Masumi Shimada et al. (2003) International Worm Meeting "Possible roles of MOE-1/OMA-1 CCCH-type zinc-finger family proteins in C. elegans oogenesis and embryogenesis"

Hiroyuki Kawahara, Masumi Shimada

[International Worm Meeting, 2003]

Oocyte maturation is an important prerequisite for the production of progeny. Although several germ-line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation had not been fully investigated. Using a multiple RNAi technique, we have identified three redundant CCCH-type zinc-finger genes, named by us moe-1, -2 and moe-3, as a group related by functions and nucleotide sequence1. Similar results were reported by Detwiler et al.(2001), and they gave the names MOE-1 and MOE-2 to OMA-1 and OMA-2, respectively2. We and Detwiler et al. have found that they have overlapping functions that are crucial for oocyte maturation; i.e. simultaneous removal of these proteins by RNAi induces arrest and expansion of growing oocytes. The results of our in situ hybridization have revealed that each of the moe/oma transcripts is expressed from the distal to proximal region of the gonad, while their corresponding proteins accumulate specifically in the cytoplasm of growing oocytes as well as on P granules. Thus, these gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH-type zinc-finger family proteins thus far discovered. Although MOE-2/OMA-2 protein is rapidly removed from P granules after fertilization, we found that MOE-2/OMA-2 associated with the centrosome-peripheral structure in dividing blastomeres. Our results suggest that moe/oma gene products are unique multifunctional proteins in terms of their redundancy and characteristic behavior during the course of oocyte maturation and subsequent embryogenesis. 1 Shimada, M., Kawahara, H., and Doi, H. (2002). Genes Cells 7, 933-947. 2 Detwiler, M.R., Reuben, M., Li, X., Rogers, E., and Lin, R. (2001). Dev. Cell 1, 187-199.

Bojanala, Nagagireesh et al. (2013) International Worm Meeting "Dissecting the mechanistic insights through which EGL-26 controls C. elegans vulva tubulogenesis."

Seraj, Nishat, Crook, Matt, Sun, Hongliu, Chang, Sarah, Goncalves, Jimmy, Upadhyay, Avni, Fine, Ryan, Bojanala, Nagagireesh, Hanna-Rose, Wendy, Detwiler, Ariana, Serra, Nick

[International Worm Meeting, 2013]

Generation of tissue architecture and biological lumen formation during development occurs through various morphogenetic processes, and defective morphogenesis results in tissue malfunctions and disease. C. elegans vulva tubulogenesis involves formation of distinct dorsal and ventral lumens and is a good model for analyzing morphogenesis in vivo. Previously we established the role of EGL-26 (Lecithin Retinol Acyl Transferase family member) in vulva dorsal lumen formation. egl-26(lf) results in defective morphogenesis of the dorsal most toroid VulF. But expression analyses suggested a non-cell autonomous function for EGL-26 within the neighboring toroid VulE. To shed more insight into the mechanistic processes through which EGL-26 regulates dorsal lumen formation, we are taking a candidate RNAi screen approach to identify additional genes/egl-26 targets regulating dorsal lumen. So far, our screen identified interesting candidates involved in spindle formation, cytoskeleton, transcriptional, translational, and membrane transport processes. These observations suggest various gene regulatory modules working in conjunction with EGL-26 during morphogenesis. To further analyze cytoskeletal changes during vulva tubulogenesis, we are carrying out gamma tubulin staining in wild type and egl-26 (lf) animals. Our preliminary observations in wild type animals identified strong microtubule presence around the dorsal toroidal membrane, and hence providing a cytoskeletal signature during vulva morphogenesis. Any possible changes occurring within this signature in egl-26 (lf) situation will identify its potential direct and indirect targets regulating cytoskeleton during lumen formation. In addition our analyses of cis-regulatory modules that govern egl-26 expression within VulE and F lineages identified a 200bp region within egl-26 promoter that is sufficient and necessary for VulE-specific expression and contains a binding site that negatively regulates VulF expression. Thus, the findings from this study will provide more details about proper maintenance of biological lumens during development.

