Keiko Hirono et al. (2001) International C. elegans Meeting "Systematic RNAi analysis of maternal genes reveals the function of proteasome in oocyte maturation and fertilization."

Yuji KOHARA, Yumiko Ueta, Mina Iwata, Masahiro Ito, Kyoko Nakata, Keiko Hirono

[International C. elegans Meeting, 2001]

Our cDNA/expression pattern project has provided unique sets of limited numbers of genes that possibly participate in specific stages, lineages, tissues and so on. Such sets of genes are suitable to systematic analysis to elucidate the molecular mechanisms governing the biological processes. Since we are particularly interested in early embryogenesis, we have focused on a set of genes whose mRNA are maternally supplied and disappear or localize to specific cells during early stages (2-cell~early gastrulation). We found out 477 (about 10%) such genes when the expression analysis of about 5,000 genes was finished, and have done RNAi (RNA mediated interference) analysis on the 477 genes. We microinjected double-stranded RNA into the gonads of N2 worms. Phenotypic analyses were performed on the injected worms themselves, F1 embryos, larvae and adults that survived (called "escapers") and F2 embryos, with respect to embryogenesis, larval growth, sterility, morphogenesis, and so on. Out of the 477 genes, 5% showed the reduction of the number of F1, 33% showed F1 embryonic lethality, 27% showed one or various phenotypes with only escapers and 35% did not show any phenotype. Interestingly, genes in X chromosome showed much higher ratio of no-phenotype than those in autosomes (X chromosome: 42/71, autosomes: 125/404). We further analyzed early cell division using the 4D microscope for the genes that showed more than 50% embryonic lethality (61 genes) in the RNAi screening. Out of them, 12 genes showed the arrest at fertilized egg and no division, 29 genes showed aberrant process until 8-cell stage and 20 genes showed no phenotype until 8-cell stage. Interestingly, 5 out of the 12 genes of the severest phenotype (arrest at fertilized egg) related to proteasome; 2 genes are proteasome components and 3 genes are proteasome regulatory subunits. Proteasome is known to participate in the regulation of meiotic maturation and fertilization in fish and frog oocytes. Thus, we examined all possible proteasome genes. In C.elegans , 14 genes for proteasome components are predicted. cDNA are available in our collection for 13 genes including the 2 genes in the above analysis. Their mRNA were detected in gonads (12/13), strongly detected in extreme early stage embryo and disappear by early gastrulation (6/13) or localize to specific cells (6/13). We did RNAi experiments for these genes, and they showed high F1 embryonic lethality (12/13) and strong reduction of the number of F1 (12/13). The affected embryos did not show any cell divisions and seemed to be arrested during meiosis like the previous ones. Immunostaining for one of the genes showed strong staining at nuclei of oocytes. These results indicate that proteasome plays important roles during meiosis in C.elegans . The experiments on the genes of proteasome regulatory subunits are in progress. Some of them show similar results to that of proteasome components.

Hung, Wesley L. et al. (2013) International Worm Meeting "Attenuation of insulin signaling contributes to FSN-1-mediated regulation of synapse development."

Gao, Shangbang, Li, Hang, Hung, Wesley L., Hwang, Christine, Bessereau, Jean-Louis, Wang, Ying, Zhen, Mei, Liao, Edward H, Chitturi, Jyothsna, Stigloher, Christian

[International Worm Meeting, 2013]

The conserved neuronal F-box protein FSN-1 regulates neuromuscular junction development by negatively regulating DLK-mediated MAPK signaling in the presynaptic terminal (Liao et al., 2004; Nakata et al., 20005; Wu et al., 2007). We show here that attenuation of insulin/IGF signaling also contributes to FSN-1-dependent synaptic development and function. Loss of fsn-1 leads to aberrant synapse growth and a significant decrease in spontaneous vesicle release frequency from the neuromuscular junctions. These synaptic defects are partially and specifically rescued by decreasing insulin/IGF signaling activity in postsynaptic muscles, as well as by reducing the activity of EGL-3, a proprotein convertase that processes neuronal agonistic insulin/IGF ligands INS-4 and INS-6. FSN-1 interacts with, and potentiates the ubiquitination of EGL-3 in vitro, and reduces the level of EGL-3 in vivo. A constitutively activated MAPK, MKK-4(DD), can revert the suppression effect of fsn-1 by daf-2, indicating that MAPK pathway may act genetically downstream of the insulin pathway. We propose that FSN-1 negatively regulates insulin/IGF signaling and MAPK pathways to coordinate synapse growth signals from both pre- and postsynaptic terminals. Liao, EH, Hung, W, Abrams, B, Zhen, M. (2004) Nature 430:345 Nakata, K, Abrams, B, Grill, B, Goncharov, A, Huang, X, Chisolm, A, Jin, Y (2005) Cell 120:407 Wu, C, Daniels, RW, DiAntonio, A (2007) Neural Dev. 2:16.

