIN-31's functions. Thus, this approach will allow us to identify the roles of specific domains or sites in the LIN-31 protein.