Wang, Wenqing et al. (2015) International Worm Meeting "Metabolic profiling and metabolic flux analyses in NAD+ salvage biosynthesis mutants."

Hanna-Rose, Wendy, Pacella, Marisa, McReynolds, Melanie, Braeckman, Bart, Detwiler, Ariana, Wang, Wenqing, Dhondt, Ineke, Lange, Stephanie, Goncalves, Jimmy, Shu, Muya, Kho, Kelvin

[International Worm Meeting, 2015]

Temporal developmental progression is highly coordinated in C. elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical temporal progression relative to the soma. Nicotinamidases mediate salvage NAD+ synthesis. There has been significant recent interest in boosting NAD+ biosynthesis for therapeutic benefit in aging-related diseases and inhibiting biosynthesis for therapeutic benefit in cancer. However, we haven't fully elucidated how perturbing the availability of NAD+ impacts whole organism physiology. Using LC-MS we show that pnc-1 mutants do not salvage nicotinamide released by NAD+ consumers to resynthesize NAD+, and as a result NAD+ availability is reduced. By manipulating NAD+ levels using genetics, we show that a reduction in NAD+ bioavailability is incompatible with a normal pace of gonad development. We use the pnc-1 mutant phenotype to explore physiological requirements for reproductive development. First we demonstrate that the deficit in NAD+ production compromises NAD+ consumer activity as would be expected. Even though effects on NAD+ consumer activity have been the primary focus for probing the functional consequences of manipulation of NAD+ bioavailability, we revealed no functional link between loss of NAD+ consumer activity and reproductive development in genetic experiments. We used metabolomics profiling to seek explanations for the observed phenotype. The deficit in NAD+ availability from lack of salvage biosynthesis has wide-spread metabolic consequences, including perturbations in glycolysis. Using flux analysis, we demonstrate that glycolysis is blocked at the NAD+-dependent step, and we functionally link the block to the reproductive phenotype using genetics and pharmacological approaches. Interestingly, mitochondria are protected from the deficiency in NAD+ biosynthesis and the effects of reduced glycolytic output, also revealing compartment specific regulation of NAD+ biosynthesis. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the cell division requirements for reproductive development. Our work has implications for understanding mechanisms that mediate therapies that target NAD+ salvage synthesis for the purposes of inhibiting tumor growth.

Priah Nadarajan et al. (2007) International Worm Meeting "Oocyte Growth and Meiotic Maturation are Coordinately Controlled."

Priah Nadarajan, David Greenstein

[International Worm Meeting, 2007]

During oogenesis, meiotic progression is accompanied by oocyte growth, driven by the actomyosin-dependent transport of cytoplasmic organelles and materials from the rachis (Wolke et al., 2005 Worm Meeting Abstracts) and receptor-mediated endocytosis of yolk protein particles (Grant and Hirsh, 1999). To understand the molecular mechanisms regulating oocyte growth, we conducted a genetic screen to isolate mutants with abnormally large oocytes. We screened a collection of 1200 temperature-sensitive lethal mutants and isolated thirteen large oocyte (looc) mutations that define at least eight genes. Six looc loci exhibit low rates of oocyte meiotic maturation in hermaphrodites, suggesting that these genes define positive regulators of MSP signaling. A similar observation was made for ptp-2 mutants (Miller et al., 2004), which also exhibit a Looc phenotype (Gutch et al., 1998). We have begun analyzing the phenotype of looc mutants in a female background to assess the coordination between oocyte growth and MSP signaling. Thus far, three mutants exhibit elevated meiotic maturation rates in a fog-2 female background, as compared to the fog-2 control, suggesting that these loci also act as negative regulators of meiotic maturation in the absence of sperm. Interestingly, for mutant alleles of two of five genes analyzed, a Looc phenotype was observed only in the presence of sperm. We made a similar observation for a ptp-2 null allele, and Detwiler et al. (2001) reported that oma-1;oma-2 mutant oocytes grow abnormally large only in the presence of sperm. These data suggest that there are at least two pathways regulating oocyte growth: a sperm-dependent pathway and a sperm-independent pathway. One of the sperm-dependent looc mutations is a novel allele of emb-30, which encodes APC4, a component of the anaphase-promoting complex or cyclosome (APC/C). This result suggests that an APC/C target contributes to the sperm-dependent oocyte growth pathway. Progress in the genetic, molecular, and cell biological analysis of the looc genes will be presented in this poster. Detwiler et al. Dev Cell. 1:187, 2001. Grant and Hirsh. Mol. Biol Cell. 10:4311, 1999. Gutch et al. Genes Dev 12:571, 1998. Miller et al. Curr Biol 14:1284, 2004.