Yuji Kohara et al. (2001) International C. elegans Meeting "Developmental expression map of C.elegans genome"

Tadasu SHIN-I, Masahiro Ito, Yuji KOHARA, Ikuko Sugiura, Mina Iwata, Masumi Obara, Kyoko Nakata, Michiko Serizawa, Takami Suzuki, Keiko Hirono, Yohei Minakuchi, Yumiko Ueta, Tokie Oba

[International C. elegans Meeting, 2001]

We have been performing whole mount in situ hybridization using the cDNAs that have been classified in our EST project (See Thierry-Mieg et al.) as gene probes. Although there was a big delay in the project last year because we needed almost half a year to fix problems due to which our standard protocol suddenly didn't work, now we get the project back on the track and have finished some 7,600 genes. We have given minimal annotation to the in situ results; 10 stages for embryogenesis, 4 stages for larval-adult stage, on average 10 patterns (cell(s), tissue, region) per stage, 3 levels of relative intensity of signals per each pattern. Using the information, clustering analysis and extraction of consensus sequences in the upstream regulatory regions are in progress (see Ito et al.). Subsets of genes whose expression patterns are identical are being subjected to various analysis including RNAi, immunostaining and bioinformatic analysis (motif search, for example). This is a unique and powerful approach and, for example, we identified the function of proteasome in oocyte maturation and fertilization (see Hirono et al.). All the relevant data are integrated in NEXTDB and we are preparing to make them open by the worm meeting (See the demo by Shin-i et al.). In this report, we will show the content and the results of studies based on the database. Furthermore, we are planning "Annotation Relay" in which experts in various fields are invited to this lab by turns (a week or two per round and several people per round depending on the number of microscope) and look at the in situ slides on the microscope with the expert eyes to give more precise and deeper annotations to the in situ data than those we gave.

Joseph Watson et al. (2004) West Coast Worm Meeting "RPM-1 regulates neuron specific gene expression."

Joseph Watson, Stuart Kim, Peter Roy, David M Miller III, Steve Von Stetina

[West Coast Worm Meeting, 2004]

With its well-defined neuronal architecture and powerful genetics, C. elegans is a useful model system for studies of neuronal differentiation. In an effort to identify genes that specify motor neuron fate, we screened for mutations that selectively reduce expression of acr-5 ::YFP in B-class motor neurons. Three of these isolates ( wd55, wd67, wd72 ) are new alleles of rpm-1 (Regulator of Presynaptic Morphology). The RPM-1 protein and its Drosophila homolog, Hiw, are localized to perisynaptic regions adjacent to the active zone. Mutations in rpm-1/hiw disrupt synaptic structure and function. This effect may result from disregulation of ubiquitin-dependent protein degradation (Liao, Hung et al. 2004) (McCabe, Hom et al. 2004). Genetic epistasis experiments indicate that RPM-1 function depends on downregulation of pmk-3 (p38 MAPK) signaling (K. Nakata and Y. Jin, pers. comm.). We have now shown that pmk-3 also suppresses disregulation of B-class motor neuron markers in rpm-1 mutants. This finding suggests that RPM-1, a component of the presynaptic apparatus, regulates transcription of specific target genes via a MAPK signaling pathway. We have used the mRNA tagging method (see Von Stetina et al., this meeting) to identify downstream targets of rpm-1 . We used a pan neural mRNA tagging line ( F25B3.3 ::FLAG::PAB-1) to isolate mRNA from all neurons in the rpm-1 deletion mutant, ok364 . From 4 independent data sets, we determined that 443 genes are upregulated and 260 genes are downregulated in rpm-1 mutant neurons. We are currently testing candidate genes from these lists for roles in RPM-1 function. Liao, E. H., W. Hung, et al. (2004). "An SCF-like ubiquitin ligase complex that controls presynaptic differentiation." Nature. McCabe, B. D., S. Hom, et al. (2004). "Highwire regulates presynaptic BMP signaling essential for synaptic growth." Neuron 41(6): 891-905.

