Kounosu, Asuka et al. (2021) International Worm Meeting "Chromosome dynamics in sex determination of the parthenogenetic nematode Strongyloides ratti"

Sugimoto, Asako, Maruyama, Haruhiko, Murase, Kazunori, Yoshida, Akemi, Hunt, Vicky, Sun, Simo, Kikuchi, Taisei, Kounosu, Asuka

[International Worm Meeting, 2021]

The genus Strongyloides is unique among nematodes in having alternate free-living and parasitic generations. The parasitic female (PF) inhabits the small intestine of its mammal host and produces clonal offspring by mitotic parthenogenesis. Eggs produced by PF are excreted with the feaces and develop into either infective larvae (iL3) directly or free-living males and females. The free-living adults mate and produce eggs which develop into iL3. It was reported that Strongyloides ratti, a rat parasite, has three chromosomes and one of them is a sex chromosome, which determines their sex by XX/XO system. However, it remains unclear how mitotic parthenogenetic PF produces XX and XO eggs though it is presumed that the male karyotype (XO) is generated by missegregation of the sex chromosome like the C. elegans male generation. In this study we investigated details of the chromosome structure of each developmental stages of S. ratti using the single-worm sequencing technique and the multiplex colored FISH observation. In the single-worm analysis, we expected a half depth of mapping coverage in sex chromosome compared to autosomes in free-living males (XO). However, the sex-chromosome of the male exhibited ~1/3 depth of the autosomes. Furthermore PF and free-living female (XX) showed lower coverage depth of the sex-chromosome and the auto:sex ration varied from 1:0.8 to 1:0.5. Additionally, in germline depleted PF, sex-chromosome showed almost equal coverages as autosomes. These results indicate that chromosome manipulations occurs in the PF germline to produce free-living males and the S. ratti sex determination system is not a simple XX/XO. Multiplex colored FISH analyses, recently developed for C. elegans in-detail chromosome observation, provided visual clues to understanding the dynamics of S. ratti chromosomal structure and the puzzling sex-determination system of Strongyloides.

RJ Eustance et al. (1999) International C. elegans Meeting "UNC-13 and Synaptic Neurotransmission"

J Duerr, RJ Eustance, I Maruyama, J Rand, A Duke, H Maruyama, J McManus

[International C. elegans Meeting, 1999]

unc-13 is essential for normal neuronal function and is involved in neurotransmitter release; C. elegans mutants are uncoordinated and resistant to the anti-cholinesterase aldicarb. UNC-13 has several regions homologous to PKC regulatory domains conferring it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88 , 1991). These binding properties indicate that UNC-13 may regulate neurotransmitter release in response to calcium and as part of a signal transduction pathway. A 5.9kb transcript coding for a 200kDa protein was initially identified (Maruyama and Brenner, PNAS 88, 1991). We identified two additional types of transcripts using cDNA library screens and RACE. One transcript includes a 1kb alternatively spliced exon and another transcript lacks the 5' region included in the other two transcripts. All three transcripts are identical at the 3' end. 21 of the 40 unc-13 alleles in our collection are sequenced. Mutations in the 5' end alter two types of transcripts resulting in an uncoordinated coily phenotype. A 2.8kb deletion at the 3' end removes all unc-13 transcripts, resulting in a lethal phenotype. This 2.8kb deletion was identified by Bob Barstead using PCR analysis. Antibodies recognizing different regions of UNC-13 indicate that protein products from the three transcripts have different expression and localization patterns. Antibodies recognizing the N-terminal region label synapses, but not synaptic vesicles, of most or all neurons. Many unc-13 alleles have decreased staining with this antibody. Antibodies against the region coded for by the alternatively spliced exon stain some sensory neurons and some neuronal processes. Antibodies recognizing the C-terminal region, common to all predicted UNC-13 proteins, label synapses and neuronal processes of most or all neurons. This staining pattern decreases in only 3 alleles, all of which are lethal or partially lethal. The existence of several unc-13 transcripts, the alternate antibody staining patterns, and the phenotypes resulting from elimination of some or all transcripts suggest that unc-13 protein products have different expression and localization patterns and possibly different functions.

Chih-Chien Cheng et al. (2007) International Worm Meeting "The cortical anchor EGG-3 is a likely target of the Anaphase-Promoting Complex (APC) during the oocyte-to-zygote transition."