Omichi, Yukari et al. (2011) International Worm Meeting "An altered method of feeding RNAi that knocks down multiple genes simultaneously in the nematode Caenorhabditis elegans."

Gouda, Kenji, Matsunaga, Yohei, Omichi, Yukari, Iwasaki, Takashi, Kawano, Tsuyoshi

[International Worm Meeting, 2011]

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene's function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. When knockdown of two genes (double knockdown), animals are fed a mixture of two sorts of RNAi bacteria at the same time (double feeding). However, in some cases, only one gene's function is significantly inhibited, or both genes are only slightly knocked down. This is due to the lesser reliability of double feeding than single feeding. Given this background, we have established a method for more convenient, efficient, and reliable knockdown of more than two genes using feeding RNAi. We expected that bacteria producing two distinct dsRNAs bound to each other could induce simultaneous knockdown. For a model experiment, we chose oma-1 and oma-2 genes, which are functionally redundant to each other and responsible for embryonic development [1]. Only when both genes loss their function simultaneously, embryonic lethal occurs. We utilized this system for establishing double knockdown. In addition, for establishing triple knockdown, we attempted to suppress expression of GFP as well as OMA-1/OMA-2 in a transgenic animal expressing GFP. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one [2]. Our method is useful for investigating genes' functions in C. elegans. References: [1] Detwiler et al. (2001) Dev. Cell 1:187-199. [2] Gouda et al. (2010) Biosci. Biothech. Bochem. 74:2361-2365.

Spike, Caroline et al. (2011) International Worm Meeting "Translational regulation and the control of meiotic maturation."

Coetzee, Donna, Spike, Caroline, Greenstein, David

[International Worm Meeting, 2011]

C. elegans oocytes undergo meiotic maturation and ovulation in response to major sperm proteins (MSPs), hormonal signals released from sperm. MSPs also function as cytoskeletal elements in the actin-independent motility of nematode sperm. The follicle-like gonadal sheath cells, which form gap junctions with oocytes and contract to drive ovulation, function as the initial MSP sensors. G protein-stimulated cAMP-dependent protein kinase A activity in the gonadal sheath cells is required for all germline responses to MSPs. In the germ line, the meiotic maturation response requires the combined activities of two Tis11-like CCCH zinc-finger proteins, OMA-1 and OMA-2 [Detwiler et al. (2001) Dev. Cell 1: 187]. To elucidate the mechanisms by which the OMA proteins promote meiotic maturation, we developed methods for purifying OMA ribonucleoprotein particles (OMA RNPs) from oocytes. OMA RNPs were purified in both the presence and absence of MSP signaling and proteins were identified using mass spectrometry. OMA RNPs contain a large number of germline-expressed RNA-binding proteins including translational activators and repressors. To distinguish core OMA RNP components from those tethered by RNA, we purified OMA complexes after treatment with RNase A. Core OMA RNP components include multiple subunits of the GLD-2 poly(A) polymerase and CCR4/NOT1 deadenylase complexes and many RNA-binding proteins, including MEX-3, MEX-1, and LIN-41. OMA RNP components that appear to be tethered primarily via RNA associations include the P-body proteins CGH-1/p54 and CAR-1/Rap55. OMA RNPs are cytoplasmically dispersed in oocytes when MSP is present but are found in foci when MSP is absent, as has been observed for a number of germline-expressed RNA-binding proteins [Schisa et al. (2001) Development 128: 1287; Jud et al. (2008) Dev. Biol 318: 38]. These data are consistent with the hypothesis that OMA-1/2 might regulate the translation of mRNAs encoding proteins involved in meiotic resumption or the oocyte-to-embryo transition. To test the role of OMA RNP components in translational regulation, we used microarrays to define mRNA components of OMA RNPs. This analysis identified a set of approximately 400 germline-expressed mRNAs that are enriched in OMA RNPs. We are testing the 3'UTRs of candidate OMA target genes and have found several that appear to mediate OMA-1/2-dependent translational repression in oocytes. We will investigate whether OMA-1/2 targets are also regulated by OMA-associated proteins, potentially in response to the MSP signal.