Benjamin S Abrams et al. (2005) International Worm Meeting "A Cellular Analysis of Peri-active Zone Function at Synapses"

Benjamin S Abrams, Yishi Jin

[International Worm Meeting, 2005]

RPM-1 (Regulator of Pre-synaptic Morphology) is a large, evolutionarily conserved protein that functions in the organization and generation of pre-synaptic termini in C. elegans. In a typical presynaptic terminal, synaptic vesicles are neatly clustered around the electron-dense active zone. Loss of function mutations in rpm-1 cause alterations in the size and overall arrangement of presynaptic termini (Zhen et al., 2000). RPM-1 is localized to a region called the periactive zone, referring to a subsynaptic domain that is adjacent to synaptic vesicle clusters and the active zone. Based on our recent findings, we have proposed that the negative regulation of a MAP kinase pathway by RPM-1 may create a subsynaptic boundary at the periactive zone, which facilitates the formation and stabilization of pre-synaptic architecture (WCWM 04 Abstract 155, Nakata et.al. 2005). To further test this hypothesis and to gain insight into how this boundary is established we are looking at how the localization of RPM-1 is affected by synaptogenesis mutants. We are performing immunocytochemical analysis to examine the localization of endogenous RPM-1, in relation to the active zone marker UNC-10/RIM and vesicle domain marker Synaptotagmin/SNT-1. We have focused our analysis on synapses in the dorsal cord, ventral cord and SAB neurons. The stereotypic morphology of the SAB neurons provides a particularly good system to observe the effects of synaptic mutants at single synapse resolution. In these neurons, RPM-1 forms a gradient that is brightest at the nerve terminus and tapers off posteriorly. We have quantitated the effects of syd-1, syd-2, sad-1 and fsn-1 mutations on RPM-1s localization in these neurons. We are also using this strategy to investigate the structure/function relationship of RPM-1 using transgenes.

Kurup, Naina et al. (2015) International Worm Meeting "Temporal regulation of MT dynamics drives synapse remodeling."

Yan, Dong, Jin, Yishi, Goncharov, Alexandr, Kurup, Naina

[International Worm Meeting, 2015]

Connectivity changes in developing neuronal circuits involve large and small scale rearrangements of the underlying cytoskeleton. While changes to the microtubule (MT) cytoskeleton during dendritic plasticity have been well characterized, much less is known about how MT dynamics contributes to structural plasticity of pre-synaptic terminals. In C. elegans, the GABAergic DD neurons undergo synapse remodeling in the L1-L2 molt, termed DD remodeling, where pre-existing ventral synapses are eliminated and new synapses are formed along the dorsal neurite, without neurite growth or pruning (White et al., 1978). Here we characterize the role of the MT cytoskeleton in DD remodeling, using a double mutant animal containing a missense mutation in alpha-tubulin (tba-1(gf)) (Baran et al., 2010) and loss of the conserved MAPKKK DLK-1 (dlk-1(0)) (Nakata et al., 2005). DD remodeling is completely blocked in tba-1(gf) dlk-1(0) animals, and temporal activation of DLK-1 in the L2 stage restores DD remodeling. MT polarity is largely unchanged before and after DD remodeling, while a loss in dynamic MTs causes the failure in DD remodeling in tba-1(gf) dlk-1(0). We hypothesized that an increase in dynamic MTs is critical for axonal transport during new synapse formation. In support of this hypothesis, 4-D imaging during DD remodeling shows a significant reduction in SNB-1::GFP labeled vesicles trafficked from the ventral to the dorsal neurite in tba-1(gf) dlk-1(0) animals. Moreover, we identify gain-of-function alleles in Kinesin-1/UNC-116 that suppress tba-1(gf) dlk-1(0), through an increase in MT-motor binding affinity that promotes synaptic vesicle transport during DD remodeling. In summary, our data provides in vivo evidence of the importance of temporal regulation of dynamic MTs, under the control of DLK-1, in facilitating circuit plasticity.