CHIH-CHIEN CHENG, Geraldine Seydoux

[International Worm Meeting, 2007]

The DYRK kinase MBK-2 phosphorylates oocyte proteins shortly after fertilization to target them for degradation in the embryo (Nishi and Lin, 2005 and Stitzel et al., 2006). Phosphorylation of MBK-2 substrates correlates with relocalization of MBK-2 from the egg cortex to the cytoplasm. We identified EGG-3 in a screen for proteins required to localize MBK-2 to the cortex. EGG-3 is a cortical protein required for egg activation (Maruyama and Singson, personal communication). Using GST-pull down and immunoprecipitation assays, we have demonstrated that MBK-2 and EGG-3 interact in vitro and in vivo. EGG-3 is degraded during meiotic exit and this degradation correlates with MBK-2 release in the cytoplasm. EGG-3 contains several D-boxes typical of proteins targeted for degradation by the Anaphase-Promoting Complex (APC). EGG-3 degradation is blocked when the first two D-boxes are mutated or when APC or the proteasome are depleted by RNAi. These results suggest that EGG-3 is an APC target, and that the oocyte-to-zygote transition is directly regulated by cell cycle proteins.

Eustance RJ et al. (1998) West Coast Worm Meeting "UNC-13 AND SYNAPTIC NEUROTRANSMISSION"

Duke A, Rand JB, McManus JR, Eustance RJ, Maruyama H, Maruyama IN, Duerr JS

[West Coast Worm Meeting, 1998]

We are investigating the roles of UNC-13 in synaptic transmission and signal transduction. Unc-13 is essential for normal neuronal function and is thought to be involved in neurotransmitter release; C. elegans mutants are uncoordinated and resistant to the anti-cholinesterase aldicarb. UNC- 13 has several regions homologous to PKC regulatory domains, which confer UNC-13 with phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88:5729, 1991). We have identified at least two types of unc-13 transcripts. They appear to be transcribed from different promoters and encode proteins with different N-terminal domains. On Northern transfers, these transcripts appear to be 5.9 kb and 4.9 kb in length. To date, ten of the >40 unc-13 alleles in our collection have been sequenced. Two of the mutations (Maruyama and Brenner, PNAS 88:5729, 1991) are located in the 5' end of the gene (both mutants are paralyzed and one has a slightly coily phenotype). Eight newly sequenced mutations in the 3' end of the gene represent a different phenotypic class. These mutants are paralyzed and most are more coily and slower growing than alleles with mutations in the 5' region. A lethal phenotype results from a 2.7 kb deletion at the 3' end of unc-13; L1 larvae hatch and remain tightly coiled, but do not grow, and eventually die. Antibodies recognizing the N terminal region of unc-13 label synapses (but not synaptic vesicles) of most or all neurons. Many unc-13 alleles have decreased staining with this antibody. Antibodies recognizing the C terminal region (common to all predicted UNC-13 proteins) label synapses and neuronal processes of most or all neurons. This staining pattern decreases in only three alleles, all of which are lethal or partially lethal. The existence of more than one unc-13 transcript, the different phenotypic classes resulting from mutations in either the 5' or 3' end of unc-13 and the alternate staining patterns with anti-UNC-13 antibodies all suggest that protein products from unc-13 have different expression or localization patterns and possibly different functions.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

CS Walker et al. (1999) International C. elegans Meeting "glr-5 and glr-7: Two Glutamate Receptor Subunits That May Have a Role in Thermotaxis."

CS Walker, AV Maricq, PJ Brockie, DM Madsen

[International C. elegans Meeting, 1999]