Melanie Reuben et al. (2001) International C. elegans Meeting "A gain-of-function mutation in the oma-1 gene results in C to EMS fate transformation"

Melanie Reuben, Rueyling Lin

[International C. elegans Meeting, 2001]

OMA-1 and OMA-2 are two CCCH zinc-finger-containing proteins whose functions are redundantly required for oocyte maturation in C. elegans (see abstract by Detwiler et al). With antibodies specific to either OMA-1 or OMA-2, we have shown that both proteins are expressed at a very high level in the cytoplasm of maturing oocytes and in one-cell embryos. The embryonic expression drops to below detectable levels by immunofluorescence in 2-cell embryos. However, with an OMA-1-GFP fusion construct, we could detect GFP up to approximately the 4-cell stage. OMA-1-GFP is preferentially segregated to the germline precursors (P1 and P2) and is P-granule-associated, in addition to being cytoplasmic. We believe that detection with OMA-1-GFP is more sensitive than our antibody staining. Because oma-1;oma-2 mutant worms are sterile, we are unable to examine the function, if any, of OMA-1 and OMA-2 in early embryos. A gain-of-function mutation in oma-1, zu405 , results in a temperature-sensitive, maternal-effect, embryonic lethality. We can show with antibody staining that OMA-1 protein is detected in zu405 embryos from the 1-cell to approximately the 8-cell stage. The staining is specific to the germline precursors and is cytoplasmic as well as P-granule-associated. The level of OMA-2 protein is not changed in zu405 animals. Embryos from zu405 homozygous mothers produce extra intestinal cells as well as extra pharyngeal and body-wall muscles, tissue types normally derived from wild-type EMS. Laser ablation analysis indicates that the C blastomere adopts the fate of EMS in zu405. Instead of producing skin and muscles, it produces muscles and intestinal cells. We have also shown that C in zu405 expresses an EMS-specific marker: MED-1-GFP. When wild type EMS divides, intestine is derived from the posterior daughter E and this specification requires a Wnt signal from P2. When C divides in zu405 embryos, intestine is always derived from the posterior daughter Cp. We could show that the intestine formation from C is dependent on wrm-1 activity, suggesting that a Wnt-like signaling pathway is required for C to produce gut in zu405 . In wild-type embryos, the C blastomere fate is dependent on the transcription factor PAL-1 whereas the EMS blastomere fate depends on SKN-1, despite the expression of both SKN-1 and PAL-1 in both EMS and C. Normally, SKN-1 level is low in 8-cell and not detectable in 12-cell embryos but PAL-1 protein lasts past 28 cells. It has been suggested that C expresses PAL-1-dependent cell fates because PAL-1 outlasts SKN-1 in C. If this model were correct, over-expression or endurance of SKN-1 in the C blastomere would allow it to develop like a wild-type EMS blastomere. Consistent with this notion, we detect a high level of SKN-1 protein up to the 8-cell and a low level in the 12-cell stage zu405 embryos. SKN-1 protein appears to last one cell division longer in zu405 than in wild type embryos. In 30% of zu405 embryos, the P2-to-EMS signaling is abnormal and both daughters of EMS produce mesoderm. The C to EMS fate transformation and the defect in P2 to EMS signaling are similar to what was described with the gsk-3(RNAi) embryos. The expression of PIE-1 and APX-1 appears unaffected in zu405 embryos. We are currently investigating the expression patterns of other maternal factors in zu405 mutants.