Joseph D Watson et al. (2005) International Worm Meeting "The synaptic protein, RPM-1, regulates neuronal gene expression."

David M Miller, Stephen E Von Stetina, Joseph D Watson

[International Worm Meeting, 2005]

With its well-defined neuronal architecture and powerful genetics, C. elegans is a useful model system for studies of neuronal differentiation. In an effort to identify genes that specify motor neuron fate, we screened for mutations that selectively reduce expression of acr-5::YFP in B-class motor neurons. Surprisingly, three of these isolates (wd55, wd67, wd72) are new alleles of rpm-1 (Regulator of Presynaptic Morphology). The RPM-1 protein, and its homologues Highwire (Hiw) (Drosophila) and PHR1 (vertebrates) are localized to presynaptic regions adjacent to the active zone where they are believed to function as E3 ubiquitin ligases (Zhen, Jin et al 2004) (Wan, Goodman et al 2000). Mutations in rpm-1/hiw/Phr1 disrupt synaptic structure and function. This effect may result from the loss of ubiquitin-dependent protein degradation (Liao, Hung et al. 2004) (McCabe, Hom et al. 2004). Genetic epistasis experiments indicate that RPM-1 normally downregulates pmk-3 (p38 MAPK) signaling (K. Nakata and Y. Jin, et al. 2005). We have now shown that pmk-3 also suppresses disregulation of B-class motor neuron markers in rpm-1 mutants. This finding suggests that RPM-1, a component of the presynaptic apparatus, regulates transcription of specific target genes via a MAPK signaling pathway. We have used the mRNA tagging method (Roy and Kim, et al. 2002) to identify downstream targets of rpm-1. A pan neural mRNA tagging line (F25B3.3::FLAG::PAB-1) was used to isolate mRNA from all neurons in the rpm-1 deletion mutant, ok364; ~100 genes are enriched (> 2x) and ~50 genes are depleted (> 2x) relative to wildtype in rpm-1 mutant neurons. We have confirmed these effects for selected genes by quantitative RT-PCR. We are currently using RNAi in a sensitized genetic background to test candidate genes from these lists for roles in RPM-1 function. This work is expected to reveal downstream targets of RPM-1 with key roles in synaptogenesis.

Alexander Bounoutas et al. (2007) International Worm Meeting "mec-15 encodes an F-box protein with WD repeats required for proper touch receptor neuron mechanosensation, morphology, and synapse development."

Alexander Bounoutas, Kun Zheng, Michael L. Nonet, Martin Chalfie

[International Worm Meeting, 2007]

The mec-15 gene was originally identified in a screen for touch insensitive mutants. mec-15 mutations cause partial touch insensitivity that is temperature sensitive. The sam-3 mutation was identified in an independent screen for animals with mislocalized presynaptic markers in the touch receptor neurons; this phenotype is also temperature sensitive. mec-15 and sam-3 are the same gene, since mec-15 and sam-3 alleles fail to complement each other for partial touch insensitivity and presynaptic marker mislocalization. All alleles also affect touch receptor neuron morphology, causing an enlargement of the cell body. We used Snip-SNP mapping and gene sequencing to identify T01E8.4 as mec-15/sam-3. T01E8.4 encodes a 406 a.a. protein containing an F-box domain and WD repeats. Interestingly, F-box proteins, which act as protein substrate adaptors in ubiquitin ligase complexes, have been implicated in C. elegans ventral cord synapse formation (Liao et al. Nature 430: 345-350, 2004). All mec-15/sam-3 alleles save one are nonsense mutations in the T01E8.4 coding sequence; the remaining allele has two missense mutations. The mutant alleles in trans to a deletion of the region do not produce an enhanced phenotype, suggesting that the alleles are functional nulls. Transforming mec-15 mutants with the T01E8.4 gene rescues their mechanosensitive abnormal phenotype. The protein-GFP fusion is expressed in several neurons including the touch receptor neurons, where its expression is cytoplasmic, absent in adults, and dependent on the MEC-3 transcription factor. We are examining gene interactions to identify proteins that are needed for mec-15 action. Since a p38 MAP kinase pathway has been implicated in ventral cord synapse development (Nakata et al. Cell 120: 407-420, 2005), we examined mec-15 phenotypes in a pmk-3 background; all the phenotypes were enhanced. In addition, loss of one copy of the mec-7 <font face=symbol>b</font>-tubulin gene suppresses mec-15 phenotypes, suggesting an interaction between MEC-15 and the touch receptor neurons'' specialized microtubules.