At least ten ionotropic glutamate receptors have been identified in C. elegans. Of these, two non-NMDA receptor subtypes show a limited expression in the nervous system. In transgenic strains that express chimeric GLR-5::GFP or GLR-7::GFP fusion proteins, fluorescence is observed in only the RIA neuron. This neuron functions downstream of AIY and AIZ and is required for normal thermotactic responses. We would like to determine the subcellular distribution of GLR-5 and GLR-7 and gain some understanding of how they may contribute to the function of RIA. An especially interesting opportunity for the study of localization of glutamate receptors is presented by our finding that GLR-5 and GLR-7 are co-expressed in only one neuron. This permits the non-ambiguous assignment of synaptic localization. We can now directly test whether GLR-5 and GLR-7 co-localize and determine whether they co-localize with presynaptic inputs from specific sensory neurons, e.g., ASH, AIY and AIZ using YFP, CFP and GFP constructs. We will present data from our localization studies. Also, we can directly test in Xenopus oocytes whether GLR-5 and GLR-7 form homomeric or heterometic receptors. We are in the process of screening the Barstead and Maruyama cDNA libraries in order to clone full-length cDNAs encoding these two receptors. Finally, we would like to understand the behavioral consequences of deleting these two genes. We are now attempting to generate deletion mutations in both glr-5 and glr-7 using a Tc1 based approach.

Eustance B et al. (1996) Midwest Worm Meeting "UNC-13 EXPRESSION IN WILD-TYPE AND MUTANT C. ELEGANS."

Eustance B, Duke A, Duerr JS, Rand JB

[Midwest Worm Meeting, 1996]

We are interested in understanding the processes underlying synaptic neurotransmission. The unc-13 gene is important for normal neuronal function and may be part of a mechanism regulating neurotransmitter release; mutations in unc-13 result in aldicarb resistance. UNC-13 contains a region homologous to the regulatory region of PKC (Maruyama and Brenner, 1991) with phorbol ester and phospholipid binding properties, but its function in neurons is currently unknown. We have generated antibodies against UNC-13 using fusion proteins representing amino acids 25-578 (A) or 903-1286 (B) of the 1734 amino acid UNC-13 protein. These two fragments exclude the PKC homology region. Anti-UNC-13 sera appear to stain most or all neurons. However, the staining pattern differed for sera generated against the two regions. Antibodies generated against fusion protein A (anti-A) label synaptic regions; antibodies against fusion protein B (anti-B) label neuronal processes uniformly. Mutations in unc-104, which cause mislocalization of synaptic vesicles, do not profoundly alter either staining pattern. We have examined twenty unc-13 alleles; most show a severe decrease in staining with anti-A, but none show any change with anti-B. Our current hypothesis is that anti-A recognizes bona fide UNC-13 protein and that UNC-13 is enhanced in synaptic regions (but is not localized to synaptic vesicles). We also hypothesize that anti-B recognizes UNC-13 and at least one other (possibly related) protein that is distributed uniformly throughout neurons. We are interested in identifying this mystery protein. After further characterization of the unc-13 mutants, we plan to conduct suppressor screens and two-hybrid analyses to look for genes that interact with the unc-13 gene or its product.

Eustance RJ et al. (1997) International C. elegans Meeting "Analysis of UNC-13 expression in C. elegans"

Duerr JS, Rand JB, Eustance RJ, Maruyama H, Maruyama IN, Duke A

[International C. elegans Meeting, 1997]

unc-13 mutants are extremely uncoordinated and are resistant to aldicarb, an anti-cholinesterase. Regions of UNC- 13 homologous to regulatory domains found in PKC confer it with phorbol ester and phospholipid binding properties (Maruyama & Brenner, P.N.A.S. 88: 5729, 1991). We are investigating the roles of UNC-13 proteins in signal transduction and synaptic transmission.. An unc-13 transcript coding for a >200 kD protein was originally identified. Another recently identified transcript includes sequences found within a large intron in the original transcript. The 3' region of both transcripts is identical and we are screening a cDNA library to determine whether the two transcripts result from alternative splicing or from multiple promoters. To examine subcellular localization, we generated antibodies recognizing the predicted UNC-13 proteins. Antibodies against the N-terminal region, A, of the original UNC-13 label synapses but not synaptic vesicles of most or all neurons. Mutations in many unc-13 alleles lead to decreased staining with this antibody and most known unc-13 mutations may be in the A region. Staining with an antibody recognizing protein sequence from the second transcript, region C, labels a subset of neurons including some sensory neurons. Antibodies recognizing a C-terminal region common to both predicted proteins, B, label synapses and neuronal processes of most or all neurons and staining with this antibody decreases in only one of over 20 alleles. Anti-UNC-13 B staining cannot be explained by a combination of the other two staining patterns. It is possible that antibodies are labelling related proteins from other genes, that protein processing affects the staining pattern or that there are more unc-13 transcripts or splice forms. Supported by grants from the NIH and OCAST.