Ikuko Yamamoto et al. (2004) East Coast Worm Meeting "The conserved DEAD-box helicase CGH-1 negatively regulates MAP kinase activation in C. elegans oocytes"

Ikuko Yamamoto, David I Greenstein

[East Coast Worm Meeting, 2004]

Oocyte meiotic cell cycle progression must be precisely coordinated with ovulation and fertilization in order to form a diploid zygote. In C. elegans , fully-grown oocytes arrest at diakinesis of meiotic prophase I, and this arrest is released by an extracellular signal provided by the sperm, the major sperm protein (MSP). MSP signaling activates the conserved MAP kinase (MAPK) pathway in oocytes and promotes diverse meiotic responses, including M-phase entry, cortical cytoskeletal rearrangement, and gonadal sheath cell contraction. To clarify the mechanisms of MSP signal transduction and to identify downstream components in meiotic maturation regulatory pathways, we took a genetic approach. We isolated a dominant mutant std-1(tn691d,ts) (stuck in diakinesis) that affects MSP signaling responses, interferes with normal oocyte meiotic maturation processes, and disrupts meiotic chromosome segregation. Positional cloning of the gene revealed that std-1 corresponds to cgh-1 (1), which encodes a member of a highly conserved small subfamily of DEAD-box RNA helicases associated with germline development and meiotic progression. According to phenotypic and molecular analyses, cgh-1(tn691d,ts) possesses dominant-negative character. Analysis of a protein null mutant, cgh-1(ok492) , indicates cgh-1 is a negative regulator of MAPK activation in oocytes. MSP signaling activates MAPK in the most proximal one to three oocytes in the wild type. In contrast, in the cgh-1 mutants, MAPK activation is observed in not only proximal but also distal oocytes. In females, in which the MSP signal is absent, MAPK activation is not observed; however, in feminized cgh-1 mutants [ cgh-1(tn691d,ts);fog-2(q71) and cgh-1(ok492);fog-3(q443) ] signal independent MAPK activation is detected. Thus, cgh-1 is required for: (i) the establishment of the response threshold in the presence of the MSP signal; and (ii) the inhibition of MAPK activation in the absence of sperm. OMA-1 and OMA-2 are two zinc finger proteins redundantly required for meiotic maturation (2). Without oma-1/oma-2 function, MAPK activation in proximal oocytes is not observed. In either cgh-1(RNAi);oma-1(te33);oma-2(te51) or oma-1(RNAi);oma-2(RNAi);cgh-1(ok492) , MAPK activation is also not observed. Therefore, cgh-1 functions upstream or in parallel to oma-1 and oma-2 for MAPK activation. In addition to germline phenotypes, cgh-1 mutants exhibit elevated gonadal sheath cell contractions in both the presence and absence of sperm. Elevated sheath cell contractions are also observed in cgh-1(RNAi);rrf-1(pk1417) hermaphrodites and females, indicating that cgh-1 functions in the germ line to modulate sheath cell response to MSP. CGH-1 protein is specifically expressed in the germ line and localizes to the cytoplasm in proximal oocytes. In the wild type, CGH-1 is localized to a subcortical band that encircles the oocyte. In contrast, in the absence of sperm, CGH-1 localizes subcortically especially to large distinctive cytoplasmic foci. Similarly, in the dominant-negative mutant, cgh-1(tn691d,ts) , CGH-1 localizes to large subcortical cytoplasmic foci with and without sperm. Thus, CGH-1 could be in different kind of complexes or associated with different factors in the presence and absence of the MSP signal, and the dominant-negative mutation may alter such associations. (1) Navarro et al. Development 128: 3221, 2001. (2) Detwiler et al. Dev. Cell 1: 87, 2001.