Gloriana Gallegos et al. (2007) International Worm Meeting "Identifying Additional Components of the RPM-1 Pathway in Synapse Formation."

Yishi Jin, Gloriana Gallegos

[International Worm Meeting, 2007]

In the nervous system, communication between neurons and their targets is facilitated by a structure known as the synapse. The process of stimulated neurotransmitter release requires a variety of proteins, all of which must be precisely organized to create a properly functioning synapse. We have taken an unbiased genetic approach to identify the structural components and associated proteins responsible for shaping the presynaptic terminal in the nematode C. elegans. One key regulator identified is the RPM-1 (Regulator of Presynaptic Morphology) protein.1 RPM-1 is a highly conserved protein and one of its functions is to act as an E3 ubiquitin ligase to downregulate the MAPKKK DLK-1.2 Loss of function mutations in MAPKKK DLK-1, and the downstream MAPKK MKK-4 and MAPK PMK-3, all suppress the synaptic phenotypes in rpm-1 mutants. However, the suppression by the MAPK mutants is not complete, suggesting that other pathways and/or substrates of RPM-1 may exist. I have taken both forward and reverse genetic approaches to identify additional components of the RPM-1 pathway. I have analyzed about 20 suppressor mutations of rpm-1. By non-complementation tests, four novel mutations were found that are not members of the MAPK cascade, one of which is defined by ju600. Homozygous ju600 appears grossly normal and suppresses rpm-1 defects similar to the MAPK mutants. I have mapped ju600 to chromosome III using the snip-SNP method. Further genetic tests will be conducted to determine whether ju600 functions in the same pathway as the MAP kinases. 1 Zhen, M., et al., Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain. Neuron, 2000. 26(2): p. 331-43. 2 Nakata, K., et al., Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell, 2005. 120(3): p. 407-20.

Ya Dai et al. (2005) International Worm Meeting "Identification and mapping of two genes involved in synaptic development"

Yishi Jin, Ya Dai

[International Worm Meeting, 2005]

The function of a neuron depends on the formation of synaptic connections with its partners. Synapses are highly organized subcellular junctions with close apposition of specialized regions of plasma membranes of two participating cells. The presynaptic compartment accumulates neurotransmitter-filled synaptic vesicles in the vicinity of active zones, and is aligned with postsynaptic specializations to ensure the precise signal transmission in a neural network.Previous work from our lab has identified several genes that regulate the assembly of presynaptic terminals. To look for additional genes, we performed enhancer and suppressor screens in selected mutant background [1, 2]. The ju541 mutation was isolated as an enhancer in rpm-1 background. Homozygous ju541 mutants are slightly uncoordinated, and SNB-1::GFP puncta show variable defects as diffused or aggregated. ju541; rpm-1 double mutants show strong uncoordination, severe defasiculation in nerve cords and commissural branching. We mapped ju541 to the left arm of chromosome I.The ju487 mutation was identified as a partial suppressor of syd-1.syd-1 mutants show sluggish movement and Egl. The egg laying behavior is regulated by HSN neurons. The HSN neurons form en passant synapses onto the VC neurons and vulva muscles. HSN synapses can be visualized by Punc-86-SNB-1::GFP [3]. In syd-1 mutants, the SNB-1::GFP is completely retained in the cell body of HSN cell. In syd-1; ju487 mutant, 75% of the HSN neurons show normal localization of SNB-1::GFP. The ju487 mutant alone doesnt have any abnormality of HSN cell morphology and synapse formation. The suppression of syd-1 by ju487 is dependent on UNC-104, a kinesin that transports synaptic vesicles from the cell body to the synapses. These results suggest that ju487 may define a gene functioning downstream of syd-1. We mapped ju487 on the right arm of chromosome X.Further mapping and cloning of these two genes will be presented in the meeting.[1] Dai et al., 2004. WCWM ab148. [2] Nakata et al., 2005. Cell 120, 407. [3] Shen and Bargmann, 2003. Cell 112, 619.