Michael Stitzel et al. (2007) International Worm Meeting "The meiotic cell cycle regulates the EGG-3/MBK-2 cortical complex essential for the oocyte-to-zygote transition."

Geraldine Seydoux, Michael Stitzel

[International Worm Meeting, 2007]

The oocyte-to-zygote transition transforms a differentiated germ cell into a totipotent zygote. The signals that activate this transition remain poorly understood. Here we report that regulation by the meiotic cell cycle of the MBK-2 kinase and its cortical anchor EGG-3 is one of the signals that initiates the oocyte-to-zygote transition in C. elegans. The DYRK kinase MBK-2 marks oocyte proteins for degradation shortly after fertilization during the second meiotic division (Nishi & Lin, 2005; Stitzel et al., 2006). GFP:MBK-2 accumulates on the cortex of oocytes and relocalizes to the cytoplasm during the second meiotic division. Cortical localization of MBK-2 depends on EGG-3, a cortical oocyte protein also essential for the oocyte-to-zygote transition (Maruyama and Singson, pers. comm). MBK-2 and EGG-3 interact directly in vitro, suggesting that EGG-3 may function as a transient cortical anchor for MBK-2 (see abstract by Cheng & Seydoux). Using cell cycle and egg-3 mutants, we found that MBK-2 is regulated both positively by regulators of meiotic M phase and negatively by EGG-3. Activators of meiotic M phase activate MBK-2 kinase activity upon oocyte maturation. EGG-3 then sequesters active MBK-2 at the cortex until the second meiotic division. This system ensures that oocyte proteins become marked for degradation only at the end of the meiotic divisions, when they are no longer needed and the egg is ready to embark on embryonic development. Remarkably, the meiotic cell cycle appears to be driving these changes independently of fertilization. We propose that, in addition to regulating chromosome segregation, the meiotic cell cycle controls egg-wide developmental changes essential for the initiation of embryonic development.

Karashima, T. et al. (2009) International Worm Meeting "Germ cell development and a Deleted in Azoospermia homolog."

Otori, M., Hasegwa, E., Karashima, T., Yamamoto, M.

[International Worm Meeting, 2009]

The DAZ (Deleted in Azoospermia) family of RNA-binding proteins, which are highly enriched in the germ line, bind to the 3'' UTR of specific mRNAs and up-regulate their translation. Deletions of the DAZ gene cluster on the human Y chromosome are associated with azoospermia, oligozoospermia, and testicular germ cell tumors. DAZ-1, the single DAZ homolog in the nematode C. elegans, is expressed in the distal part of germ line (Maruyama et al. 2005). Depletion of DAZ-1 blocks the transition from pachytene to diakinesis during female meiosis and abolishes oocyte production (Karashima et al. 2000). We previously reported that DAZ-1 protein can bind to the 3'' UTR of gld-1 mRNA and promotes the accumulation of GLD-1 protein, a translational repressor essential for the progression of female pachytene (Francis et al. 1995; Jones et al. 1996). Interestingly, oogenesis can be restored in the daz-1 mutant by attenuating the activity of the translational regulator GLD-3 (Otori et al. 2006). GLD-3 promotes spermatogenesis and/or inhibits oogenesis, is translationally repressed by FBF, and shows relatively high accumulation in the proximal germ line in the wild-type hermaphrodite (Eckmann et al. 2002 and 2004). Although DAZ-1 protein can bind to the 3'' UTR of fbf mRNA (Otori et al. 2006), FBF amount was not reduced in the daz-1 adult. Instead, we found that GLD-3 accumulation is repressed by GLD-1 in the wild-type female pachytene germ cells, and is derepressed in the daz-1 mutant. Biochemical and genetic analyses suggested that DAZ-1 promotes GLD-1 accumulation independently of FBF/PUF, which both negatively and positively regulates the translation of gld-1 mRNA by polyadenylation and deadenylation (Crittenden et al. 2002; Suh et al. 2009), and that DAZ-1 directly recruits PAB-1/poly(A)-binding protein to the gld-1 3'' UTR. On the other hand, CPB-3/cytoplasmic polyadenylation element-binding protein, an in vitro interactor of DAZ-1, is not likely involved in the poly(A) elongation and thus the translational activation of gld-1 mRNA.