Ikuko Yamamoto et al. (2004) Mid-west Worm Meeting "The conserved DEAD-box helicase CGH-1 negatively regulates MAP kinase activation in C. elegans oocytes"

Ikuko Yamamoto, David I Greenstein

[Mid-west Worm Meeting, 2004]

Oocyte meiotic cell cycle progression must be precisely coordinated with ovulation and fertilization in order to form a diploid zygote. In C. elegans , fully-grown oocytes arrest at diakinesis of meiotic prophase I, and this arrest is released by an extracellular signal provided by the sperm, the major sperm protein (MSP). MSP signaling activates the conserved MAP kinase (MAPK) pathway in oocytes and promotes diverse meiotic responses, including M-phase entry, cortical cytoskeletal rearrangement, and gonadal sheath cell contraction. To clarify the mechanisms of MSP signal transduction and to identify downstream components in meiotic maturation regulatory pathways, we took a genetic approach. We isolated a dominant mutant std-1(tn691d,ts) (stuck in diakinesis) that affects MSP signaling responses, interferes with normal oocyte meiotic maturation processes, and disrupts meiotic chromosome segregation. Positional cloning of the gene revealed that std-1 corresponds to cgh-1 (1), which encodes a member of a highly conserved small subfamily of DEAD-box RNA helicases associated with germline development and meiotic progression. According to phenotypic and molecular analyses, cgh-1(tn691d,ts) possesses dominant-negative character. Analysis of a protein null mutant, cgh-1(ok492) , indicates cgh-1 is a negative regulator of MAPK activation in oocytes. MSP signaling activates MAPK in the most proximal one to three oocytes in the wild type. In contrast, in the cgh-1 mutants, MAPK activation is observed in not only proximal but also distal oocytes. In females, in which the MSP signal is absent, MAPK activation is not observed; however, in feminized cgh-1 mutants [ cgh-1(tn691d,ts);fog-2(q71) and cgh-1(ok492);fog-3(q443) ] signal independent MAPK activation is detected. Thus, cgh-1 is required for: (i) the establishment of the response threshold in the presence of the MSP signal; and (ii) the inhibition of MAPK activation in the absence of sperm. OMA-1 and OMA-2 are two zinc finger proteins redundantly required for meiotic maturation (2). Without oma-1/oma-2 function, MAPK activation in proximal oocytes is not observed. In either cgh-1(RNAi);oma-1(te33);oma-2(te51) or oma-1(RNAi);oma-2(RNAi);cgh-1(ok492) , MAPK activation is also not observed. Therefore, cgh-1 functions upstream or in parallel to oma-1 and oma-2 for MAPK activation. In addition to germline phenotypes, cgh-1 mutants exhibit elevated gonadal sheath cell contractions in both the presence and absence of sperm. Elevated sheath cell contractions are also observed in cgh-1(RNAi);rrf-1(pk1417) hermaphrodites and females, indicating that cgh-1 functions in the germ line to modulate sheath cell response to MSP. CGH-1 protein is specifically expressed in the germ line and localizes to the cytoplasm in proximal oocytes. In the wild type, CGH-1 is localized to a subcortical band that encircles the oocyte. In contrast, in the absence of sperm CGH-1 localizes subcortically, especially to large distinctive cytoplasmic foci. Similarly, in the dominant-negative mutant, cgh-1(tn691d,ts) , CGH-1 localizes to large subcortical cytoplasmic foci with and without sperm. Thus, CGH-1 could be in different kind of complexes or associated with different factors in the presence and absence of the MSP signal, and the dominant-negative mutation may alter such associations. (1) Navarro et al. Development 128: 3221, 2001. (2) Detwiler et al. Dev. Cell 1: 87, 2001.